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Hum Gene Ther:T细胞激活及衰竭在AAV基因治疗中的角色?

研究人员对使用腺相关病毒(AAV)输送基因进行基因治疗导致的免疫反应(活化免疫反应或免疫耐受)产生的方式的了解越来越深入。最近一篇发表在HumanGeneTherapy上的综述性文章对此进行了详细的解...即将发布

日期:2017年5月2日 - 来自[技术要闻]栏目
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‘Long Life‘ Gene Might Make Some Smarter, Too

By Dennis Thompson

HealthDay Reporter

TUESDAY, Jan. 27, 2015 (HealthDay News) -- A gene variant believed to "wire" people to live longer might also ensure that they keep their wits about them as they age, a new study reports.

People who carry this gene variant have larger volumes in a front part of the brain involved in planning and decision-making, researchers reported Jan. 27 in the Annals of Clinical and Translational Neurology.

These folks performed better on tests of working memory and the brain's processing speed, both considered good measures of the planning and decision-making functions controlled by the brain region in question.

"The thing that is most exciting about this is this is one of the first genetic variants we've identified that helps promote healthy brain aging," said study lead author Jennifer Yokoyama, an assistant professor of neurology at the University of California, San Francisco (UCSF). She noted that genetic research has mainly focused on abnormalities that cause diseases such as Alzheimer's and Parkinson's.

The gene involved, KLOTHO, provides the coding for a protein called klotho that is produced in the kidney and brain and regulates many processes in the body, the researchers said.

Previous research has found that a genetic variation of KLOTHO called KL-VS is associated with increased klotho levels, longer lifespan and better heart and kidney function, the study authors said in background information. About one in five people carries a single copy of KL-VS, and enjoys these benefits.

For this study, the researchers scanned the healthy brains of 422 men and women aged 53 and older to see if having a single copy of KL-VS affected the size of any brain area.

They found that people with this genetic variation had about 10 percent more volume in a brain region called the right dorsolateral prefrontal cortex, Yokoyama said.

This region is especially vulnerable to atrophy as people age, and its age-related decline may be one reason why older people can be easily distracted and have difficulty juggling tasks, she said.

Referring to the region as the "conductor of the brain's orchestra," Yokoyama said that it helps people "pay attention to certain types of things, to appropriately shift your attention and to engage working memory," which is the ability to keep a small amount of newly acquired information in mind.

日期:2015年1月28日 - 来自[Health News]栏目

3‘-Untranslated region of the type 2 bradykinin receptor is a potent regulator of gene expression

【关键词】  bradykinin

    Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee

    ABSTRACT

    Regulation of the constitutively expressed type 2 bradykinin (B2) receptor, which mediates the principal actions of bradykinin, occurs at multiple levels. The goal of the current study was to determine whether the human B2 3'-untranslated region (UTR) has effects on gene expression, with particular focus on the variable number of tandem repeats (B2-VNTR) polymorphic portion of the 3'-UTR and its flanking AU-rich elements (AREs). When inserted downstream of the luciferase coding region of the pGL3-Promoter vector, the B2-VNTR reduced reporter gene activity by 85% compared with pGL3-Promoter alone (promoter control; P < 0.001), an effect that was not appreciably affected by mutation of the flanking AREs. The negative regulatory effects of the B2-VNTR region were position and orientation dependent and strongly positively correlated with the number of tandem repeats in the B2-VNTR region (r = 0.85, P < 0.001). With respect to mechanism, quantitative RT-PCR revealed that the B2-VNTR mRNA level was 32% of that of promoter control (P = 0.008), whereas the number of polyadenylated transcripts was 4% (P = 0.02). In contrast, the mRNA half-life of the B2-VNTR was increased (B2-VNTR: 14.9 vs. promoter control: 12.2 h, P = 0.009). Transient transfection of human kidney-derived tsA201 cells with the B2-VNTR construct increased transcription of the native B2 receptor mRNA by 43% (P < 0.05), supporting an endogenous B2 receptor-regulatory capacity of the B2-VNTR. In conclusion, these results identify novel pretranslational effects of the B2-VNTR region to act as a potent negative regulator of heterologous gene expression and support the notion that the bradykinin B2 3'-UTR may impact endogenous receptor regulation.

    gene regulation; variable number of tandem repeats; polymorphism

    BRADYKININ HAS AN INTEGRAL relevance to a range of biological processes including vasodilatory control (25, 26), sodium homeostasis (1), fibrinolysis (6), thrombosis (18), pain (7, 12), allergy (19, 31), and inflammation (7, 19). The principal actions of bradykinin and the kinin system are mediated by the constitutively expressed type 2 (B2) receptor, which has extensive tissue distribution, notably in the kidney, heart, skin, and peripheral vasculature (17, 26). The use of bradykinin receptor antagonists specific for the B2 receptor as well as knockout and transgenic mice has established beneficial effects of kinins to reduce blood pressure (15, 25), oppose vasoconstrictive and growth effects of ANG II (20, 35), and stimulate fibrinolysis (6). Other studies support the involvement of the kinin system in the pathogenesis of asthma (19, 31), arthritis (10), and angioedema (19).

    Several levels of regulation of the B2 receptor have been described (4) of which receptor level regulation has been the most well characterized (30). With respect to transcriptional control, the rat B2 promoter has been extensively studied. Pesquero et al. (29) reported the capacity of the rat B2 receptor to be upregulated by the action of cAMP, bradykinin, phorbol esters, and by coexpression of an activated ras oncogene. A silencer element that occurs in a putative sterol responsive element (SRE-1) in the rat B2 promoter has been associated with a cell-specific downregulation of the B2 receptor (2). Marks et al. (27) demonstrated that p53 acts on an upstream sequence-specific transcriptional enhancer in the rat B2 promoter, which is conserved in rats and humans. The finding of B2 receptor upregulation which is dependent on the tumor suppressor protein, 53, suggests the importance of transcriptional control of the rat B2 receptor in renal epithelial cell development (13). In a recent study, Saifudeen et al. (14, 33) demonstrated the complexity of the p53 regulation of the B2 receptor by showing that p53 can physically interact with both cAMP response element binding protein (CREB) and Kruppel-like factor (KLF-4) to regulate B2 promoter function in the developing kidney.

    Whether significant posttranscriptional aspects of B2 regulation exist is less well studied (4, 30), whereas the 3'-untranslated region (UTR) mediates significant posttranscriptional regulation of the B1 receptor, largely through a reduction of stability of mRNA (41). The presence of regulatory elements in the noncoding exon 1 of the human B2 receptor has been postulated based on the finding of increased mRNA levels in subjects with a nine-base deletion variant in that region (23). Lung et al. (24) showed that interferon- caused an upregulation of bradykinin receptor mRNA and protein expression, which could be blocked by either transcriptional or protein synthesis inhibition, suggesting the existence of both transcriptional and posttranscriptional effects.

    The capacity for posttranscriptional regulation of the human B2 receptor exists by virtue of the existence of AU-rich elements (AREs) in the 3'-UTR, which can act in a context-specific manner to increase or decrease mRNA stability and otherwise have effects on translation (3). Specifically, two AREs which contain the core pentad, AUUUA, occur in the B2 3'-UTR and are located immediately upstream and downstream of a variable number of tandem repeats (VNTR)-type polymorphism, which was previously reported by Braun et al (5). The role of the AREs in B2 receptor regulation has not been tested.

    The goal of the current study was to determine the effects of the human B2 3'-UTR on gene expression, with particular focus on the VNTR polymorphic portion (B2-VNTR) of the 3'-UTR including the flanking ARE segments. In this report, we provide evidence that the B2 3'-UTR harbors a functional element within the B2-VNTR region that potently regulates gene expression which occurs through a non-ARE-dependent mechanism. We demonstrated that the B2-VNTR, which is not conserved among species, acts in a position- and orientation-dependent fashion in a reporter plasmid to reduce gene expression in human and nonhuman cell lines. We further showed that the transfection of the minimal B2-VNTR element in a human kidney-derived cell line, which expresses the bradykinin receptor, modulates the regulation of the endogenous B2 receptor.

    MATERIALS AND METHODS

    3'-UTR constructs. Segments of the human B2 receptor 3'-UTR were PCR-amplified using oligonucleotide primers containing flanking SacII and NdeI recognition sequences. For antisense constructs, the flanking restriction endonuclease sequences for the primers were interchanged. The PCR products were gel-purified and ligated downstream of the firefly luciferase coding region of the pGL3-Promoter vector (Promega, Madison, WI). The XbaI restriction site in this vector was reengineered with a linker segment to allow unidirectional insertion of the 3'-UTR. The pGL3-Promoter vector backbone was chosen because it contains the SV40 promoter without enhancers; therefore, changes in luciferase activity can be attributed to the effect of 3'-UTR inserts. The following primers were used to amplify the B2-VNTR region: forward: 5'-ctcagcaaccaagggattgt-3'; reverse: 5'-GGTCAGGATTTATGGGCTCTT-3'. The sequence of the most commonly occurring variant (43 repeats) of the B2-VNTR region was used in this experiment (5). All constructs were confirmed by direct sequencing.

    Cell culture/transient transfections. Human kidney-derived tsA201 cells, a subclone of the HEK293 cell line, were cultured in a defined medium made by mixing equal parts of DMEM and Coon's modified Ham's F-12 medium, 10% fetal bovine serum, and 100 U/ml penicillin-streptomycin in a humidified chamber maintained at 37°C containing 5% CO2. tsA201 cells were plated at 5060% confluence in 24-well dishes 24 h before transfection. Pilot studies were performed to optimize the ratio of DNA to the FuGENE 6 transfection agent (Roche Diagnostics, Indianapolis, IN) and demonstrate independent expression of the luciferase reporter and Renilla coreporter vector signals. For these transfections, a 3:2 ratio of FuGENE:cDNA was used. Before transfection, cells were washed three times with phosphate-buffered saline. The FuGENE-DNA mixture was divided equally among two wells, the second of which was used as a duplicate. Four-hundred microliters of serum-free DMEM were added to each well followed by incubation as described above for 4.5 h. Cells were then washed with phosphate-buffered saline and DMEM and 10% fetal bovine serum was added. Forty-eight hours later, cells were harvested and assayed for firefly and Renilla luciferase activity.

    Measurement of luciferase activity. To determine whether the B2 3'-UTR could confer transcriptional or posttranscriptional control of gene expression in a heterologous reporter system, we compared luciferase activity among chimeric luciferase constructs which contained either 1) the B2-VNTR which encompasses the midsegment of the B2 3'-UTR and flanking class I AREs (defined as the occurrence of an AUUUA pentamer in a non-U-rich region), 2) the 3'-UTR from the smooth muscle myosin light chain (SMMLC) (used as a length control), or the 3) insertion-less pGL3 Promoter (designated promoter control).

    Luciferase reporter assays were performed using the Dual-Glo Luciferase Reporter Assay System (Promega). At the time of harvest, the culture medium was removed, and cells were washed with phosphate-buffered saline. Passive lysis buffer (100 μl of 1x buffer) was added to each well, and plates were placed in a shaking incubator for 15 min at room temperature. For additional lysis, two freeze-thaw cycles were performed in which the cells were frozen to 80°C. The lysate was placed in a 96-well luminometer plate (Packard Bioscience, Billerica, MA) with an equal volume of Dual-Glo Luciferase Reagent and incubated for 10 min. Firefly luciferase luminescence was measured. Before measurement of Renilla luciferase (pRL-TK vector, Promega), 100 μl of the Stop and Glo reagent (Promega) were added to each well to quench the firefly luciferase reaction. Renilla luciferase luminescence was measured after an incubation of 10 min. All luciferase measurements represent an average of readings performed in triplicate. Fresh reagents were used for entire sets of samples and their duplicates to ensure equivalent reaction conditions. Relative firefly luciferase light output was normalized by Renilla luciferase output after appropriate subtraction of background light output. In most cases, data points represent the means ± SE of two or three iterations of three to seven independent experiments.

    Mutation assay. In the experiments regarding the role of the AREs in gene expression, the core AUUUA pentamer of each element was altered using site-directed mutagenesis performed according to the manufacturer's instructions (Stratagene, La Jolla, CA). Mutagenesis oligonucleotide primers (1st ARE forward: 5'-gggcagcactcattcacttgataaatgaataGGGattagctggttgg-3' and 2nd ARE forward: 5'-gggctagaacctggagaatgagaAAGGGTtacatggcaaagagccc-3'; mutated bases underlined) were added to purified plasmid DNA and extended during temperature cycling using Pfu Turbo DNA polymerase. The resultant product was digested with DpnI and transformed into DH5.1- cells. Mutagenesis products were confirmed by direct sequencing.

    Total RNA extraction, cDNA synthesis, and quantitative PCR. The levels of chimeric luciferase transcripts were determined using real-time quantitative reverse transcriptase PCR using SYBR Green chemistry. Total RNA was extracted from tsA201 cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to instructions from the manufacturer. To eliminate amplification of reporter plasmid DNA and genomic DNA, total RNA was treated with DNase RQI (Promega, 10 U) at 37°C for 30 min, followed by reisolation of RNA with phenol/chloroform treatment. RNA (1.0 μg) was reverse transcribed with SuperScript II RNase H reverse transcriptase (Invitrogen) using either random hexamer or oligo dT primers. After first-strand synthesis, the cDNA was quantified using the DNA-binding dye SYBR green I (Qiagen, Valencia, CA). Fluorescence was detected using an iCycler iQ sequence detection system (Bio-Rad, Richmond, CA). Luciferase amplification primers were forward: 5'-GCCTGAAGTCTCTGATTAAGT-3' and reverse: 5'-ACACCTGCGTCGAAGATGT-3'. Renilla luciferase primers were forward: 5'-ATGGGATGAATGGCCTGATA-3' and reverse: 5'-CAACATGGTTTCCACGAAGA-3'. Amplification primers for human glyceraldehyde-3-phosphate (GAPDH) were 5'-ACTCTGGTAAAGTGGATATTGTTGC-3' for the forward primer and 5'-GAAGATGGTGATGGGATTTCC-'3 for the reverse primer. For detection of the B2 receptor, the following primers were used: forward: 5'-TCAATGTTTCTGTCTGTTCGTG-3'; reverse 5'-AAAGGTCCCGTTAAGAGTGG-3'.

    Melting curves were generated to maximize fluorescence from SYBR green I binding to the desired amplicon. The starting quantity (SQ) of each sample was calculated using a standard curve derived from a 1-, 9-, and 27-fold dilution series of RNA for both pGL3-Promoter without insert and Renilla luciferase. The correlation coefficient for each standard curve was greater than 0.99. Real-time PCR reactions were performed in triplicate. The SQ mean was calculated for each time point and normalized for transfection efficiency by dividing by the Renilla SQ mean from the same RNA sample.

    EMSA. tsA201 nuclear and cytoplasmic extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL). RNA was generated using an in vitro transcription kit (Promega) and labeled by T4 PolyA polymerase (USB, Cleveland, OH) using biotinylated ATP. RNA was cleaned using an RNA Cleanup Kit (Qiagen) and quantified using RiboGreen (Molecular Probes, Eugene, OR). Binding was performed with 5 μg of protein (nuclear or cytoplasmic) and 23.5 ng of probe in binding buffer (Promega). For competition experiments, cold B2-VNTR or nonspecific competitor was added in 1025 x molar excess to protein extracts and incubated for 20 min followed by the addition of labeled probe and incubated 20 additional min on ice. Complexes were resolved using 5% polyacrylamide electrophoresis, and gel was transferred to a positively charged nylon membrane. Chemiluminescent signals were detected using BrightStar BioDetect (Ambion, Austin, TX) after X-ray film exposure.

    Statistical analysis. Differences in luciferase activity were compared using 2, Mann-Whitney, or general linear models with repeated-measures testing, where appropriate. All statistical tests were two-sided, and a value of P < 0.05 was considered statistically significant.

    RESULTS

    Characteristics of the B2-VNTR region. As shown in Fig. 1, the B2-VNTR contains the purine-rich tandem repeats polymorphism which is composed of four alleles, the most common of which is the 43-repeat allele (5), and also includes the flanking class I flanking AREs which contain the core AUUUA pentad (upstream: AAUAUUUAUUA; downstream: AAAAUUUACAU). With respect to species conservation, the B2-VNTR segment is also found in the chimpanzee (P. troglodytes) and contains 32 repeats but otherwise shows little conservation among species (Table 1), such that no identifiable VNTR exists in the B2 3'-UTR for the rat (R. norvegicus), mouse (M. musculus), or dog (C. familiaris). In humans and chimpanzees, the B2-VNTR segment is a purine-rich pocket (68 and 64%, respectively) in the 3'-UTR which otherwise has a purine percentage of 54%. Despite the lack of sequence identity in the B2 3'-UTR among species with respect to the VNTR, a purine-rich tandem repeat structure with considerable similarity to the B2-VNTR was identified in the rat, mouse, and dog in alternate chromosomal locations (Table 2).

    B2 3'-UTR is a potent regulator of gene expression. The tsA201 cells were transiently transfected with luciferase vectors (pGL3-Promoter) in which the B2-VNTR or SMMLC 3'-UTRs were inserted downstream of the luciferase coding region. Renilla-normalized luciferase activity for each construct was compared with the insertion-less pGL3-Promoter vector (promoter control). As shown in Fig. 2, the construct containing the B2-VNTR reduced luciferase expression by 85% compared with promoter control (85 ± 1%, Z = 4.4, P < 0.001). Gene expression of the construct containing the SMMLC 3'-UTR, used as a length control, was increased over that of promoter control (31 ± 1%; Z = 4.0, P < 0.001).

    AREs in the B2 3'-UTR do not affect gene expression. To determine the importance of the AREs to gene expression, one or both AREs were mutated by site-directed mutagenesis. As shown in Fig. 3, compared with the nonmutated B2-VNTR construct, normalized luciferase activity was minimally different for constructs in which the first (difference = 2%, Z = 2.9, P = 0.004) or both AREs were mutated (difference = 3%, Z = 2.9, P = 0.004). These data suggest that the AREs do not have appreciable functional effects on gene expression in the context of the B2 3'-UTR.

    Effects of B2-VNTR on gene expression are position and orientation dependent. To assess whether the B2-VNTR region could operate in the inverse orientation, the reverse complement of the B2-VNTR segment was cloned into the pGL3-Promoter vector downstream of the luciferase coding region (B2-VNTR-INVERSE). This construct reduced luciferase activity to 56% of promoter control (P < 0.001) or approximately two-thirds of the reduction attributable to the sense orientation of the B2-VNTR construct (Fig. 4).

    To determine whether the location of the B2-VNTR in the luciferase reporter plasmid influenced its capacity to alter reporter gene expression, both the B2-VNTR and B2-VNTR-INVERSE segments were inserted into the multiple cloning region (MCR) adjacent to the SV40 promoter of the pGL3-Promoter vector. As shown in Fig. 4, transcription attributable to these constructs was not significantly different from that of promoter control, nor was there a difference when the B2-VNTR was inserted in a position downstream of the SV40 late polyA signal in the pGL3-Promoter vector (Fig. 4). These results support that contention that the effects of the B2-VNTR on transcription are strictly position dependent but are also orientation dependent such that the maximal effect is observed in the sense orientation.

    Multiple cis-elements are associated with a reduction in gene expression by the B2 3'-UTR. To test the hypothesis that cis-acting elements, other than the AREs, within the B2-VNTR region mediated the effects on gene expression, we compared luciferase expression among a series of 3'-UTR deletion constructs (Fig. 5). The maximal reduction of gene expression was observed with the B2-VNTR, which includes upstream and downstream flanking regions. The upstream flanking region tested alone significantly reduced gene expression (58%; Z = 3.1; P = 0.002), whereas the effect of the downstream flanking region was modest (27%; Z = 3.1; P = 0.002) compared with promoter control. The number of tandem repeats in the B2-VNTR region correlated positively with the reduction in gene expression (r = 0.85, P < 0.001). Taken together, these data support the presence of elements both within and upstream of the B2-VNTR region that reduce gene expression but suggest that the regulatory potential of this region depends on a critical number of tandem repeats.

    B2-VNTR is associated with reduced mRNA levels but not accelerated mRNA degradation. To determine whether accelerated mRNA decay can contribute to the observed reduction in luciferase activity associated with the B2-VNTR, real-time quantitative PCR was used to compare mRNA levels (normalized log SQ mean) between the B2-VNTR and promoter control at various timepoints after the addition of transcriptional inhibitor actinomycin D, which was initially added at 48 h following transfection, denoted as time 0 (Fig. 6). These data were normalized by Renilla to control for transfection efficiency. With respect to mRNA decay, the calculated half-life of the B2-VNTR construct was significantly greater than that of the promoter control (B2-VNTR: 14.9 vs. promoter control: 12.2 h, Z = 2.6; P = 0.009). These data were qualitatively similar to that obtained for the respective constructs after transfection in Madine-Darby canine kidney (MDCK-2) cells (data not shown). These data do not suggest that an accelerated degradation of mRNA contributed to the observed reduction in reporter gene expression associated with the B2-VNTR.

    B2-VNTR is associated with decreased polyadenylation of heterologous transcripts. To determine more precisely whether a posttranscriptional mechanism contributes to the effect of B2-VNTR to reduce luciferase activity, we measured total RNA and polyadenylated mRNA levels for each of the constructs. As summarized in Table 3, total RNA (normalized SQ mean) for the B2-VNTR construct was 32% of that of promoter control (B2-VNTR vs. promoter control: 0.27 ± 0.12 vs. 0.86 ± 0.04; P = 0.008) at 48 h following transfection. The B2-VNTR construct was associated with reduced polyadenylation efficiency which resulted in a polyadenylated B2-VNTR mRNA level that was 4% of promoter control (B2-VNTR vs. promoter control: 0.03 ± 0.01 vs. 0.74 ± 0.11; P = 0.02). These results support the notion that the B2-VNTR affects processes involved in effective 3'-end formation of mRNA.

    EMSA of B2-VNTR using nuclear and cytoplasmic protein extracts. To assess the capacity of the B2-VNTR region to bind proteins, we performed gel shift assays in which a biotinylated minimal B2-VNTR element (the 203-bp segment designated 12-REPEATS in Fig. 5) was exposed to both nuclear and cytoplasmic tsA201 protein extracts. As shown in Fig. 7A, the addition of the minimal B2-VNTR element to the nuclear proteins resulted in a specific band shift that was not observed with the addition of a comparably sized nonspecific competitor. In contrast, the pattern of banding was similar between the B2-VNTR and the competitor using cytoplasmic protein extracts (Fig. 7B). These data suggest that the regulatory effect associated with the B2-VNTR may involve protein binding which occurs in the nuclear compartment.

    Native B2 receptor transcription is increased with cotransfection of the B2-VNTR. To determine whether the B2 3'-UTR has the potential to affect endogenous B2 receptor transcription, the B2-VNTR and promoter control were transiently transfected separately (n = 5) in tsA201 cells, and the native B2 receptor mRNA was measured using real-time quantitative PCR. Following normalization with GAPDH, native B2 mRNA was increased by 43% (Z = 2.0; P < 0.05) in cells transfected with the B2-VNTR compared with those transfected with promoter control (Table 4). These data suggest that the B2 3'-UTR has the capacity to influence the regulation of the endogenous B2 receptor.

    DISCUSSION

    In this study, we demonstrated that the human B2 3'-UTR harbors elements that negatively regulate gene expression. We focused on the portion of the 3'-UTR (B2-VNTR) that contained a tandem repeats-type polymorphism that is flanked by AREs. Many studies demonstrated the importance of AREs as putative mediators of posttranscriptional and translational control (37, 38). By binding trans-acting factors, such as hnRNPs and Hu proteins, AREs exert regulatory control of gene expression through rapid degradation of mRNAs and/or repression of translation (3) and operate in a context- and stimulus-specific manner (38). In the current study, however, we detected no effects of the AREs contained in the B2-VNTR region to mediate or modify the observation of reduced luciferase expression. In contrast, we found that the VNTR segment of the B2 3'-UTR itself accounted for the significant negative regulatory effect on reporter gene activity.

    Regulatory sequences downstream of the promoter have the capacity to modulate transcriptional (8, 34), posttranscriptional (24), and even translational activity (21, 32, 39, 40). For example, Carrion et al. (8) demonstrated the existence of a downstream regulatory element (DRE) sequence that mediates position-dependent, but orientation-independent, repression of prodynorphin gene transcription from its location in the 5'-UTR. Sanz et al. (34) reported an example of a DRE sequence, which is located downstream of the open reading frame in the 3'-UTR of the apoptotic hrk gene, that on binding a repressor protein results in transcriptional silencing. Analysis of the DRE sequence revealed a core GTCA sequence, similar to that found in CRE and AP-1 elements, which could also function in an inverse orientation (22). In contrast to this situation, we found no evidence for a regulatory effect of a segment containing fewer than five copies of the core tandem repeat sequence of the B2-VNTR; however, the degree of repression was strongly positively correlated with the number of tandem repeats (Fig. 5). Using a deletional analysis, we determined that the capacity of the B2-VNTR to repress gene expression was largely localized to the proximal portion of the element, and its effects, like the DRE sequence, were position dependent, but in contrast to the DRE sequence were more marked in the sense orientation.

    To address more specifically the level at which the B2-VNTR acts to regulate gene expression, quantitative RT-PCR revealed that the total RNA level of the B2-VNTR construct was 32% of that of promoter control. With respect to the genomic structure of the B2 receptor, the B2-VNTR occurs in the untranslated portion of the terminal exon, suggesting the possibility that the B2-VNTR could influence the 3'-end formation of mRNA. In support of this contention, our data showed that the B2-VNTR region had a marked effect on the efficiency of polyadenylation such that the number of polyadenylated RNA transcripts was 4% compared with control levels. Given the complexity of transcriptional regulation, additional studies are necessary to rule out the contribution of other transcript processing events. Taken together, these results are consistent with the hypothesis that the B2-VNTR acts at a point before the successful transport of transcripts into the cytoplasm. Indeed, our gel shift data showed that the B2-VNTR binds nuclear but not cytoplasmic protein extracts and supports the notion that the mechanism of the B2-VNTR to repress reporter gene expression occurs at a pretranslational level.

    Somewhat surprisingly we found that the B2-VNTR was associated with increased mRNA stability (Fig. 6), which suggests the possibility that the B2-VNTR can also affect translational events. Several studies demonstrated that untranslated regions, notably the 3'-UTR, can critically impact translational regulation (21, 32, 39, 40). By virtue of the potential to bind trans-acting factors, in some cases directed by specific structural features, the 3'-UTR can act to either enhance or repress translation, the latter of which is exemplified in the 3'-UTR of the 15-lipoxygenase (15-lox) gene. Translational repression occurs in 15-lox by a mechanism that involves protein binding of heteronuclear RNP K and -CP to a motif that is comprised of a pyrimidine-rich 19 nt tandem repeat located in the 3'-UTR (28).

    Several aspects of B2 receptor regulation have been well characterized. In studies of the rat B2 promoter, factors such as cAMP (9, 11, 29) can transcriptionally increase B2 receptor expression in a cell-specific manner and may contribute to the cytokine stimulation of the B2 receptor by interleukin-1 (36). Transcriptional control of the B2 receptor may have particular relevance in the developing kidney where a complex regulatory role of p53 has been demonstrated (33). A transcriptional silencing mechanism in the rat B2 promoter has also been reported that is mediated by a putative SRE-1 site (2). In the context of inflammation, Lung et al. (24) characterized an upregulation of human B2 receptor mRNA in response to interferon- that was dependent on both transcriptional and posttranscriptional components.

    In the current study, we addressed the potential for the B2-VNTR to affect endogenous regulation of the B2 receptor. Cotransfection of the B2-VNTR in cells expressing the B2 receptor (tsA201) was associated with a 43% increase in native B2 receptor mRNA level. These results coupled with the gel-shift data that show binding of nuclear proteins to the B2-VNTR support the speculation that a nuclear protein(s) bound by the B2-VNTR has the capacity to negatively regulate events leading to the processing of mature mRNA transcripts.

    It is unknown whether the mechanism by which the B2-VNTR segment regulates gene expression is applicable to other species or other genes. With respect to comparative genomics, the B2-VNTR segment in the human B2 receptor is similar to that of the chimpanzee but displays little sequence conservation with the rat, mouse, or canine B2 receptor sequences. Remarkably, as shown in Table 2, a tandem repeat structure which bears considerable similarity to that of the B2-VNTR occurs in these latter species in alternate chromosomal locations, the significance of which awaits further study.

    In summary, we identified a novel effect of the B2 3'-UTR to act as a potent negative regulator of heterologous gene expression and provided data to support that the B2-VNTR modulates transcript-processing events such as polyadenylation which impact the efficiency of 3'-end formation. In addition, we identified the capacity for the B2-VNTR element to participate in the regulation of the native B2 receptor in a human kidney-derived cell line. Studies involving further fine localization of regulatory sequences in the B2-VNTR and identification of binding proteins will help address the relevant questions of whether the B2 3'-UTR contributes to the endogenous regulation of the B2 receptor in vivo and participates in kinin-dependent pathophysiology and also determine the generalizability of this 3'-UTR-based mechanism to the regulation of other genes.

    GRANTS

    This work was supported by National Institutes of Health Grants HL-70754 and RR-00095 and by a Pharmaceutical Research and Manufacturers Association Foundation Career Development Award (to J. V. Gainer).

    ACKNOWLEDGMENTS

    We thank A. L. George, N. J. Brown, and G. Valadez for helpful conversations and a critical reading of this manuscript.

    FOOTNOTES

    The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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日期:2013年9月26日 - 来自[2006年第289卷第3期]栏目
循环ads

Differential renal gene expression in prehypertensive and hypertensive spontaneously hypertensive rats

【关键词】  Differential

    Division of Intramural Research and Microarray Group, National Center for Toxicogenomics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina
    Department of Biopharmaceutical Sciences, School of Pharmacy, University of California, San Francisco, California

    ABSTRACT

    Development of hypertension stems from both environmental and genetic factors wherein the kidney plays a central role. Spontaneously hypertensive rats (SHR) and the nonhypertensive Wistar-Kyoto (WKY) controls are widely used as a model for studying hypertension. The present study examined the renal gene expression profiles between SHR and WKY at a prehypertensive stage (3 wk of age) and hypertensive stage (9 wk of age). Additionally, age-related changes in gene expression patterns were examined from 3 to 9 wk in both WKY and SHR. Five to six individual kidney samples of the same experimental group were pooled together, and quadruplicate hybridizations were performed using the National Institute of Environmental Health Sciences Rat version 2.0 Chip, which contains 6,700 genes. Twenty two genes were found to be differentially expressed between SHR and WKY at 3 wk of age, and 104 genes were differentially expressed at 9 wk of age. Soluble epoxide hydrolase (Ephx2) was found to be significantly upregulated in SHR at both time points and was the predominant outlier. Conversely, elastase 1 (Ela1) was found to be the predominant gene downregulated in SHR at both time points. Analysis of profiles at 3 vs. 9 wk of age identified 508 differentially expressed genes in WKY rats. In contrast, only 211 genes were found to be differentially expressed during this time period in SHR. The altered gene expression patterns observed in the age-related analysis suggested significant differences in the vascular extracellular matrix system between SHR and WKY kidney. Together, our data highlight the complexity of hypertension and the numerous genes involved in and affected by this condition.

    soluble epoxide hydrolase; hypertension; arachidonic acid; elastase; real-time polymerase chain reaction

    HYPERTENSION IS A MAJOR RISK factor for cardiovascular disease, renal failure, and stroke and is associated with significant morbidity and mortality (6). Many systems and factors contribute to the regulation of blood pressure, such as the renin-angiotensin-aldosterone system, extracellular matrix, and endothelin. Alteration in the complex array of polygenic and environmental factors that regulate blood pressure results in hypertension. Such perturbations commonly affect salt homeostasis, intravascular volume, and systemic vascular resistance (23). Even with such a diversity of physiological systems that control blood pressure, the majority of genetic and acquired forms of hypertension involve the kidney (23, 28, 38). Indeed, renal transplantation studies demonstrating the transfer of the hypertension phenotype from donor to recipient highlight a key role of the kidney in this disease (15, 39). Investigation into the renal gene expression profiles that accompany hypertension will help identify potentially important causes of this disease and/or novel therapeutic targets.

    Increased prevalence of hypertension with age coincides with changes in blood pressure patterns and reflects differences in hemodynamics between young and old hypertensives (1012, 34, 4143). For example, a shift from peripheral arterial resistance to arterial stiffness occurs with age (1012, 34, 4143). Besides environmental factors such as diet and lack of exercise, genetic studies provide evidence that intrinsic factors may also contribute to the development of hypertension with age. A common animal model used to investigate hypertension is the spontaneously hypertensive rat (SHR) and the normotensive control Wistar-Kyoto (WKY) rat strain (31). These animals exhibit similar age-dependent and end-organ damage phenotypes as observed in humans (28, 38).

    We performed cDNA microarray analysis to investigate differences in renal gene expression between SHR and WKY rats at both a prehypertensive stage (3 wk of age) and during the developmental phase of hypertension in the SHR (9 wk of age). Although blood pressure is similar in SHR and normotensive controls at 3 wk of age, changes in glomerular function, pressure-natriuresis, and vascular structure are well documented in prehypertensive SHRs (8, 40, 46). At 9 wk of age, blood pressure is still rising rapidly in the SHR and is elevated relative to the WKY (46). Comparisons between WKY and SHR at each time point allow the detection of differentially expressed genes that might contribute to the distinct blood pressure and vascular and renal phenotypes at each age. A comparison of changes in the SHR between 3 and 9 wk of age allows for the detection of temporal gene changes that might be associated with the blood pressure changes during this period. Numerous differences in the profiles between SHR and WKY were observed and independently validated, as were temporal changes within each strain, thus identifying several potentially important genes involved in blood pressure regulation and the development of hypertension.

    MATERIALS AND METHODS

    Animals and RNA isolation. Kidney RNA was isolated from individual male and female SHR and WKY rats at ages corresponding to the prehypertensive (3 wk) and hypertensive (9 wk) stages of life. SHR and WKY rats (3 and 9 wk old) were purchased from Charles River Laboratories (Wilmington, MA). Rats were anesthetized with methoxyflurane, abdominal cavities were opened, and kidneys were perfused with ice-cold PBS solution. Kidneys were rapidly removed, cut into small pieces, and immersed immediately in liquid nitrogen. All tissues were stored at 80°C until preparation of RNA. Total RNA was isolated using an RNeasy Midi kit (Qiagen, Valencia, CA) and concentrated using a Microcon YM-30 column (Millipore, Billerica, MA). A formaldehyde agarose gel containing ethidium bromide was used to assess the quality of the RNA.

    Microarray hybridization. The National Institute of Environmental Health Sciences (NIEHS) cDNA Rat version 2.0 Chip, which contains 6,700 genes, was used for gene expression profiling experiments. A complete listing of the genes on this chip is available at the following website: http://dir.niehs.nih.gov/microarray/chips.htm. The cDNA microarray chips were prepared as previously described (7). The spotted cDNAs were derived from a collection of sequence-verified IMAGE clones that spanned the 5'-end of the genes and ranged in size from 500 to 2,000 bp (Incyte Genomics, Palo Alto, CA). M13 primers were used to amplify insert cDNAs from purified plasmid DNA in a 100-μl PCR reaction mixture. A sample of the PCR products (10 μl) was separated on 2% agarose gels to ensure quality of the amplifications. The remaining PCR products were purified by ethanol precipitation, resuspended in ArrayIt Spotting Solution Plus buffer (Telechem, San Jose, CA), and spotted on poly-L-lysine-coated glass slides using a modified, robotic DNA arrayer (Beecher Instruments, Bethesda, MD). Each total RNA sample (1575 μg), representing five to six individual animals per experimental group, was labeled with cyanine 3 (Cy3) or cyanine 5 (Cy5)-conjugated dUTP (Amersham, Piscataway, NJ) by a reverse transcription reaction using SuperScript RT (Invitrogen, Carlsbad, CA) and oligo(dT) primer (Amersham). The fluorescently labeled cDNAs were mixed and hybridized simultaneously to the cDNA microarray chip. Each RNA pair was hybridized to a total of four arrays employing a fluor reversal accomplished by labeling the control sample with Cy3 in two hybridizations and with Cy5 in the other two hybridizations. The cDNA chips were scanned with either an Axon Scanner (Axon Instruments, Foster City, CA) or an Agilent Scanner (Agilent Technologies, Wilmington, DE) using independent laser excitation of the two fluors at 532- and 635-nm wavelengths for the Cy3 and Cy5 labels, respectively. Pairwise comparisons were carried out as described in Table 1.

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    The raw pixel intensity images were analyzed using the ArraySuite version 2.0 extensions of the IPLab image processing software package (Scanalytics, Fairfax, VA). This program uses methods that were developed and previously described by Chen and coworkers (5) to locate targets on the array, measure local background for each target and subtract it from the target intensity value, and identify differentially expressed genes using a probability-based method. The data were filtered to provide a cut-off at the intensity level just above the buffer blank measurement values to remove those genes having one or more intensity values in the background range from further analyses. The ratio intensity data from all of the spots printed on the Rat Chip were used to fit a probability distribution to the ratio intensity values and estimate the normalization constants (m and c) that this distribution provides. The constant m, which provides a measure of the intensity gain between the two channels, indicated that the channels were approximately balanced near a value of 1.0. For each array, the ratio intensity values were normalized to account for the imbalance between the two fluorescent dyes by dividing the ratio intensity value by m. The other constant, c, estimates the coefficient of variation for the intensity values of the two samples. All arrays in this analysis had a c value of 0.12 or less. The probability distribution that is fit to the data was used to calculate a 95% confidence interval for the ratio intensity values. Genes having normalized ratio intensity values outside of this interval were considered significantly differentially expressed. For each of the four replicate arrays for each sample, lists of differentially expressed genes at the 95% confidence level were created and deposited in the NIEHS MAPS database (3). For each time point, a query of the database yielded a list of genes that were differentially expressed in at least three of the four replicate experiments. Any of these genes that indicated fluor bias or high variation were not considered for further analysis. Assuming that the replicate hybridizations are independent, a calculation using the binomial probability distribution indicated that the probability of a single gene appearing on this list when there was no real differential expression is 0.00048.

    Hierarchical clustering was performed using Eisen's Cluster/Treeview software package (http://rana.lbl.gov; see Ref. 9). The entire data are available at the following website: http://dir.niehs.nih.gov/microarray/seubert/.

    Independent validation by Northern analysis and RT-PCR. The identity of microarray chip outlier cDNAs was confirmed by direct sequencing. Plasmid DNA was prepared using a QIAprep Mini-prep kit (QIAGEN) and completely sequenced using an ABI Prism BigDye DNA sequencing kit (Applied Biosystems, Foster City, CA). Sequence identity was confirmed using a BLAST search (National Center for Biotechnology Information/National Institutes of Health). Northern blot analysis was used to verify altered expression of RNAs in SHR and WKY kidney as previously described (25). Briefly, blots were probed with IMAGE clones (Research Genetics/Invitrogen) identified as outliers by microarray analysis. Fragments were isolated using a QIAquick Gel Extraction Kit (Qiagen), labeled with [-32P]dCTP using a Random Primed DNA Labeling Kit (Roche Applied Science, Indianapolis, IN), and purified by NICK Columns (Amersham Biosciences). Autoradiographs were scanned, and relative RNA levels from SHR and WKY kidney were determined by normalization to -actin expression. Statistically significant differences in the relative RNA levels between SHR and WKY kidney were determined using a Student's t-test. Values were considered significantly different at P < 0.05.

    To address discrepancies between Northern blot and microarray chip results, we performed independent quantitative RT-PCR analysis of selected genes. Total RNA from individual WKY and SHR kidneys were treated with DNase I, and then 1 μg was used to prepare cDNA with the High-Capacity cDNA Archive Kit (Applied Biosystems). cDNA levels were detected using real-time PCR with the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) and SYBR Green I dye. Primers were created using the Oligo 6.4 program. (Supplemental data for this article may be found at http://ajprenal.physiology.org/cgi/content/full/00354.2004/DC1; see supplemental Table 10). For cDNA amplification, 1 ng cDNA was combined with 12.5 μl 2x SYBR Green Master Mix (Applied Biosystems), 1 μl forward and reverse primers (10 μM each), and 6.5 μl RNase-free water for a total volume of 25 μl. Samples were analyzed in triplicate, and a RT sample was included with each plate to detect contamination by genomic DNA. Amplification was as follows: 1) 50°C, 2 min (for uracil-N-glycosylase incubation); 2) 95°C, 10 min (denaturation); and 3) 40 cycles of 95°C, 15 s, 60°C, 30 s (denaturation/amplification). Dissociation curves were also created by adding the following steps to the end of the amplification reaction: 95°C, 15 s (denaturation), 60°C, 15 s, and then gradually increasing to 95°C over 20 min, and finally holding at 95°C for 15 s. The degree of induction or repression was determined by quantitation of cDNA from target (SHR) samples relative to a calibrator sample (WKY). For all samples, the gene for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the endogenous control for normalization of initial RNA levels. To determine this normalized value, 2Ct values were compared between target and calibrator samples, where Ct = target gene (crossing threshold) Ct GAPDH Ct, and Ct = CtSHR CtWKY (24).

    RESULTS

    Cluster analysis. Expression patterns were initially examined using cluster analyses generated from outlier lists of average gene expression ratio intensities at the 95% confidence level (Fig. 1). The expression patterns were remarkably similar between the quadruplicate hybridizations (data not shown). Interesting patterns emerged that highlight differences in expression profiles between the hybridization groups (Fig. 1). Region A represents genes that were downregulated from 3 to 9 wk of age in both SHR and WKY but were expressed at higher levels in SHR than WKY at both time points. In contrast, region C represents genes that were generally expressed at lower levels in SHR than WKY at 9 wk of age but were increased in expression in both SHR and WKY from 3 to 9 wk of age. Region B represents genes that do not change expression significantly in either SHR or WKY from 3 to 9 wk of age but are expressed at higher levels in SHR than WKY at 9 wk. Region D represents genes that were expressed in lower levels in SHR than WKY at 9 wk of age but did not show significant changes in expression from 3 to 9 wk in either SHR or WKY. Gene lists for each of these regions are found in the on-line data supplement. (Supplemental data for this article may be found at http://ajprenal.physiology.org/cgi/content/full/00354.2004/DC1).

    Differentially expressed genes in 3-wk-old WKY vs. SHR and 9-wk-old WKY vs. SHR. Microarray analysis comparing RNA isolated from 3-wk-old WKY vs. SHR animals identified 22 genes that were differentially expressed at the 95% confidence interval (Table 2). When analysis was performed comparing RNA from 9-wk-old WKY and SHR animals, 104 outliers were identified. A partial list containing outliers with the largest increase or decrease is found in Table 3. The complete gene lists of outliers are found in the on-line data supplement. (Supplemental data for this article may be found at http://ajprenal.physiology.org/cgi/content/full/00354.2004/DC1). Seven genes were differentially expressed between SHR and WKY at both 3 and 9 wk of age (Fig. 2). Soluble epoxide hydrolase (Ephx2), the gene with the largest increase in expression, was found to be significantly upregulated in SHR at both time points. Conversely, elastase 1 (Ela1), the gene with the largest decrease in expression, was found to be downregulated in SHR at both time points compared with WKY. Ninety-seven genes were differentially expressed between SHR and WKY only at 9 wk of age (Fig. 2). Examples include peroxisomal calcium-dependent solute carrier-like protein (Pcsc1), collagen type 1 (Col1a1), and cathespin L (Ctsl). Genes involved in lipid metabolism, such as CD36 antigen (downregulated) and apolipoprotein H (upregulated), were also differentially expressed in SHR vs. WKY at 9 wk of age. Glutathione-S-transferase mu type 2 (Yb2) exhibited lower expression in SHR at 9 wk consistent with its role in diastolic blood pressure regulation (48). The G protein binding protein critical renal failure gene exhibited higher expression levels in SHR at 9 wk and has been previously shown to be differentially expressed in renal disease (21).

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    Age-related alterations in gene expression in WKY and SHR. Separate microarray analyses were performed to compare gene expression changes between 3- and 9-wk-old WKY or between 3- and 9-wk-old SHR. A total of 508 genes were differentially expressed at the 95% confidence interval in WKY animals, whereas only 211 genes were differentially expressed in SHR. A partial list containing outliers with the largest increase or decrease in expression is found in Table 4 (for SHR and WKY). The complete gene lists of outliers are found in the on-line data supplement. (Supplemental data for this article may be found at http://ajprenal.physiology.org/cgi/content/full/00354.2004/DC1.) The most notable difference in the gene expression patterns between 3 and 9 wk of age in SHR and WKY was the greater number of changes observed in WKY animals. When comparing all the genes that were differentially expressed during this time period, only 174 were found in both outlier lists for SHR and WKY (Fig. 3). The majority of outliers observed in SHR were also present in WKY. Lysozyme (Lyz) was the predominant gene upregulated in both SHR and WKY between 3 and 9 wk of age but was upregulated to a much greater extent in WKY than in SHR. Decreased expression in collagen-related genes was prevalent in both SHR and WKY. For example, collagen type 1 (Col1a1) was the predominant gene downregulated in both SHR and WKY between 3 and 9 wk of age. However, increased expression of cathespin H (Ctsh), cathespin L (Ctsl), and elastase (Ela1) was observed only in WKY from 3 to 9 wk of age. In addition, cathespin L and elastase were found to have significantly lower expression in SHR compared with WKY (Tables 2 and 3).

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    Analysis of the expression patterns of genes that are involved in arachidonic acid metabolism or blood pressure regulation indicated few significant alterations (see supplemental Tables 2 and 3).

    Independent validation of microarray results. To validate the microarray results, we first confirmed the identity of 28 selected cDNAs used on the chip by direct sequencing (Table 5). cDNAs (100%) were found to be identical to the clone ID from the IMAGE collection. Next, we performed Northern blot analysis on RNAs prepared from individual SHR and WKY kidneys for 28 selected outliers. Actin was used to normalize relative changes on Northern blots, since no significant differences were observed in actin expression (AA859846 [GenBank] ) between SHR and WKY samples on the microarray chip. We observed excellent concordance between array data and Northern blot data with respect to the direction and magnitude of expression changes (Fig. 4 and Table 5). Our concordance rates were 73% for 3-wk samples and 77% for 9-wk samples. However, there were discrepancies between Northern blot and microarray data. Given that quantitative RT-PCR is a more sensitive method for validating array data than Northern blot, we performed RT-PCR analysis on 15 outliers. When there was a discrepancy between Northern blot and RT-PCR, we gave priority to the RT-PCR results in deciding whether or not an outlier gene was validated. We considered a gene to validate if a statistically significant difference was found, and it changed in the same direction as the microarray results. With this definition, our concordance rate was 89% (25 out of 28 genes). Of the genes we examined, the three that did not validate were expressed sequence tags.

    View this table:

    DISCUSSION

    In the present study, we investigated renal gene expression profiles in SHR and WKY animals at prehypertensive (3 wk of age) and hypertensive (9 wk of age) stages. We identified 22 genes at 3 wk of age and 104 genes at 9 wk of age that were differentially expressed in SHR compared with WKY. Our study utilized the NCrINR SHR strain and yielded a similar list of differentially expressed genes at 9 wk of age as those reported by Okuda et al. (32) in the NCrj SHR strain at 10 wk of age. Our study was unique in that it identified age-dependent differences in renal gene expression between SHR and WKY animals. Most notable was the fewer number of genes that were differentially expressed between 3 and 9 wk of age in SHR (211) compared with WKY (508) kidneys, suggesting developmental differences in renal gene expression may be responsible, at least in part, for the SHR phenotype.

    We found soluble epoxide hydrolase (sEH; EphX2) to be the predominant gene upregulated in SHR at both 3 and 9 wk of age. Consistent with other reports, these differences in sEH expression suggest an important role for eicosanoids in renal vascular homeostasis (17, 33, 45, 53, 54). Abundant expression of sEH is found in the kidney and localized to smooth muscle layers of the arterial wall (37, 50, 53). Arachidonic acid is first converted to epoxyeicosatrienoic acids (EETs) by cytochrome P-450 epoxygenases and then to corresponding diols [dihydroxyeicosatrienoic acids (DHETs)] by sEH. EETs have potent vasodilatory effects in the circulation and are thought to play a role in regulation of renal blood flow, arterial resistance, and systemic blood pressure by activating calcium-sensitive K+ channels (4, 16). Indeed, the P-450 epoxygenase metabolites are leading candidates for endothelial-derived hyperpolarizing factor (EDHF), the nitric oxide synthase- and cyclooxygenase-independent vasodilator that hyperpolarizes vascular smooth muscle cells (4, 16). In general, EETs are believed to be more biologically active than DHETs. Therefore, increased sEH expression, as occurs in SHR, would lead to enhanced EET hydrolysis and removal of vasodilatory eicosanoids. Recent studies have shown that the elevated blood pressure observed in SHR and in mouse models of hypertension was decreased after treatment with selective sEH inhibitors (17, 45, 54) and that sEH null mice have reduced blood pressure (45). Rat models of impaired renal hemodynamics have been associated with decreased EETs levels (19, 30). Together, these studies indicate that hydrolysis of the EETs to DHETs by sEH is an important mechanism for regulation of blood pressure. However, it remains unclear whether this is the key factor that initiates the hypertensive response in these animals.

    A large diversity of physiological systems influences blood pressure, and alterations of any of these can result in hypertension (23). Interestingly, data generated from our microarray analysis did not reveal significant changes in most of the genes thought to be involved in blood pressure regulation, such as baroreceptors, the renin-angiotensin-aldosterone system, or natriuretic peptides. However, a moderate increase in expression of angiotensin receptor type II was observed in SHR at both 3 and 9 wk of age. We observed a few significant differences in genes commonly associated with a hypertensive response, such as decreased expression of GST mu 1 (Gstm1; see Refs. 27 and 48) and retinol binding protein 4 (Rbp4; see Ref. 29) and increased expression of 2-glycoprotein I (apolipoprotein H; see Refs. 13 and 44). Consistent with other reports, altered expression of CD36 was identified in SHR animals, suggesting a significant change in fatty acid metabolism (1, 35, 36). These data suggest other mechanism(s) may be involved in the spontaneous hypertension in this animal model. It should be noted that our analysis was limited to genes that were represented on our microarray chip; hence, we cannot rule out the possibility that changes in the expression of other genes might contribute to the hypertensive phenotype in SHR.

    Analysis of age-related changes within each strain revealed a marked reduction in the number of genes differentially expressed in SHR animals compared with WKY. Aging is associated with many vascular changes, such as a rise in arterial stiffness, smooth muscle cell hypertrophy, and fibrosis (22). The regulation of expression of extracellular matrix proteins is important in the elastic properties of the vascular system, and an age-related increase in aortic stiffness has been observed in SHR (2, 14, 18, 26, 47, 49). Interestingly, we observed significant changes in the expression of several connective tissue genes from 3 to 9 wk of age in both WKY and SHR; however, the levels of procollagen (type I and II) and collagen (type I) were higher in SHR at both 3 and 9 wk of age. The age-related changes in metalloproteinases (e.g., Mmp14, gelatinase, TIMP2) observed in WKY were absent in SHR animals. In addition, the lower expression of elastase and cathespin L observed in SHR suggests a difference in the regulation of the vascular extracellular matrix system (20, 42). The reduction in expression of elastase in the current study is consistent with a reported reduction in blood pressure observed in SHR after injection of elastase (18). The altered expression of matrix genes appears to begin early in the SHR animals, since the differences between strains were apparent at 3 wk of age. It remains unknown whether the different gene expression patterns related to collagen and elastin contribute to development of hypertension or, alternatively, are the result mechanical factors stemming from the hypertension (26).

    Differences in the gene expression profiles found in this and other studies reflect both genetic strain differences and environmental factors, all of which result in the same phenotype of spontaneous hypertension. Many of the significant differences in gene expression between SHR and WKY rats in the present study were found to occur at the prehypertensive stage (3 wk of age). Importantly, these data suggest that alterations in gene expression patterns occur in SHR before the onset of the hypertensive phenotype. The present data set provides evidence of potentially novel targets in blood pressure regulation and the development of hypertension, notably the important roles of sEH and extracellular matrix proteins.

    GRANTS

    This study was supported by the National Institute of Environmental Health Sciences Division of Intramural Research (D. C. Zeldin) and National Heart, Lung, and Blood Institute Grant HL-53994 (to D. L. Kroetz).

    ACKNOWLEDGMENTS

    We thank Drs. Elizabeth Murphy and John Pritchard for helpful suggestions during the preparation of this manuscript.

    FOOTNOTES

    The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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日期:2013年9月26日 - 来自[2005年第288卷第10期]栏目

Kallikrein gene transfer reduces renal fibrosis, hypertrophy, and proliferation in DOCA-salt hypertensive rats

【关键词】  fibrosis

    Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina

    ABSTRACT

    In DOCA-salt hypertension, renal kallikrein levels are increased and may play a protective role in renal injury. We investigated the effect of enhanced kallikrein levels on kidney remodeling of DOCA-salt hypertensive rats by systemic delivery of adenovirus containing human tissue kallikrein gene. Recombinant human kallikrein was detected in the urine and serum of rats after gene delivery. Kallikrein gene transfer significantly decreased DOCA- and salt-induced proteinuria, glomerular sclerosis, tubular dilatation, and luminal protein casts. Sirius red staining showed that kallikrein gene transfer reduced renal fibrosis, which was confirmed by decreased collagen I and fibronectin levels. Furthermore, kallikrein gene delivery diminished myofibroblast accumulation in the interstitium of the cortex and medulla, as well as transforming growth factor (TGF)-1 immunostaining in glomeruli. Western blot analysis and ELISA verified the decrease in immunoreactive TGF-1 levels. Kallikrein gene transfer also significantly reduced kidney weight, glomerular size, proliferating tubular epithelial cells, and macrophages/monocytes. Reduction of proliferation and hypertrophy was associated with reduced levels of the cyclin-dependent kinase inhibitor p27Kip1, and the phosphorylation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). The protective effects of kallikrein were accompanied by increased urinary nitrate/nitrite and cGMP levels, and suppression of superoxide formation. These results indicate that kallikrein protects against mineralocorticoid-induced renal fibrosis glomerular hypertrophy, and renal cell proliferation via inhibition of oxidative stress, JNK/ERK activation, and p27Kip1 and TGF-1 expression.

    transforming growth factor-1; oxidative stress; nitric oxide; mitogen-activated protein kinase

    DOCA-SALT ADMINISTRATION has been reported to provoke a low-renin hypertension in rats (9). This volume-overload hypertension results in sodium and water retention in an angiotensin-independent manner. The renal kallikrein-kinin system (KKS) has been shown to play a role in suppressing the development of DOCA-salt hypertension (16, 22). The renal KKS is activated after the administration of mineralocorticoids in conscious rats and consequently enhances urinary excretion of prostaglandins (28). This compensatory augmentation of renal kallikrein found in DOCA-salt rats is most likely a response to sodium and volume retention (3). However, activation of the endogenous renal KKS may not be adequate to protect against hypertension, renal injury, and other organ damages caused by DOCA-salt treatment (31). It has been shown that potentiation of kinin formation in the renal tubules prevents the development of hypertension by inhibition of sodium retention (12, 15, 21). Moreover, in vivo transfer of antisense oligonucleotide against urinary kininase blunted DOCA-salt hypertension in rats (10). In addition, our previous study showed that adenovirus-mediated kallikrein gene delivery attenuates hypertension and protects against renal injury in DOCA-salt rats (7). Therefore, potentiation of the renal KKS may offer an intervention to protect against DOCA-salt-related renal damage.

    DOCA-salt treatment often triggers a malignant hypertension that gradually leads to damage of the kidney, heart, and vasculatures (18, 24, 34, 36). Glomerular sclerosis, tubular fibrosis, and cardiac hypertrophy and fibrosis are commonly observed in DOCA-salt-treated animals, along with activation of renal transforming growth factor (TGF)-1 expression and downregulation of endothelial nitric oxide synthase levels in the heart, kidney, and blood vessels (19, 37). Furthermore, administration of DOCA-salt induces oxidative stress by increasing the formation of superoxide (23), thereby contributing to organ injury. Based on these observations, we further evaluated DOCA-salt-induced renal injury by examining the effect and potential mechanism of human kallikrein gene delivery on renal fibrosis, hypertrophy, and cellular proliferation. In this study, we demonstrate that the KKS has protective effects on kidney damage in the DOCA-salt animal model by suppressing oxidative stress, extracellular matrix (ECM) protein expression, TGF-1 levels, and c-Jun NH2-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK) activation.

    MATERIALS AND METHODS

    Preparation of adenovirus harboring the human tissue kallikrein gene. Adenoviral vectors harboring the luciferase or human tissue kallikrein cDNA under the control of the cytomegalovirus (CMV) enhancer/promoter (Ad.CMV-Luc, Ad.CMV-TK) were constructed and prepared as previously described (7).

    Animal treatment. Left unilateral nephrectomy was performed on male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN) at 4 wk of age. After surgery (1 wk), experimental animals received weekly subcutaneous injections of DOCA (30 mg/kg body wt; Sigma) suspended in sesame oil and were provided with 1% NaCl drinking water. Each rat was injected with 1 x 1010 plaque-forming units of either Ad.CMV-Luc or Ad.CMV-TK (n = 10 each) one time via the tail vein 2 wk after the start of steroid/salt treatment. For the control group, seven rats were subcutaneously injected with sesame oil and provided with tap water. All procedures complied with the standards for care and use of animal subjects as stated in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Resources, National Academy of Sciences, Bethesda, MD). The protocol for our animal studies were approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina.

    Blood pressure measurement. Systolic blood pressure was measured with DASYlab 5.5 software (Kent Scientific, Turrington, CT) by the tail-cuff method. Unanesthetized rats were placed in a plastic holder resting on a warm pad maintained at 37°C during the measurements. Average readings were taken for each animal after the animals had become acclimated to the environment.

    Blood and urine collection. For blood collection, unanesthetized rats were placed in a 37°C incubator for 10 min. Rats were then transferred to a plastic holder, and an insulin syringe was used to withdraw blood from the tail vein. Serum was collected at days 3, 7, and 12 after virus injection. Twenty-four-hour urine was collected from rats in metabolic cages 5 days after virus delivery. To eliminate contamination of urine samples, animals received only water during the 24-h collection period. Urine was collected and centrifuged at 1,000 g to remove particles. Serum and urine were stored at 20°C until analysis.

    Morphological and histological analyses. After gene transfer (17 days), rats were anesthetized with ketamine/xylazine (90 mg?10 mg1?1 kg body wt1). Kidneys were removed, washed in saline, blotted, and weighed. Kidney sections were preserved in 4% formaldehyde solution, paraffin-embedded, and cut to a thickness of 4 μm. Kidney sections were subjected to immunohistochemistry and Sirius red staining (7), which stains collagen fibers red and cytoplasm yellow. Kidney damage was identified by Sirius red staining by counting the damaged and sclerotic glomeruli and tubular protein casts in each section. The glomerular area of 25 glomeruli of each kidney section was measured using NIH Image software under blind conditions without prior knowledge as to which section belonged to which rat. For immunohistochemistry, the following antibodies were used: mouse anti-collagen I (1:400 dilution; Sigma-Aldrich), mouse anti--smooth muscle actin (-SMA, 1:2 dilution; Sigma-Aldrich), rabbit anti-TGF-1 (1:100 dilution; Santa Cruz), mouse anti- p27Kip1 (1:500 dilution; BD Transduction), and mouse anti-proliferating cell nuclear antigen (PCNA, 1:3,000 dilution; Sigma-Aldrich). Immunohistochemistry was performed using the Vectastain Universal Elite ABC Kit (Vector Laboratories). Collagen I immunostaining was evaluated in a score of 03, based upon the amount of staining observed in a gridded area of the kidney at low magnification (x40) as follows: 0 = no staining, 1 = 13 areas, 2 = 47 areas, 3 = 8 or more areas. PCNA-positive cells were quantified in 15 fields per cross-section in the cortex, excluding glomeruli. Double immunolabeling was performed by incubating a mixture of rabbit anti-PCNA antibody (1:100 dilution; Santa Cruz) and mouse anti-ED-1 antibody (a marker for macrophages/monocytes, at 1:20 dilution; Chemicon). A mixture of anti-mouse IgG-TRITC antibody (1:200 dilution; Sigma-Aldrich) and anti-rabbit IgG-FITC antibody (1:200 dilution; Sigma-Aldrich) was used as a secondary antibody.

    Renal cytoplasmic and nuclear protein extraction. Renal tissues were homogenized in lysis buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 20 mM Na4P2O7, 2 mM Na3VO4, and 1% Triton X-100) containing 1:100 protease inhibitor cocktail (Sigma) and centrifuged at 14,000 rpm at 4°C for 40 min. After centrifugation, the supernatant (the cytosolic fraction) was removed and stored at 80°C. To extract the nuclear proteins, pellets were resuspended in an equal volume of lysis buffer, and NaCl was added to a final concentration of 0.6 M. After incubation on ice for 1 h, the lysates were centrifuged at 14,000 rpm at 4°C for 30 min, and the nuclear proteins in the supernatant were removed and stored at 80°C.

    ELISA for human tissue kallikrein and TGF-1. The levels of immunoreactive tissue kallikrein in rat serum and urine were measured by an ELISA specific for human tissue kallikrein, as previously described (4). Total TGF-1 levels in renal extracts were determined by ELISA according to the manufacturer's instructions (R&D).

    Western blot analyses for fibronectin, TGF-1, p27Kip1, and MAPKs. Kidney extracts (100 μg) were resolved by SDS-PAGE for Western blot. Proteins were electrotransferred on nitrocellulose membrane in a transfer buffer containing 25 mM Tris base, 0.2 M glycine, and 20% methanol (pH 8.5). Membranes were incubated in blocking buffer (1x Tris-buffered saline, 0.1% Tween 20 with 5% wt/vol nonfat dry milk) for 1 h and then incubated with antibodies against the following: fibronectin, phospho-p42/p44 ERK (1:1,000 dilution; New England BioLabs), total p42/p44 ERK (1:1,000 dilution; Santa Cruz), TGF-1 (1:1,500 dilution; Santa Cruz), phospho- and total p46/p54 JNK (1:1,000 dilution; Cell Signaling), p27Kip1 (1:2,500 dilution; BD Transduction Laboratories), and -actin (1:5,000 dilution; Sigma) at 4°C overnight with gentle shaking. Membranes were incubated with secondary anti-rabbit or anti-mouse antibody conjugated to LumiGLO chemiluminescent reagent. Chemiluminescence of the blot was detected by an ECL-Plus kit (Perkin-Elmer) according to the manufacturer's instruction and exposed to Kodak X-ray films.

    Assays for urinary protein, nitrate/nitrite, and cGMP levels. Total urinary protein levels were measured by MicroLowry assay. Urinary nitrate/nitrite (NOx) levels were measured by a fluorometric assay (26). Briefly, urine samples were incubated in the presence of 5 μl of 14 mU nitrate reductase (Sigma) and 5 μl of NADPH (Sigma) in 20 mM Tris buffer, pH 7.6. Next, 10 μl 2,3-diaminonaphthalene were added, resulting in the generation of a fluorescent product. Fluorescence was measured using a spectrofluorometer set at an excitation wavelength of 365 nm and an emission wavelength of 450 nm. Urinary cGMP levels were measured by RIA, as previously described (39).

    Superoxide measurements. Superoxide levels were quantified by a spectrophotometric assay based on the rapid reduction of ferricytochrome c to ferrocytochrome c. Non-superoxide-dependent reduction of cytochrome c was corrected for by deducting the activity not inhibited by superoxide dismutase (SOD).

    Statistical analysis. Results are expressed as means ± SE. Comparisons among groups were made by ANOVA followed by Fisher's protected least-significant difference or by unpaired Student's t-test. Differences were considered significant at P < 0.05.

    RESULTS

    Expression of human kallikrein and its effects on blood pressure. Expression of recombinant human tissue kallikrein in rats was measured by an ELISA. After intravenous injection of Ad.CMV-TK, immunoreactive human kallikrein expression reached a level of 3.6 ± 0.5 μg/ml (n = 10) in rat serum at day 3 postgene delivery and declined thereafter (243.1 ± 82.6 ng/ml at day 7; 137.1 ± 55.8 ng/ml at day 12, n = 10). Human tissue kallikrein was also detected in rat urine at day 5 at a level of 16.5 ± 2.2 μg?100 g body wt1?day1 (n = 10). Moreover, kallikrein gene delivery significantly reduced systolic pressure compared with rats receiving the luciferase gene 5 days after virus injection (158.1 ± 4.7 vs. 177.4 ± 3.3 mmHg, n = 10, P < 0.01).

    Effect of kallikrein gene transfer on proteinuria and renal injury in DOCA-salt rats. Delivery of the kallikrein gene significantly reduced urinary protein levels compared with the luciferase group (35.7 ± 2.8 vs. 64.3 ± 13.1 mg?day1?100 g body wt1, n = 79, P < 0.01). Morphological evaluation of the renal cortex and medulla by Sirius red staining showed beneficial effects of kallikrein gene transfer on DOCA-salt hypertensive rats (Fig. 1A). The cortex and medulla of unilaterally nephrectomized control rats appeared normal, whereas rats treated with DOCA-salt and injected with Ad.CMV-Luc developed significant renal injury, occurring in both the cortex and medulla. The damage in the renal cortex of the luciferase group included tubular dilatation, luminal protein cast formation, and glomerular sclerosis. In the medulla, DOCA-salt rats injected with the luciferase gene developed large colloidal casts within renal tubules. However, kallikrein gene transfer markedly attenuated renal damage induced by DOCA-salt treatment.

    Glomerular sclerosis was quantified in DOCA-salt hypertensive rats, as shown in Fig. 1B. It was observed that DOCA-salt hypertensive rats injected with the kallikrein gene had a significant reduction in glomerular sclerosis compared with DOCA-salt rats receiving the luciferase gene (1.44 ± 0.56 vs. 14.57 ± 7.15 damaged and sclerotic glomeruli/cross section, n = 79, P < 0.05). Kallikrein gene delivery also significantly attenuated tubular protein cast accumulation compared with DOCA-salt rats injected with Ad.CMV-Luc (1.14 ± 0.53 vs. 8.57 ± 3.31 tubular protein casts/cross section, n = 79, P < 0.05; Fig. 1C). These results indicate that kallikrein gene transfer markedly protected against DOCA-salt-induced renal injury by decreasing proteinuria, glomerular sclerosis and tubular protein cast formation, as well as renal fibrosis.

    Effect of kallikrein gene transfer on renal fibrosis in DOCA-salt rats. The protective effect of kallikrein on DOCA-salt-induced renal fibrosis was further verified by immunostaining of kidney sections for collagen I. Figure 2A shows representative images of collagen I immunostaining. Positive staining was found only in connective tissue around blood vessels in the control (Fig. 2A, left). After DOCA-salt treatment, many damaged tubules were stained in the Ad.CMV-Luc group (Fig. 2A, middle), and kallikrein gene transfer (Ad.CMV-TK) significantly reduced collagen accumulation (Fig. 2A, right) to the level comparable to the control group. Kallikrein gene transfer markedly decreased the quantitative score of collagen type I deposition in the cortex compared with the luciferase group (0.5 ± 0.39 vs. 1.88 ± 0.66, n = 5, P < 0.05; Fig. 2B). Western blot analysis showed that kallikrein gene delivery significantly reduced relative fibronectin levels normalized by -actin in the kidney of DOCA-salt rats compared with those in rats receiving the luciferase gene (Fig. 2C).

    Figure 3A shows immunostaining of -SMA, a marker of myofibroblasts, in the renal cortex and medulla. Positive staining of -SMA was found only in the blood vessels in the control group. In DOCA-salt rats receiving the luciferase gene, -SMA was mainly expressed in the interstitial space. However, the expression of -SMA was attenuated with kallikrein gene transfer. As shown in Fig. 3B, immunoreactive TGF-1 was mainly found in the glomeruli of the luciferase group, whereas kallikrein gene transfer markedly decreased TGF-1 expression in these regions. Western blot analysis showed that kallikrein gene delivery significantly reduced relative TGF-1 levels normalized by -actin in the kidney of DOCA-salt rats compared with those in rats receiving the luciferase gene (Fig. 3C). This observation was consistent with the TGF-1 ELISA results (Fig. 3D) in that kallikrein gene transfer markedly reduced total renal TGF-1 levels compared with that of the luciferase group (257.3 ± 28.3 vs. 515.6 ± 69.0 pg/mg protein, n = 56, P < 0.01).

    Effect of kallikrein gene transfer on renal hypertrophy and cellular proliferation in DOCA-salt rats. The ratio of right kidney weight to body weight was significantly reduced after kallikrein gene transfer compared with the luciferase group (0.75 ± 0.03 vs. 0.88 ± 0.06 g/100 g body wt, n = 610, P < 0.05; Fig. 4A). Kallikrein gene delivery also markedly reduced glomerular size compared with rats receiving Ad.CMV-Luc injection (9,356 ± 183 vs. 9,869 ± 177 μm2, n = 610, 25 glomeruli/kidney section for each group, P < 0.001; Fig. 4B). Figure 5, A and B, shows immunostaining of PCNA and the quantitative result indicating that kallikrein gene transfer markedly attenuated proliferating cell number in the cortical area of the kidney (excluding glomeruli) compared with that in DOCA-salt rats receiving the luciferase gene (9.9 ± 1.4 vs. 27.7 ± 6.4 PCNA-positive cells/mm2, n = 56, P < 0.01). PCNA-positive cells were observed mostly in distal tubules and collecting ducts (as indicated by arrows in Fig. 5A), and a few were in proximal tubules and glomeruli, indicating the proliferation of tubular epithelial cells after DOCA-salt treatment. In addition, some PCNA-positive cells were found in interstitial cells. To identify the specific type of these proliferating cells in interstitium, we performed double immunostaining of PCNA with ED-1 (a marker for macrophages/monocytes) or with -SMA (a marker for myofibroblasts). We found colocalization of PCNA with ED-1 (Fig. 5C) but very few with -SMA (data not shown). Therefore, the results indicate that kallikrein gene delivery reduced the proliferation of tubular epithelial cells and macrophages/monocytes but not myofibroblasts in the kidney in this animal model.

    Effect of kallikrein gene transfer on p27Kip1 levels and MAPK activation. Positive immunostaining of the cyclin-dependent kinase inhibitor p27Kip1 was mainly observed in glomeruli in DOCA-salt rats receiving Ad.CMV-Luc, but not in control or kallikrein groups (Fig. 6A). Representative Western blot of p27Kip1 in renal nuclear extracts is shown in Fig. 6B, bottom. Expression of renal p27Kip1 protein was markedly decreased after kallikrein gene transfer compared with that of the luciferase group. Figure 6, C and D, shows Western blot analyses of phospho- and total JNK and ERK and the quantitative result. Kallikrein gene delivery markedly reduced phosphorylation of JNK and ERK in the kidney of DOCA-salt rats compared with rats injected with the luciferase gene.

    Effect of kallikrein gene transfer on urinary NOx and cGMP excretion and superoxide formation. DOCA-salt induced NOx excretion in urine and kallikrein gene delivery further elevated urinary NOx levels compared with rats receiving the luciferase gene (145.1 ± 18.2 vs. 65.1 ± 13.6 mmol?day1?100 g body wt1, n = 8, P < 0.01; Fig. 7A). Also, urinary cGMP excretion was markedly increased in rats receiving kallikrein gene transfer compared with that in the luciferase group (15.67 ± 2.32 vs. 9.49 ± 2.14 nmol?day1?100 g body wt1, n = 8, P < 0.05; Fig. 7B). Kallikrein gene delivery also significantly reduced renal superoxide production in the DOCA-salt animals compared with the rats injected with the luciferase gene (0.51 ± 0.17 vs. 1.81 ± 0.68 nmol?min1?mg protein1, n = 10, P < 0.01; Fig. 7C).

    DISCUSSION

    In the present study, we demonstrated that human tissue kallikrein gene delivery protects against DOCA-salt-induced renal fibrosis, cellular proliferation, and glomerular sclerosis and hypertrophy. After systemic delivery of adenovirus containing the kallikrein gene, immunoreactive human kallikrein was detected in the serum and urine of DOCA-salt rats, indicating that recombinant human kallikrein was secreted from the liver and kidney. In addition, we have previously shown that, after kallikrein gene delivery, human kallikrein mRNA is present in key tissues involved in cardiovascular and renal function, such as the heart, aorta, and kidney (7). Expression of recombinant kallikrein in DOCA-salt rats was capable of eliciting renal protective actions via modulation of the profibrotic factor TGF-1, JNK/ERK activation, cyclin-dependent kinase inhibitor, and oxidative stress, leading to a reduction in kidney injury in these animals. The current study provides significant insights regarding the role of the KKS in mediating protective actions in volume-overload hypertension.

    DOCA-salt hypertensive rats receiving the control virus exhibited apparent renal damage, including massive tubular protein cast accumulation, collagen deposition, and glomerular sclerosis. These observations were accompanied by a rise in urinary protein excretion. Increased urinary protein levels were most likely a result of kidney injury, secondary to increased glomerular pressure in the setting of volume-overload hypertension. After kallikrein gene transfer, however, urinary protein levels were markedly reduced in the DOCA-salt-treated animals, depicting renal protection by kallikrein.

    Low-renin hypertension is an outcome of DOCA-salt administration, yet accumulating evidence indicates that a local renin-angiotensin system (RAS) exists in the kidney and contributes to renal injury of DOCA-salt hypertension by stimulating gene expression of TGF-1 and ECM components (17). TGF-1 is a key factor in fibrosis by promoting ECM protein synthesis and myofibroblast formation (14). In the present study, renal TGF-1 levels, in conjunction with fibronectin and collagen type I protein expression, were upregulated after DOCA-salt administration, whereas kallikrein gene transfer markedly attenuated their increased expression. We also observed that DOCA-salt-induced myofibroblast accumulation was reduced after kallikrein gene delivery. We found that expression of recombinant kallikrein resulted in a significant increase in urinary NOx and cGMP excretion over that observed in rats receiving the luciferase gene. It is possible that, through an NO-cGMP pathway, kallikrein is able to downregulate the production of TGF-1 and ECM components. This is supported by the fact that NO can inhibit TGF-1 and collagen expression in mesangial cells (6). Thus, by reducing TGF-1 production, tissue kallikrein could decrease collagen deposition and myofibroblast accumulation, consequently preventing the development of renal fibrosis.

    The cell cycle inhibitory protein p27Kip1 functions as a regulator of cellular proliferation and hypertrophy (32). TGF-1 promotes glomerular hypertrophy in diabetic mice through prohypertrophic mechanisms in the mesangial cell (33), and p27Kip1 has been shown to be required for the development of glomerular hypertrophy induced by TGF-1 (27). In this study, we observed that DOCA-salt rats receiving the luciferase gene had a larger kidney weight and a significant increase in glomerular size. Increased immunostaining of p27Kip1 in the glomeruli and a notable rise in renal nuclear protein levels of p27Kip1 would explain these observations. Kallikrein gene delivery, on the other hand, dramatically attenuated the development of glomerular hypertrophy and the expression of p27Kip1 in the kidney. Because p27Kip1 is necessary for TGF-1-induced glomerular hypertrophy, downregulation of TGF-1 by kallikrein may be the cause for the decrease in glomerular size. TGF-1 and p27Kip1 were primarily expressed in the glomeruli of DOCA-salt rats, signifying that both play a role in the development of glomerular hypertrophy. JNK activity has been shown to be involved in cardiac hypertrophy (13) and, as we have observed in this study, may also play a part in the enlargement in glomerular size. Cellular proliferation, localized in distal tubules and collecting ducts, was also observed in the DOCA-salt rats receiving control virus. Proliferating cells, however, were not prevalent in the glomeruli, indicating that p27Kip1 may be regulating the cell cycle in addition to promoting hypertrophy in mesangial cells of the glomeruli. This finding is also in agreement with the observation that p27Kip1 was not localized in tubular and collecting duct cells. Although both elevated proliferation and hypertrophy were observed in our study, our results showed that increased levels of p27Kip1 accompanied glomerular hypertrophy. This suggests that p27Kip1 contributes to the development of hypertrophy, whereas if a relationship between p27Kip1 and proliferation existed, then the levels of these proteins would be reduced or undetectable in rats treated with DOCA-salt.

    Ample evidence has shown that, in cultured cell lines, mitogenic stimulation by various extracellular agonists correlates with activation of ERK (2). Dominant-negative mutants of Ras or Raf-1, components upstream of mitogen/extracellular cell-regulated kinase in the ERK signaling cascade, were shown to inhibit growth factor-induced cell proliferation, whereas constitutively activated Raf-1 induces cell proliferation (25, 30). Moreover, catalytically inactive mutants of ERK and its antisense cDNA inhibit proliferation (29). As shown in the present study, activation of ERK in the kidney of DOCA-salt rats is a novel finding and points to ERK as a putative regulator of the proliferative response to mineralocorticoid injury in vivo. More intriguingly, we found that phosphorylation of ERK was completely blocked in the kidney after kallikrein gene transfer. Because ERK plays a pivotal role in cellular proliferation, inhibition of ERK activation by kallikrein gene transfer may account for the reduction of cell proliferation in the kidney, thereby protecting against DOCA- salt-induced renal fibrosis and glomerular hypertrophy.

    It is known that, after administration of DOCA-salt, oxidative stress is mainly induced in the vasculature (35), although the local RAS has been implicated in stimulating NADH/NADPH oxidase activity and superoxide formation in the kidney as well (1). Reduced Cu/Zn SOD activity is also considered to contribute to oxidative stress in the aorta of DOCA-salt rats (40). In the present study, increased superoxide formation in the kidney was markedly reduced after kallikrein gene transfer. This observation may be because of an increase in NO production. NO can directly react with superoxide to form peroxynitrite, thereby inactivating superoxide (5). Moreover, NO can inhibit the assembly of NADH/NADPH oxidase subunits, thus reducing superoxide formation (8). A previous study also showed that NO blockade enhances renal responses to SOD inhibition in dogs, suggesting that NO serves a renoprotective effect against these actions of superoxide (20). It would also be interesting to investigate whether kallikrein gene delivery could partly restore SOD activity in DOCA-salt rats and subsequently facilitate the protective effect of kallikrein/kinin on oxidative stress-induced renal damage.

    Our present study showed that unilateral nephrectomized rats after DOCA administration and high-salt loading for 19 days and injected with control virus for 5 days had an average systolic blood pressure value of 177 mmHg, whereas rats receiving the kallikrein gene had an average blood pressure value of 158 mmHg, which was 40 mmHg higher than control rats. The renoprotective effect of kallikrein gene transfer cannot be explained solely based on blood pressure reduction. In support of this notion, it has been shown that a long-term infusion of rat urinary kallikrein in Dahl salt-sensitive rats on high-salt intake was able to attenuate renal injury without affecting systolic blood pressure (38). Furthermore, infusion of the B2 receptor antagonist icatibant (HOE-140) in salt-loaded Dahl salt-sensitive rats abolished kallikrein's protective effect in the kidney but had no effect on the time-dependent rise in blood pressure (11). The mechanism by which kallikrein exerts renal protection in DOCA-salt-induced renal damage and the role of kinin B2 receptor in mediating this protective effect awaits further investigation.

    In summary, we have shown that adenovirus-mediated gene delivery of human kallikrein protected against oxidative stress-induced renal damage, fibrosis, and glomerular hypertrophy in volume-overload hypertensive rats. The ability of kallikrein gene delivery to produce these beneficial effects could be mediated by NO production, subsequently leading to attenuation of oxidative stress, reduction of renal TGF-1 expression, and inhibition of MAPK activation in DOCA-salt hypertension.

    GRANTS

    This work was supported by National Institutes of Health Grant HL-29397 and DK-066350.

    ACKNOWLEDGMENTS

    We thank Dr. Jo Anne Simson for critical evaluation of tissue sections with histological and immunostaining analyses.

    FOOTNOTES

    The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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日期:2013年9月26日 - 来自[2005年第288卷第10期]栏目
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Micropuncture gene delivery and intravital two-photon visualization of protein expression in rat kidney

【关键词】  Micropuncture

    Department of Cellular and Integrative Physiology and Division of Nephrology, Department of Medicine, Indiana University School of Medicine, Indianapolis
    Roudebush Veterans Affairs Administration Medical Center, Indianapolis, Indiana
    Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado

    ABSTRACT

    Understanding molecular mechanisms of pathophysiology and disease processes requires the development of new methods for studying proteins in animal tissues and organs. Here, we describe a method for adenoviral-mediated gene transfer into tubule or endothelial cells of the rat kidney. The left kidney of an anesthetized rat was exposed and the lumens of superficial proximal tubules or vascular welling points were microinfused, usually for 20 min. The microinfusion solution contained adenovirus with a cDNA construct of either 1) Xenopus laevis actin depolymerizing factor/cofilin [XAC; wt-green fluorescent protein (GFP)], 2) actin-GFP, or 3) GFP. Sudan black-stained castor oil, injected into nearby tubules, allowed us to localize the microinfused structures for subsequent visualization. Two days later, the rat was anesthetized and the kidneys were fixed for tissue imaging or the left kidney was observed in vivo using two-photon microscopy. Expression of GFP and GFP-chimeric proteins was clearly seen in epithelial cells of the injected proximal tubules and the expressed proteins were localized similarly to their endogenously expressed counterparts. Only a minority of the cells in the virally exposed regions, however, expressed these proteins. Endothelial cells also expressed XAC-GFP after injection of the virus cDNA construct into vascular welling points. An advantage of the proximal tubule and vascular micropuncture approaches is that only minute amounts of virus are required to achieve protein expression in vivo. This micropuncture approach to gene transfer of the virus cDNA construct and intravital two-photon microscopy should be applicable to study of the behavior of any fluorescently tagged protein in the kidney and shows promise in studying renal physiology and pathophysiology.

    actin; actin depolymerizing factor; adenovirus; green fluorescent protein; intravital microscopy

    THE TRANSFER OF GENETIC MATERIAL into kidney cells in vivo is widely recognized as a potentially useful tool for studying renal pathophysiology and for gene therapy of renal diseases (46, 8). This approach can be used to express specific proteins in kidney cells. Our interest has been in examining the effects of ischemia on the kidney and in observing the behavior of specific protein molecules in response to an ischemic insult (1, 10). We hypothesized that expression of these molecules, labeled with a fluorescent tag, could permit study of their intracellular localization and behavior before, during, and after renal ischemia.

    In unpublished studies (Ashworth S and Boyd-White J), we attempted to express protein chimeras with green fluorescent protein (GFP) by injection of an adenovirus vector containing the cDNA construct into the renal artery of the rat kidney. We saw modest levels of expression of the GFP-tagged proteins in these experiments but encountered several problems. First, the injection method involved a period of renal ischemia, which could have had consequences or altered the response to a second ischemic insult induced after protein expression. Second, the intra-arterial injection of a large dose of adenovirus caused renal vascular inflammation and possible systemic effects. Third, we only saw expression of GFP in the vasculature, not in kidney tubule cells.

    In this study, we present a novel approach to accomplish gene transfer and visualization of a protein product in the rat kidney. We used micropuncture methods to infuse adenovirus containing 1) Xenopus laevis actin depolymerizing factor/cofilin isoform (wild type) linked to GFP [XAC(wt)-GFP], 2) actin-GFP, or 3) GFP cDNA constructs (7) into the lumens of single proximal tubules or vascular welling points on the kidney cortex surface of anesthetized rats. The ADF/cofilins are actin-binding proteins that regulate actin filament dynamics and participate in proximal tubule microvillus disruption after an ischemic insult (1, 10). Two days after recovery from anesthesia and surgery, expression of GFP-tagged proteins was observed in proximal tubule and endothelial cells. In vivo imaging with a two-photon microscope (3) was used to visualize the GFP-tagged proteins. This approach should have wide applicability in studying the behavior of fluorescently tagged proteins in vivo.

    MATERIALS AND METHODS

    Microinjection of adenovirus. Micropuncture experiments were done in a biosafety level 2-certified facility on 32 male Sprague-Dawley rats, weighing 210430 g. The rats were fasted overnight, with free access to drinking water, and anesthetized intraperitoneally with 40 mg/kg pentobarbital sodium. The animal was placed on a heated animal board. With the use of sterile techniques, the left kidney was exposed via a subcostal flank incision and was supported in a micropuncture cup. Surgery and kidney micropuncture were done in a biological safety cabinet (model SG-603 SterilGARD III, Baker, Sanford, ME). The hood was fitted with a Leica MZ-6 microscope with ergotube head (W. Nuhsbaum, McHenry, IL). A fiberoptic light source was used to illuminate the kidney. The exposed kidney was kept moist by periodically dripping sterile 0.9% NaCl solution onto its surface.

    The replication-incompetent adenovirus containing the cDNA for expressing XAC(wt)-GFP, actin-GFP, or GFP (used as a control) was suspended in DMEM at a concentration of 35 x 108 pfu/ml and was kept frozen at 80°C before use (7). The adenovirus constructs had been previously tested for efficient infection and transgene expression in LLC-PK cultured renal cells (2). We found that a higher virus titer was necessary for adenoviral infection and transgene expression via the vascular route, and in these experiments the viruses were concentrated to 35 x 1011 pfu/ml using the Viraprep Adenovirus Purification Kit (Virapur, Carlsbad, CA) and then suspended in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, 1 mM CaCl2, 2 mM MgCl2, pH 7.4) just before use. The injection solutions were colored with lissamine green (30 mg/dl) or FD & C green dye no. 3 (100 mg/dl).

    The virus suspension was aspirated into a sharpened micropipette (tip diameter 78 μm) that was filled with Sudan black-stained heavy mineral oil. We used constant bore, 0.55-mm (SD 0.02) inner diameter, capillary tubing to prepare these micropipettes. The solution volume injected was estimated from the length of the fluid column before and after injection and the relation 1-mm length = 0.23 μl. The tip of the micropipette was filled with light mineral oil or a tiny amount of Sudan black-stained castor oil. The micropipette was mounted in a holder on a Leitz micromanipulator and was connected to a mercury leveling bulb which could be used to change the pressure in the micropipette and inject its contents in a controlled manner. The manipulator was clamped to a metal plate on the hood surface for better control of its movements.

    The usual procedure for tubule injections was as follows. The kidney was observed at a magnification of x96. A surface proximal tubule was selected, micropunctured, and the small droplet of castor oil in the micropipette tip (about 3 tubule diameters in length) was injected into the tubule lumen. The oil droplet flowed downstream and disappeared from view. The purpose of the oil droplet was to reduce dilution of the virus suspension by freshly formed glomerular filtrate. This was achieved by impeding flow through the narrow loop of Henle with the viscous oil; this causes proximal tubular pressure to rise and single nephron glomerular filtration rate to fall. In this "stopped-flow" condition, the injected virus suspension was concentrated by tubular water reabsorption, as was readily apparent from the increased dye intensity in the lumen of blocked tubules. The virus suspension was injected slowly, under visual observation, for an average of 17 min (SD 5), n = 33 tubules (range 522 min, median = 20 min). During the injection, colored injection solution usually appeared in a surface distal tubule after a delay of many minutes. The injected oil droplet occasionally appeared in a distal tubule segment, and the injection rate then had to be increased.

    We also used a "free-flow" approach to tubule injections. In this case, all oil was expelled from the micropipette tip before puncturing the tubule and no oil was injected into the tubule lumen. The infusion rate was adjusted so as to prevent retrograde flow of the colored solution toward the glomerulus. In these experiments, microinfusion rates averaged 107 nl/min (SD 64; range 26194 nl/min) and lasted 18 min (SD 4; range 1121 min; n = 11 tubules).

    Vascular injections of colored virus suspension were done by microinjection into welling points, the place where a superficial efferent arteriole breaks up into the peritubular capillaries on the kidney surface. The infusion rates ranged from 180 to 700 nl/min and lasted 918 min (4 welling points).

    We usually injected one or two tubules or blood vessels per animal. To aid subsequent identification of the injected tubules or blood vessels, a nearby proximal tubule was micropunctured, its lumen was completely filled with Sudan black-stained castor oil, and a map of the puncture sites was drawn. The flank incision was closed by apposing the muscle layers with sterile sutures and by using metal clips on the skin. The rat was allowed to recover from anesthesia and housed in an individual cage. No mortality or morbidity was observed.

    Observations on the kidneys 2 days after adenovirus microinjection. Two days later, the rat was studied after an overnight fast. The animal was anesthetized with Inactin (130 mg/kg body wt) intraperitoneally. For observations with the two-photon microscope, the animal was placed on the microscope stage and kept warm with a circulating water heating blanket. The left kidney, which had been exposed by a small flank incision, was under the animal in a dish of isotonic saline (3). The nephron that had been filled with Sudan black-stained castor oil was located, and nearby GFP-expressing tubules were identified. Images were collected using a Bio-Rad two-photon microscope, with a titanium-sapphire laser set at a wavelength of 860 nm (3). To visualize proximal tubule cells in vivo more clearly, we injected a bolus of Texas red-conjugated folate (100200 μg dissolved in isotonic saline) into a tail vein before collecting images in two rats (9). These rats had been on a folate-deficient diet for 67 days. Folate accumulates in the brush border and is endocytosed by proximal tubule cells (9). In two experiments, we injected a bolus of 1.6 mg Texas red-labeled neutral dextran 3,000 (Molecular Probes, Eugene, OR) to outline the tubular lumens.

    In 18 experiments, the kidneys in the anesthetized rats were fixed for tissue imaging using retrograde aortic perfusion. The fixation solution contained 3% paraformaldehyde, 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 4 mM Na2HPO4, and 2 mM picric acid (pH adjusted to 7.4 with NaOH) and was delivered at a perfusion pressure of 150170 mmHg for 20 min. The kidney was kept in the fixative solution at 4°C for several days, and then a block of kidney tissue containing the micropunctured area was cut with a scalpel. The kidney block was sectioned with a vibrotome and 100-μm-thick sections were stained for actin using Texas red-phalloidin diluted 1:200 in blocking buffer (2% bovine serum albumin and 0.1% Triton X-100 diluted 1:200 in phosphate-buffered saline). The sections were imaged using confocal and two-photon microscopes.

    Experiments were conducted in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals, and protocols were approved by the Indiana University School of Medicine Animal Care and Use Committee.

    RESULTS

    Two days after microinjection of adenovirus cDNA constructs for XAC(wt)-GFP, actin-GFP, or GFP into the lumen of single proximal tubules, GFP was expressed in tubule cells in 23 of 28 experiments. Failure to express the proteins or very weak expression appeared to be associated with low virus titer, short durations of injection (<10 min), or free-flow, as opposed to stopped-flow, injections. Figure 1 shows a typical example of expression of XAC(wt)-GFP in the renal cortex. Expression of GFP appeared to be restricted to the injected tubule, as shown in the low-power images (Fig. 1, AC), but only a minority of its cells expressed the GFP signal, as shown in the higher magnification images (Fig. 1, CF). The XAC(wt)-GFP complex appeared to be distributed throughout the cytoplasm as was previously observed for the distribution of endogenous cofilin (2). Texas red-phalloidin staining of the actin cytoskeleton appeared to be unaffected by XAC(wt)-GFP expression in the infected cells, compared with uninfected cells (Fig. 1).

    Expression of actin-GFP (Fig. 2) was induced by microinjecting an adenovirus suspension containing a cDNA construct of actin-GFP. GFP fluorescence was distributed throughout the cytoplasm but was significantly increased in the dense F-actin bundles found in the apical microvilli of the brush border and also at the base of the cell.

    Figure 3 shows results obtained from an experiment in which an adenovirus-GFP construct was injected 2 days earlier. Because this construct did not contain XAC or actin, these experiments served as a control for the XAC or actin experiments. Although the GFP was present throughout the cell cytoplasm, protein expression levels varied from cell to cell.

    With two-photon microscopy, we next imaged XAC(wt)-GFP in vivo. We often had difficulty visualizing non-GFP-expressing proximal tubule cells in the absence of a fluorescent marker. In Fig. 4, the brightness level of autofluorescence was increased digitally to see the outlines of non-GFP-expressing proximal tubule cells. Two GFP-expressing proximal tubule cells are clearly visible. Intravenous injection of a bolus of Texas red-labeled dextran 3,000, a freely filtered molecule, allowed us to visualize the lumen of the microinjected nephron (Fig. 5). The presence of an open lumen with dextran in it indicated that the glomerulus of the nephron that had been microinjected 2 days earlier continued to filter the plasma. Another approach was to inject intravenously a Texas red-folate conjugate (Fig. 6). Filtered folate accumulated in the brush-border region and was incorporated into endocytic vesicles in proximal tubule cells, greatly improving visualization of these cells. Qualitatively, the uptake of folate did not appear to differ in cells which did or did not express the XAC(wt)-GFP.

    Infusion of a high titer adenovirus-XAC(wt)-GFP construct into vascular welling points on the kidney cortex surface led to expression of XAC(wt)-GFP in peritubular capillary endothelial cells (Fig. 7A). With the increased adenovirus titer, GFP signal was sometimes observed in adjacent proximal tubule cells (Fig. 7B), demonstrating that tubular cells can also be infected and labeled by the vascular route. This limited GFP expression may be a result of extravasation of the virus, possibly due to pressure effects on the endothelium, and labels far fewer epithelial cells than tubule lumen microinjection.

    DISCUSSION

    Delivery of adenovirus constructs directly into kidney proximal tubules and microvasculature resulted in expression of GFP-labeled proteins in kidney tubule and endothelial cells. Several conclusions can be made from the present experiments. First, infusion of the adenovirus over 1020 min appears to be necessary for adequate cellular uptake, as protein expression was not achieved when the virus suspension was infused into tubule lumens for <10 min. This is why most infusions lasted 20 min. Second, tubule infusions in which a stopped-flow condition existed gave more consistent expression than free-flow infusions into tubules. We do not know whether this results from greater concentration of the virus suspension under stopped-flow conditions, raised intratubular pressure, or persistent tubule blockade (12, 13). In many of the stopped-flow tubule injections, we did see colored dye solution in a distal tubule segment or the sudden appearance of oil in a distal segment, but usually only after many minutes of injection. This suggests that the tubules were not completely or permanently blocked. Third, successful gene expression of XAC(wt)-GFP in vascular endothelial cells can be achieved by injection of a high titer adenovirus suspension, but not with the much lower titers used for tubular injections.

    The present study raises a number of issues, some of which will need to be answered by further experiments.

    First, we do not know why only a minority of cells in the injected tubules visibly expressed the GFP-tagged proteins, and why these particular cells, and not others, did so. It is difficult to estimate the exact percentage of tubule cells that expressed the GFP signal, because tubule cross sections that belong to the injected nephron might not be labeled and hence would not be recognized. Furthermore, in some experiments we could not find the injected nephron on day 2, possibly due to lack of GFP expression. In 31 tubule cross sections that contained at least one GFP-expressing cell, we estimated that an average of 43% of the cells (SD 29) expressed the GFP; the true frequency of GFP expression is likely much lower. We also noted in cell culture that not all cells in the monolayer became infected with the adenovirus and expressed the transgene protein (2). This may be due to the adenovirus titer or to the length of infection time. In one additional rat (unpublished observations), we saw robust expression of actin-GFP 16 days after microinjection of the adenovirus construct. Thus gene expression persists in vivo and its extent may be time dependent.

    Second, whether the procedure described injures the tubule or affects tubule function needs further study. Our observations on fixed tissue suggest that the microinjection procedure was usually innocuous and did not cause cell injury, but occasionally we saw debris in the tubule lumen. Potentially, the virus proteins could initiate an immune response or the expressed protein, if at high enough levels, might be toxic. We did not see any evidence of an immune response (e.g., edema or white cells), but no staining for leukocytes was done.

    The intratubular microinfusion rates in the present study (average 107 nl/min) were high, exceeding a normal single nephron glomerular filtration (30 nl/min in a rat) several-fold. The high intratubular infusion rate minimized dilution of the injected virus suspension by glomerular filtrate, but at the same time would have increased intratubular pressure to values close to, but not above, the stopped-flow pressure. We saw no evidence (e.g., dye uptake by the cells) that these high pressures acutely damaged the tubule cells. It has been common practice in micropuncture studies to elevate, for short periods of time, tubular pressures (e.g., during stopped-flow pressure measurements or in split oil-droplet experiments), with no detrimental effects reported. There was no apparent relationship between the rate of infusion and the expression of the GFP protein.

    The vascular microinfusion rates were similar to those used by Spitzer and Windhager (11). These authors noted that adequate capillary perfusion rates could be achieved only at infusion rates of 400800 nl/min, which is higher than the normal blood flow in a single efferent arteriole (200 nl/min). The capillary network perfused clearly exceeds the number of peritubular capillaries originating directly from a single efferent arteriole.

    It is possible to study the functional properties of the injected structures and their individual cells using imaging techniques. Limited observations suggest that the proximal tubule cells in the microinjected nephrons continue to endocytose folate and small molecular mass dextran.

    Finally, we do not know whether cellular expression of the injected molecules will alter the response to an insult such as ischemia.

    In this report, we highlighted observations made on fixed kidneys 2 days after microinjection (Figs. 13). The main advantages of the fixed tissue are that it provides an opportunity for staining and allows a clearer picture of cell morphology. Observations on fixed tissue sections, however, provide only a snapshot at one point of time. As a result of our ability to express specific protein molecules in vivo, we will now be able to make sequential observations on molecule behavior using live-animal imaging and the two-photon microscope.

    The methods described in this paper allow the study of expression, cellular localization, and behavior of fluorescently tagged proteins in the living kidney. Both tubule cells and capillary endothelial cells can be made to express GFP-tagged proteins and on rare occasions we also saw expression in cortical collecting duct cells. A clear advantage of this approach is that only minute quantities of virus are injected into the lumen of a single tubule or microscopic blood vessel, so that the whole kidney or animal is not labeled or infected. By varying the injection site and virus dose, it might be possible, in future experiments, to label specifically glomerular cells, peritubular capillary endothelial cells, proximal tubules, or distal tubules. This approach should be generally useful in studying the in vivo behavior of a variety of molecules in numerous pathophysiological conditions.

    GRANTS

    This research was supported by National Institutes of Health (NIH) Grants PO1-DK-53465 and P50-DK-061594 (B. A. Molitoris), by an NIH O'Brien Center award, and by an Indiana Genomics Initiative grant (INGEN) from the Lilly Endowment to Indiana University School of Medicine. These images were obtained with microscopes provided by the Indiana Center for Biological Microscopy. Adenoviruses used for these experiments were produced through funding by NIH Grant GM-35126 and NS-40371 (J. R. Bamburg).

    FOOTNOTES

    The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

    G. A. Tanner and S. L. Ashworth contributed equally to this study.

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    Ashworth SL, Southgate EL, Sandoval RM, Meberg PJ, Bamburg JR, and Molitoris BA. ADF/cofilin mediates actin cytoskeletal alterations in LLC-PK cells during ATP depletion. Am J Physiol Renal Physiol 284: F852F862, 2003.

    Dunn KW, Sandoval RM, Kelly KJ, Dagher PC, Tanner GA, Atkinson SJ, Bacallao RL, and Molitoris BA. Functional studies of the kidney of living animals using multicolor two-photon microscopy. Am J Physiol Cell Physiol 283: C905C916, 2002.

    Imai E and Isaka Y. Strategies of gene transfer to the kidney. Kidney Int 53: 264272, 1998.

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    McDonald GA, Zhu G, Li Y, Kovesdi I, Wickham TJ, and Sukhatme VP. Efficient adenoviral gene transfer to kidney cortical vasculature utilizing a fiber modified vector. J Gene Med 1: 103110, 1999.

    Meberg PJ and Bamburg JR. Increase in neurite outgrowth mediated by overexpression of actin depolymerizing factor. J Neurosci 20: 24592469, 2000.

    Moullier P, Friedlander G, Calise D, Ronco P, Perricaudet M, and Ferry N. Adenoviral-mediated gene transfer to renal tubular cells in vivo. Kidney Int 45: 12201225, 1994.

    Sandoval RM, Kennedy MD, Low PS, and Molitoris BA. Uptake and trafficking of fluorescent conjugates of folic acid in intact kidney determined using intravital two-photon microscopy. Am J Physiol Cell Physiol 287: C517C526, 2004.

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    Tanner GA and Evan AP. Glomerular and proximal tubular morphology after single nephron obstruction. Kidney Int 36: 10501060, 1989.

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日期:2013年9月26日 - 来自[2005年第288卷第10期]栏目

Renal medullary gene expression in aquaporin-1 null mice

【关键词】  mice

    Department of Physiology, College of Medicine, and Arizona Research Laboratories, Genomics Research Laboratory, University of Arizona, Tucson, Arizona

    ABSTRACT

    Mice that lack the aquaporin-1 gene (AQP1) lack a functional countercurrent multiplier mechanism, fail to concentrate the inner medullary (IM) interstitium, and present with a urinary concentrating defect. In this study, we use DNA microarrays to identify the gene expression profile of the IM of AQP1 null mice and corresponding changes in gene expression resulting from a loss of a hypertonic medullary interstitium. An ANOVA analysis model, CARMA, was used to isolate the knockout effect while taking into account experimental variability associated with microarray studies. In this study 5,701 genes of the possible 12,000 genes on the array were included in the ANOVA; 531 genes were identified as demonstrating a >1.5-fold up- or downregulation between the wild-type and knockout groups. We randomly selected 35 genes for confirmation by real-time PCR, and 29 of the 35 genes were confirmed using this method. The overall pattern of gene expression in the AQP1 null mice was one of downregulation compared with gene expression in the renal medullas of the wild-type mice. Heat shock proteins 105 and 94, aldose reductase, adenylate kinase 2, aldolase B, aldehyde reductase 6, and p8 were decreased in the AQP1 null mice. Carboxylesterase 3, matrilin 2, lipocalin 2, and transforming growth factor- were increased in IM of AQP1 null mice. In addition, we observed a loss of vasopressin type 2 receptor mRNA expression in renal medullas of the AQP1 null mice. Thus the loss of the hyperosmotic renal interstitium, due to a loss of the concentrating mechanism, drastically altered not only the phenotype of these animals but also their renal medullary gene expression profile.

    osmolarity; aquaporin; microarray; real-time PCR; vasopressin

    IN THE KIDNEY, AQUAPORIN-1 water channels (AQP1) have been shown to be expressed in the apical and basolateral epithelial cell membranes of proximal tubules, thin descending limbs of Henle's loop, and in endothelial cells of descending vasa recta (2527). Countercurrent multiplication relies on active solute transport in the thick ascending limb of Henle (TAL) and the rapid osmotic equilibration along the thin descending limb of Henle (20); therefore, the presence of AQP1 in the thin descending limbs plays a vital role in this mechanism. A study of AQP1 null mice demonstrated their failure to produce concentrated urine on water deprivation (8). Further studies demonstrated that fluid reabsorption along the proximal tubule of the AQP1 null mice was reduced, due to the impairment of near-isosmolar fluid reabsorption in this segment (36). The reduction in proximal tubule fluid reabsorption in these mice did not translate to an increase in the distal delivery of fluid (32). Moreover, the loss of an effect of a V2 receptor-specific agonist, 1-deamino-8-D-arginine vasopressin (dDAVP), which should equalize urine and medullary interstitial osmolality, also indicated that the medullary interstitium of AQP1 null mice is not appropriately hypertonic. Therefore, the diuresis seen in AQP1 null mice resulted primarily from reduced fluid absorption in the collecting ducts (32). Taken together, these results suggest that the primary renal defect in AQP1 null mice is the inability to generate a hypertonic medullary interstitium by countercurrent multiplication (22). Thus these animals can be considered to be "countercurrent multiplier knockouts," providing a tool to investigate how the loss of high local osmolality affects inner medullary cell gene expression in vivo, and the present study uses DNA microarrays to identify this effect. These animals could also provide us with a tool to investigate the independent effects of vasopressin and high local osmolality on gene expression in renal cells in vivo.

    METHODS

    Animals. Mice lacking aquaporin-1 (AQP1) were generated by homologous recombination in embryonic stem cells as previously reported (22). Animals were bred and maintained in the animal facility of the University of Arizona Health Sciences Center under National Institutes of Health guidelines. AQP1 genotypes designated (+) for the wild-type allele and () for the targeted allele were determined by PCR analysis of genomic DNA isolated from tail biopsies. F2 generation AQP1 +/+ and / male animals derived from crosses of AQP1 +/ were used in our studies. We used mice that were 10 wk old. Mice received regular food and water ad libitum.

    RNA isolation, amplification, and cDNA purification. Full methodology for the RNA purification, amplification, and cDNA production can be found in supplemental data C. RNA was isolated from mouse inner medullas using a Qiagen RNeasy Mini Kit (cat. no. 74104) according to the manufacturer's protocol for isolation from tissue. The RNA was quantified using a spectrophotometer and run on a 1% agarose gel to determine both its quality and purity. Throughout the following procedures, all samples were kept as individual samples (i.e., from a single mouse) and termed C1C3 for wild-type mice and E1E3 for AQP1 null mice. RNA was amplified using a MessageAmp kit (Ambion cat. no. 1750) according to the manufacturer's protocols. Three micrograms of total RNA from each individual mouse were used as a template for each amplification reaction, and this gave a yield of 50 μg of amplified RNA (aRNA). This amplified RNA was then reverse transcribed to cDNA using an EndoFree RT kit (Ambion cat. no. 1740) according to the manufacturer's protocol. Amino allyl-modified cDNA was purified using PCR purification columns (Qiagen cat. no. 28104) according to the manufacturer's protocol. The modified cDNA was labeled with Alexa dyes via free amine modification (Molecular Probes, Eugene, OR, A20002 [GenBank] Alexa Fluor 546 and A20006 [GenBank] Alexa Fluor 647).

    Microarray slide preparation and hybridization. Microarrays for our study were prepared within the Genomic Research Laboratory (GRL) at the University of Arizona using the NIA mouse 15K clone set http://lgsun.grc.nia.nih.gov/cDNA/15k.html. Full methodology for the production of the microarrays and the hybridization protocols can be found in supplemental data C. We randomly selected 15 clones from our array results for sequence confirmation, and all 15 were identical to that published for the clone set on http://lgsun.grc.nia.nih.gov/, confirming the accuracy of the print and the clone set used to make the arrays. Labeled modified cDNA in hybridization buffer was loaded onto a slide and set to hybridize at 47°C for a minimum of 16 h. After completion, a short wash was run in the hybridization station after which the slide was removed and dipped in 0.05x SSC, to remove any residual nonhybridized cDNA. The slide was dried and analyzed using the arrayWORx eCCD-based microarray scanner from Applied Precision, capable of multichannel fluorescence scanning.

    Microarray analysis. A multivariate experimental approach was used in the analysis of microarray data. This approach enables us to analyze a variety of variables in a microarray study (i.e., time course, treatment, condition, genotype) as well as identify and eliminate sources of experimental variance inherent to microarray data (i.e., array variation, dye performance). This approach is based on an ANOVA statistical model using a custom software package, CARMA (1). CARMA permits a robust characterization and classification of the data as well as provides outputs of residuals and other statistical parameters as references for users. Data were reduced and evaluated by CARMA and the results were submitted to the Gene Expression Omnibus (GEO) and can be found under GEO reference number GSE1298 [NCBI GEO] .

    Real-time quantitative PCR. Real-time quantitative PCR was carried out using the RotorGene RG3000 (Corbett Research) sequence detection system and SYBR Green reagents from Qiagen (Quantitect Sybr Green PCR Kit, cat. no. 20414). Primers were designed using Primer3 software (29) and are listed in supplemental data B along with the gene accession number for the target gene. Three micrograms of total or amplified RNA were reverse transcribed with the Endofree RT kit, according to the manufacturer's protocol (Ambion). The cDNA was diluted to 8 ng/μl and the PCR reaction mixture contained 5 μl of Sybr master mix, 0.4 μl 25 mM MgCl2, 0.6 μl RNAse-free water, 100 pmol of forward and reverse primers, and 16 ng cDNA, in a volume of 10 μl. Each reaction was performed in triplicate at 95°C, 15 min; then 95°C, 15 s, and 58°C, 15 s, and 20 s at 72°C for 40 cycles. This was followed by a melt cycle that consisted of stepwise increase in temperature from 72 to 99°C.

    A single predominant peak was observed in the dissociation curve of each gene, supporting the specificity of the PCR product. Ct numbers (threshold values) were set within the exponential phase of the PCR and were used to calculate the expression levels of the genes of interest and were normalized to endogenous cellular dynactin RNA. The level of dynactin RNA was measured in parallel samples using dynactin-specific primers.

    RESULTS

    Microarray analysis. We isolated total RNA from the medullas of wild-type (samples C1, C2, C3) and AQP1 null mice (samples E1, E2, E3) for microarray analysis of gene expression. Total RNA from each sample was amplified before being labeled in the cDNA reaction. The hybridization scheme we used for microarray analysis is based on the interwoven loop design as previously described (19). Each sample from an individual mouse was hybridized to an array four times, generating four independent replicate measures of that particular RNA sample (see MIAME document, supplemental data). An ANOVA model was used to isolate the knockout effect while taking into account experimental effects (array, dye, spot) and thus determined the effect of AQP1 expression on gene expression patterns in the renal medulla. The two channels for each array were normalized using intensity- and location-dependent Lowess regression. Data were first transformed using a Loglin function, which performs a log transformation for higher intensities, and a linear transformation for lower intensities (16). The ANOVA was performed on a gene by gene basis and was limited to genes that were measured confidently on a minimum of three of the four hybridizations, for at least one sample. In this study 5,701 genes of thepossible 12,000 genes on the array were included in the ANOVA. We considered genes to be significantly differentially expressed if the ANOVA P value was <0.05. Genes that exhibited small changes in gene expression but were identified as significant because of unusually small variance were excluded based on a cutoff of >1.5-fold up- or downregulation between the wild-type and AQP1 null mice; 531 genes were in this selected group. An output file showing individual measurements, gene name (if known) and links to the NIA and Genbank databases was generated (see supplemental data A). A large proportion of the genes selected as significantly differentially expressed were genes showing a downregulation in expression in the renal medullas of the AQP1 null mice compared with wild-type controls; of 531 genes in the microarray output, 465 genes were downregulated and 66 genes were upregulated in the AQP1 null mice compared with wild-type mice.

    Multiple genes identified as encoding members of the mitochondrial electron transport chain were downregulated in the renal medullas of the AQP1 null mice (see supplemental data A). Among those with the largest decrease in expression in AQP1 null mice, compared with wild-type mice, were cytochrome c oxidase (Cox) subunits (Cox I, 5-fold decrease; Cox IVa, 3-fold decrease), mitochondrial H+-ATP synthase F1 and F0 complex subunits (ATP5C1, 2.5-fold decrease; ATP5L, 2.4-fold decrease), NADH dehydrogenase (ubiquinone) (NADH, 2.4-fold decrease), and malate dehydrogenase (MDH; 2-fold decrease). Several heat shock and stress genes were identified as being significantly decreased in expression in the AQP1 null mice; heat shock protein 105 kDa (HSP105; 5-fold decrease), osmotic stress protein 94 kDa (OSP94; 2.7-fold decrease), heat shock protein 30 kDa (HSP30; 1.7-fold decrease), heat-responsive protein 12 kDa (HRP12;, 1.7-fold decrease), aldose reductase (AR; 4-fold decrease), and the reactive oxygen species scavenger, superoxide dismutase 1 (SOD1; 2.4-fold decrease).

    Validation of array results via real-time quantitative PCR. Quantitative real-time PCR was used to confirm the validity of our array analysis. We confirmed our results for a randomly selected number of genes that were either up- or downregulated in the AQP1 null mice, compared with wild-type mice, using the same amplified RNA samples that were used for the microarrays. Additionally, we confirmed the up- and downregulation of specific genes in a second set of RNA samples from wild-type and AQP1 null mice; these RNA samples were not amplified before use in the real-time PCR in order to eliminate any potential error associated with amplification. The amplified RNA is considered a measure of the validity of the microarray analysis, and the total RNA a confirmation of the mRNA expression levels in a biological replicate. We used dynactin as an internal standard for the real-time analysis as it was unchanged on the microarray data. Initial observations for -actin and GAPDH in our array output demonstrated that both of these commonly used housekeeping genes were differentially expressed, with a greater than 1.5-fold decrease in expression in the AQP1 null animals compared with wild-type (see supplemental data A).

    The data collected from the real-time PCR on amplified RNA samples were compared with that obtained from the microarray experiments and can be found in supplemental data D. The real-time PCR results confirmed the microarray data in 29 of 35 randomly selected genes. The data collected from the real-time PCR using the unamplified RNA, from a separate set of mice, were compared with that obtained from the microarray experiments and are presented graphically in Figs. 1, 2, 3, and 4. The real-time PCR results from the unamplified RNA confirmed the microarray data in 25 of 28 genes selected for confirmation. Figures 13 show the real-time PCR results for selected genes that decreased in the renal medullas of AQP1 null mice compared with wild-type mice. Figure 4 shows the results for the randomly selected genes that increased in the renal medullas of AQP1 null mice.

    As previously mentioned, multiple genes encoding members of the mitochondrial electron transport chain were found to decrease in the microarray data and we selected several genes in order to reexamine the mRNA levels by real-time PCR in a separate set of mice. Figure 1 presents the data from the real-time PCR assay (3 wild-type vs. 3 AQP1 null mice) plotted against the corresponding microarray values. NADH dehydrogenase (0.63 ± 0.13 compared with 1.00 ± 0.1 in wild-type medullas, P < 0.05), the mitochondrial proton pump, H+-ATP synthase subunits ATP 5C1 (0.24 ± 0.01 compared with 1.00 ± 0.20 in wild-type medullas, P < 0.05), and ATP 5L (0.12 ± 0.03 compared with 1.00 ± 0.23 in wild-type medullas, P < 0.05) all demonstrated a significant decrease in mRNA abundance in AQP1 null compared with wild-type mice. We also demonstrated a significant decrease in the abundance of the Na+-K+-ATPase -subunit in the AQP1 null mice (0.53 ± 0.13 compared with 1.00 ± 0.16 in wild-type medullas, P < 0.05) and confirmed that adenylate kinase 2 (AK2), a metabolic enzyme often linked to mitochondrial metabolic rate, was significantly decreased in the AQP1 null mice (0.24 ± 0.06 compared with 1.00 ± 0.03 in wild-type medullas, P < 0.05).

    Renal medullary cells are usually exposed to hyperosmotic conditions and many stress genes and heat shock proteins have been studied in this region of the kidney. Figure 2 presents the results for the real-time PCR assay (3 wild-type vs. 3 AQP1 null mice) plotted against the corresponding microarray values for several genes that fell into this functional category of heat and stress related genes. Heat shock protein 105 (HSP105) and osmotic stress protein 94 (OSP94) were confirmed as decreasing in abundance in the renal medullary cells of AQP1 null mice (HSP105, 0.18 ± 0.01 compared with 1.00 ± 0.10, P < 0.05; OSP94, 0.11 ± 0.01 compared with 1.00 ± 0.16, P < 0.05 in wild-type medullas). Aldose reductase was significantly decreased in the renal medullas of the AQP1 null mice (0.06 ± 0.01 compared with 1.00 ± 0.58 in wild-type medullas, P < 0.05). We also demonstrated that mRNA abundance for the serum and glucocorticoid regulated kinase 1 (SGK1), a signaling molecule known to mediate the effects of aldosterone on epithelial sodium transport, via the epithelial sodium channel (ENaC) (28) was decreased in abundance in the AQP1 null mice (0.12 ± 0.04 compared with 1.00 ± 0.35 in wild-type medullas, P < 0.05). Also shown in Fig. 2 are data for the mRNA expression of both P8, a gene whose function is unknown, and tissue plasminogen activator (tPA). Both were both significantly decreased in the AQP1 null mice (P8, 0.15 ± 0.03 compared with 1.00 ± 0.24, P < 0.05; tPA, 0.23 ± 0.03 compared with 1.00 ± 0.21, P < 0.05 in wild-type medullas).

    Several genes, whose function in the kidney and more specifically function in the renal medulla are unknown, were reexamined by real-time PCR assay and these data are presented in Fig. 3. For example, mRNA levels for both cell growth-regulating nucleolar protein (LYAR) (0.31 ± 0.02 compared with 1.00 ± 0.06 in wild-type medullas, P < 0.05) and lymphocyte antigen 6 complex (Ly6) (0.4 ± 0.06 compared with 1.00 ± 0.11 in wild-type medullas, P < 0.05) were decreased in the AQP1 null mice.

    Unlike the genes observed to decrease in the AQP1 null mice, there was no obvious pattern to be seen in the types of genes observed to increase in the AQP1 null mice. Of the 66 genes that showed a significant increase in the microarray results, we reexamined seven by real-time PCR and the results are presented in Fig. 4. Several of the genes we studied by real-time were classed in the array annotation as structural or matrix protein-encoding genes; carboxylesterase (CES3), matrilin 2 (MATN2) and mouse fat tumor suppressor homolog (mFAT1). The mRNA levels of all three were confirmed as significantly increased in the AQP1 null mice (CES3, 3.75 ± 0.57 compared with 1.00 ± 0.17, P < 0.05; MATN2, 7.77 ± 2.45 compared with 1.00 ± 0.41, P < 0.05; mFAT1, 1.97 ± 0.35 compared with 1.00 ± 0.09 in wild-type medullas, P < 0.05).

    Several genes, known to be present in collecting duct cells, were not represented on our microarrays; therefore we used real-time PCR to investigate their relative expression levels in the AQP1 null mice. Figure 5 presents the mean relative expression of these selected genes in the AQP1 null mice relative to the wild-type mice plotted on a log scale (± SE). We observed a significant increase in mRNA abundance in the AQP1 null mice for all three of the epithelial sodium transporter subunits, ENaC- (4.92 ± 1.04 compared with 1.00 ± 0.56 in wild-type medullas, P < 0.05), ENaC- (3.98 ± 0.48 compared with 1.00 ± 0.31 in wild-type medullas, P < 0.05), and ENaC- (4.77 ± 0.77 compared with 1.00 ± 0.37 in wild-type medullas, P < 0.05). There was a significant increase in the abundance in the mRNA of AQP3 in the renal medulla of the AQP1 null mice (2.04 ± 0.38 compared with 1.00 ± 0.04 in wild-type medullas, P < 0.05). We also demonstrated that there was no significant difference between the abundance of AQP2 mRNA in the renal medulla of the two sets of animals (0.88 ± 0.49 compared with 1.00 ± 0.25 in wild-type, P = 0.26, not significant). AQP2 expression is known to be regulated in collecting duct principal cells following activation of type 2, vasopressin receptors (V2R) located on the basolateral membrane of collecting duct cells. We examined the levels of V2R messenger RNA in the medulla of the AQP1 null animals and observed a significant decrease in V2R abundance compared with wild-type animals (0.61 ± 0.06 compared with 1.00 ± 0.14 in wild-type medullas, P < 0.05). In contrast, vasopressin 1a receptor (V1aR) mRNA abundance was significantly increased in AQP1 null animals (4.77 ± 0.47 compared with 1.00 ± 0.11 in wild-type medullas, P < 0.05).

    DISCUSSION

    AQP1 null mice manifest a severe defect in urinary concentrating ability (22). When deprived of water, the mice are unable to concentrate their urine and conserve fluid. The increased urinary flow rates observed with these mice result from reduced fluid absorption in the collecting duct, due to the low medullary interstitial osmolality, and not as a result of an increase in distal delivery of fluid following the decrease in proximal tubule fluid reabsorption (32). The reduction of interstitial osmolality in the AQP1 null mice suggests that the cells of the renal medulla are not exposed to the high NaCl and urea concentrations normally associated with the environment of the renal medulla. The aim of this study was to investigate the gene expression profile of the renal medullary cells of AQP1 null mice compared with those of wild-type mice. We proceeded to analyze the gene expression profile via microarray analysis and confirmed, by real-time quantitative PCR analysis, many of the genes identified as differentially expressed. A dramatic downregulation of gene expression was observed in the renal medulla of the AQP1 null mice, compared with wild-type medulla, and included genes known to be localized to renal medullary cells and previously suggested to function in the protection of cells against osmotic stress. We also identified many genes whose localization and function in the renal medulla are not yet known. We will discuss a few of these observations in the context of renal function.

    Heat shock/stress genes. The cells of the renal medulla are normally exposed to high concentrations of NaCl and urea that vary depending on whether the urine is dilute or concentrated. When exposed to hyperosmolar NaCl, renal epithelial cells are known to increase the expression of stress proteins such as HSP70 (31), along with proteins such as aldose reductase and the betaine transporter that allow the cells to accumulate organic osmolytes (14). Aldose reductase is an enzyme that catalyses the synthesis of sorbitol from glucose. In renal cells, hypertonicity is known to increase the transcription of the aldose reductase gene (33), which results in corresponding increases in both mRNA abundance and enzyme activity, thus allowing an increase in the accumulation of renal cell sorbitol (6), suggesting that these proteins help to stabilize cellular biochemical processes during osmotic stress (5). In our study, we demonstrate a decrease in the abundance of several transcripts known to encode heat- and stress-induced proteins, and we confirmed the decrease in mRNA levels for HSP 105, OSP 94 and aldose reductase in the AQP1 null animals, perhaps reflecting the loss of high interstitial osmolality in the kidneys of these mice. OSP94 is a member of the HSP110/SSE stress protein subfamily and likely acts as a molecular chaperone (21). In mouse kidney, previous studies showed that OSP94 mRNA expression paralleled the known corticomedullary osmolality gradient, with highest expression in the inner medulla. Moreover, inner medullary OSP94 expression was increased during water restriction when osmolality was known to increase (21). In concordance with our observations, studies in mIMCD3 cells demonstrated a decrease in the expression of OSP94 and aldose reductase mRNA when osmolarity was decreased (7).

    It was reported recently that HSP105, which is known to be induced by heat stress and able to bind to p53 in a temperature-sensitive manner, is located in the renal medulla and has a similar pattern of distribution as OSP94 (23). In fact, the authors suggested that HSP105 and OSP94 are the same protein. Heat-responsive protein 12 (HRP12) is a 12-kDa protein of unknown function with significant sequence similarity to HSP70 and DnaK. It is endogenously expressed at high levels in the liver and the kidney, and its expression is known to be upregulated in response to heat shock (30). A recent proteomic study identified it as having a mitochondrial localization (24).

    Our findings confirm and extend previous studies indicating a role for stress protein in renal medullary function. A detailed study into all known heat shock protein family members in the AQP1 null mice would be an interesting future study, as only two members of the family were selected in this study (as a measure to confirm our microarray results). Moreover, the specific roles for this family of proteins in the maintenance of cell integrity in the face of osmotic stress need to be elucidated.

    Mitochondrial pathway genes. The kidney is an organ known to have high basal ATP turnover rates. In the kidney and other organs with high energy demands, the postnatal development of ATP-requiring functions is linked to the biogenesis of mitochondria (10, 35). In the thick ascending limb of Henle's loop, the main oxidative cell type of the renal medulla, this mitochondrial biogenesis occurs between 16 days after birth and adulthood, a time frame that corresponds to when the concentrating mechanisms develop in the renal medulla (11). Mitochondrial biogenesis in the kidney is characterized by a doubling of mitochondrial density as well as an increase in the activity of several mitochondrial oxidative enzymes. The rise in postnatal circulating glucocortocoids was shown to be responsible for changes in the numbers of mitochondria but not for the increase in enzyme activity (12).

    We observed a decrease in expression of a range of transcripts that encode mitochondrial enzymes and proteins in our microarray study, confirming this general trend by real-time PCR. The F1/F0 ATPase subunits were significantly decreased in the renal medullas of the AQP1 null mice, suggesting that the respiratory capacity per mitochondrium may be decreased in these mice compared with wild-type mice. Malate dehydrogenase, the enzyme that catalyzes the final step of the citric acid cycle, was decreased. In addition, in the AQP1 null mice there was a significant decrease in genes that encode the cytochrome c oxidase complex of enzymes, along with an observed decrease in NADH dehydrogenase (ubiquinone), all proteins associated with the oxidative phosphorylation pathway of the inner mitochondrial membrane. Overall, our results suggest that either the total number of mitochondria in the renal medulla of the AQP1 null mice are reduced or that the activity of individual mitochondria is significantly altered. Interestingly, we also observed a decrease in the mRNA abundance of the Na+-K+-ATPase -subunit in the AQP1 null mice, which also suggests that active sodium transport across the basolateral membranes of the renal medullary cells in these mice is decreased. Because Na+-K+-ATPase activity is the primary ATP-consuming process in all renal cells, a decrease in oxidative capacity could be in response to the decrease in energetic load. Further studies, such as mitochondrial activity assays, and calculation of the number of mitochondria in the renal medullary cells of the AQP1 null mice are required to differentiate between these possibilities.

    Vasopressin receptors. Vasopressin receptors are expressed in several cell types along the nephron. V2R are expressed on the basolateral membrane of collecting duct cells, and via activation of adenylyl cyclase mediate an increase in water, urea and sodium permeabilities of these cells. V1aR have been found on the apical membrane of cortical collecting duct cells (2, 18). V1aR activation in collecting duct cells of rabbits has been shown to increase prostaglandin synthesis, leading to the stimulation of phosphodiesterases (4) that would reduce cAMP levels in the cells and blunt the V2R-dependent stimulation of adenylyl cyclase (3). Receptors for V1a are also expressed in the vasa recta of the kidney and stimulation of the V1aR reduces renal blood flow in the medulla during antidiuresis (9).

    We observed a significant decrease in the mRNA abundance for the V2R in the inner medulla of the AQP1 null mice. In contrast, the mRNA abundance of the V1aR was significantly increased in these mice compared with wild-type controls. The decrease in V2R message in the renal medulla suggests that vasopressin activation of the cAMP pathway in the renal medulla of these mice may be reduced. Our general observation from the microarray data was that the pattern of gene expression in the renal inner medulla of the AQP1 null mice was one of downregulation compared with genes expressed in the wild-type mice. This raises several questions about the role of AQP1 in establishing the hyperosmotic environment of the renal medulla. Are the genes downregulated in response to the loss of the hyperosmolarity in the renal medulla or is the presence of AQP1 required as an upstream regulator of several gene pathways P8, a transcription factor whose function is currently unknown, was shown to be induced in rat diabetic kidneys (15). P8 was significantly decreased in the cells of the AQP1 null mice. mRNA abundance for all three ENaC subunits was significantly increased in the AQP1 null mice. The abundance of this protein is known to be increased in diabetic kidneys (13, 34). Future studies are planned to compare the gene expression profiles of renal medullary cells in animal models of diseases that have been associated with defects in the handling of salt and water, such as diabetes. Additionally, the effect of water restriction on the gene expression profile of the medullary cells of the AQP1 null mice will be dissected by microarray studies. These studies will allow us to identify the effect of an increase in vasopressin on renal medullary gene expression in the absence of an increase in osmolarity.

    In summary, gene array analysis, using a new approach for statistical identification of expression changes, CARMA, suggested that several families of proteins are downregulated in the renal medullas of AQP1 null mice. These global gene profile findings were verified by real-time quantitative PCR demonstrating the validity of our approach. Limitations due to the limited number of genes and gene families confirmed remain an issue, but the combination of gene array studies with subsequent verification clearly provides an approach for evaluating general changes in gene expression associated with models of renal dysfunction.

    GRANTS

    This work was funded by a grant from the University of Arizona Foundation (H. L. Brooks) and by National Institutes of Health Grant DK-064706 (H. L. Brooks).

    ACKNOWLEDGMENTS

    We are grateful to A. Verkman (UCSF) for providing the AQP1 null mice and to C. Weber (UA) for technical assistance.

    Supplemental data sets AD can be found at http://ajprenal.physiology.org/cgi/content/full/00207.2004/DC1.

    FOOTNOTES

    The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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日期:2013年9月26日 - 来自[2005年第288卷第2期]栏目
循环ads

The human paracellin-1 gene (hPCLN-1): renal epithelial cell-specific expression and regulation

【关键词】  cell-specific

    Laboratory of Developmental Nephrology, Faculty of Medicine, Technion-Israel Institute of Technology, and  Pediatric Nephrology Unit, Department of Nephrology, Rambam Medical Center, Haifa, Israel

    ABSTRACT

    Tubular reabsorption of Mg2+ is mediated by the tight junction protein paracellin-1, which is encoded by the gene PCLN-1 (CLDN16) and exclusively expressed in the kidney. Tubular Mg2+ reclamation is modulated by many hormones and factors. The aim of this study was to define regulatory elements essential for renal tubular cell-specific expression of human PCLN-1 (hPCLN-1) and to explore the effect of Mg2+ transport modulators on the paracellin-1 gene promoter. Endogenous paracellin-1 mRNA and protein were detected in renal cell lines opossom kidney (OK), HEK293, and MDCT, but not in the fibroblast cell line NIH3T3. A 7.5-kb hPCLN-1 5'-flanking DNA sequence along with seven 5'-deletion products were cloned into luciferase reporter vectors and transiently transfected into the renal and nonrenal cells. The highest levels of luciferase activity resulted from transfection of a 5'-flanking 2.5-kb fragment (pJ2M). This activity was maximal in OK cells, was orientation dependent, and was absent in NIH3T3 cells. Mg2+ deprivation significantly increased pJ2M-driven activity in transfected OK cells, whereas Mg2+ load decreased it compared with conditions of normal Mg2+. Deletion analysis along with electrophoretic mobility-shift assay demonstrated that OK cells contain nuclear proteins, which bind a 70-bp region between 1633 and 1703 of major functional significance. Deleting this 70-bp segment, which contains a single peroxisome proliferator-response element (PPRE), or mutating the PPRE, caused a 60% reduction in luciferase activity. Stimulating the 70-bp sequence with 1,25(OH)2 vitamin D decreased luciferase activity by 52%. This effect of 1,25(OH)2 vitamin D was abolished in the absence of PPRE or in the presence of mutated PPRE. We conclude that the PPRE within this 70-bp DNA region may play a key role in the cell-specific and regulatory activity of the hPCLN-1 promoter. Ambient Mg2+ concentration and 1,25(OH)2 vitamin D may modulate paracellular, paracellin-1-mediated, Mg2+ transport at the transcriptional level. 1,25(OH)2 vitamin D exerts its activity on the hPCLN-1 promoter likely via the PPRE site.

    magnesium; renal tubule; transcription; promoter; gene expression; gene regulation; peroxisome proliferator response element; 1,25(OH)2 vitamin D

    MAGNESIUM IS THE MOST ABUNDANT divalent cation in the intracellular fluid. It plays a critical role in a wide variety of metabolic and cellular processes, including cellular energy storage, DNA/RNA processing, ion transport, membrane stabilization, and nerve conduction (33). Abnormalities in Mg2+ homeostasis are relatively common in clinical practice and may lead to neuromuscular disturbances, central nervous system manifestations, and cardiovascular dysfunction (16, 30). In mammals, the kidney is the principal organ responsible for Mg2+ balance (16, 30). Normally, >95% of the filtered Mg2+ is reabsorbed by the renal tubule. Ten to fifteen percent of the filtered Mg2+ is reabsorbed in the proximal tubule and 10% in the distal tubule. The major site of Mg2+ reabsorption is the thick ascending limb of the loop of Henle (TAL), where 6070% of the filtered load is reclaimed (16, 30). Mg2+ transport in this tubule segment occurs primarily through paracellular conductance driven by the lumen positive electrical potential (30). While renal Mg2+ handling has been thoroughly investigated at the tubular and cellular levels (8, 16, 30), the molecular mechanisms of tubular Mg2+ reabsorption are poorly understood.

    Recently, Simon et al. (38) using positional cloning, have identified a human gene, hPCLN-1 (also known as CLDN16, NCBI accession no. NM-006580), mutations in which cause familial hypomagnesemia-hypercalciuria syndrome. hPCLN-1 consists of five exons and resides on chromosome 3q27. The gene encodes a protein, paracellin-1, which is composed of 305 amino acids (38). Northern blot analysis of human tissues has shown that the 3.5-kb PCLN-1 mRNA transcript is expressed exclusively in the kidney (38). RT-PCR analysis of mRNA from nephron segments of the rabbit (38) and rat (42) has demonstrated that PCLN-1 is expressed in the TAL and the distal convoluted tubule (DCT). The paracellin-1 protein, which is located in the paracellular tight junctions of the TAL and DCT, is a member of the claudin family of tight junction proteins (27) and appears to mediate resorption of both Mg2+ and Ca2+ (38).

    In the kidney, the specialized reabsorptive and/or secretory function of each tubule segment depends upon its structural arrangement and upon the specific pattern of gene expression in each tubular cell type. The promoters of several transporter and channel genes including aquaporin (28), the Na+-phosphate cotransporter (36), the Na+-K+-Cl cotransporter (39), and chloride channels (40) as well as the promoters of nephrin (46) and cadherin (13, 43) genes have been cloned and shown to direct kidney-specific expression in vitro and/or in transgenic mice. Several transcription factors including myc-associated zinc finger proteins and Krüppel-like factor (41), hepatocyte nuclear factor-3 (39), and hepatocyte nuclear factor-1 (2), were found to be involved in kidney-specific expression of the ClC-K1 chloride channel, thiazide-sensitive Na-Cl cotransporter, and cadherin genes, respectively. However, very little is known about the regulatory elements responsible for cell-specific expression of transporter and channel genes in the kidney. As yet, the promoter region of the human paracellin-1 gene has not been characterized, and the molecular mechanisms for renal epithelial-specific activity of this gene have not been investigated.

    Many factors are known to modulate Mg2+ reabsorption in various nephron segments. These factors include hormones such as insulin, 1,25(OH)2 vitamin D, aldosterone, and parathyroid hormone as well as nonhormonal factors such as Mg2+ restriction and load and acid-base changes (8, 30). Most of these factors influence both transcellular magnesium transport in the DCT as well as paracellular transport of this cation in the TAL (8, 30). However, very little is known about the molecular mechanisms of this modulation and whether it occurs at the protein or DNA/RNA level. The magnesium restriction (30)- and 1,25(OH)2 vitamin D (32)-induced increase in Mg2+ uptake by renal MDCT cells was diminished by pretreatment of cells with actinomycin D, suggesting that this stimulation occurs through transcriptional activation. Nevertheless, the molecular mechanisms whereby hormones and other factors modulate Mg2+ transport across the paracellular pathway in the renal tubule are unknown.

    The purpose of this study was to define cis-acting promoter regulatory elements and to examine trans-acting factors essential for renal tubular epithelial-specific expression of hPCLN-1. We also explored the effect of modulators of Mg2+ transport on the hPCLN-1 gene promoter. We show that a 70-bp 5'-flanking region of the paracellin-1 gene may determine renal epithelial-specific expression of this gene. We demonstrate an increase in hPCLN-1 promoter activity in response to Mg2+ depletion and a decrease in response to Mg2+ load. In addition, we show that 1,25(OH)2 vitamin D may modulate Mg2+ transport at the transcriptional level, probably via the peroxisome proliferator response element (PPRE) contained within the 70-bp region.

    MATERIALS AND METHODS

    Opossum kidney (OK) cells (provided by Dr. J Green, Technion, Haifa, Israel), human embryonic kidney (HEK293) cells (provided by Dr. K. Skorecki, Technion, Haifa, Israel), mouse distal convoluted tubule (MDCT) cells (provided by Dr. P. Friedman, University of Pittsburgh, Pittsburgh, PA), and mouse embryonic fibroblast (NIH3T3) cells were grown and maintained in DMEM/F-12 supplemented with 10% fetal calf serum, 2 mM glutamine, 50 IU/ml penicillin, and 50 μg/ml streptomycin at 37°C in a humidified atmosphere of 95% air-5% CO2.

    RT-PCR followed by Southern blot analysis. Total RNA isolated with Tri-reagent (MRB, Cincinnati, OH) from the cell lines mentioned above was reverse-transcribed using OmniscriptRT (Qiagen, Hilden, Germany) with random hexamers (Promega, Madison, WI). PCR was performed using HotStarTaq DNA polymerase (Qiagen) with two sets of primers (Table 1). The first (F1,R1) complementary to exon 1 of hPCLN-1 and the second (F2,R2) complementary to a region between exons 3 and 5 of PCLN-1 (spanning two introns) highly homologous among human, rat, and mouse (mPCLN-1). The resultant DNA was separated on 1% agarose gels and transferred to nylon membranes (Osmonics, Minnetonka, MN). The membranes were probed with PCLN-1 cDNA probes generated by PCR using primers F1,R1 for hPCLN-1 and primers F3,R2 (Table 1) from exon 5 for mPCLN-1, with human genomic DNA as a template. A similar procedure was carried out with actin primers (F4,R4) as control. Probes were 32P labeled by random priming (Biological Industries, Beit Ha’Emek, Israel) using [-32P]dCTP (DuPont-New England Nuclear, Boston, MA).

    View this table:

    Western blot analysis. Protein extracts from HEK293, OK, and NIH3T3 cells were prepared using standard protocols (35). Proteins were separated on 10% SDS polyacrylamide gels, transferred to nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany), and probed with anti-human paracellin-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) using chemiluminescense (Biological Industries). To verify antibody specificity, a peptide competition assay was carried out with 100-fold excess of paracellin-derived peptide.

    Cloning of hPCLN-1 5'-flanking DNA. Using the published sequence of human paracellin-1 cDNA (NCBI accession no. NM-006580), we localized the hPCLN-1 gene on chromosome 3, drafts of which have recently been deposited in NCBI (accession no. NT-00962). A DNA fragment of 7.5 kb in the 5'-flanking sequence region, to but not including the PCLN-1 translation start site (Fig. 1), was synthesized using PCR with human genomic DNA as a template and primers F5/R5 (Table 1). To minimize the possibility of PCR-generated mutations, the High-Fidelity Long-Range PCR system (Roche, Mannheim, Germany) was used. The fragment was TA cloned into pCR-XL-TOPO vector (Invitrogen, Carlsbad, CA) and sequence verified. Computer analysis (Wisconsin Package version 8.0, Genetic Computer Group) of the 5'-flanking region of hPCLN-1 from the gene’s translation start site was carried out, and putative transcription factor binding sites were identified.

    Generation of promoter/reporter constructs. The 7.5-kb 5'-flanking sequence described above was subcloned into pGL3-basic vector (Promega) upstream of a luciferase reporter gene (and named pJ12) (Fig. 1). In addition, a set of seven deletion fragments decreasing in size from the 5'-end of the 7,514-bp genomic fragment were produced and cloned into pGL3-basic vector. Five inserts were prepared using PCR-based strategies with genomic DNA as template with primers F6, F7, F8, F9, F10, and R5 (Table 1). The additional two were prepared by removing specific 5'-segments from cloned hPCLN-1 sequences in pGL3-basic, using restriction enzymes. All eight constructs were sequenced to verify the orientation and integrity of the inserts. As illustrated in Fig. 1, the 5'-ends of the deletion fragments were positioned at 4687 (pJ2L), 3986 (pJ2/3.9), 3317 (pJ2/3.3), 2554 (pJ2M), 1982 (pJ2/1.9), 1458 (pJ2.1/4), and 733 (pJ2.7), respectively. All fragments extended to the ATG site, but did not include it. All eight constructs were cloned into the vector in sense orientation, and the 2.5-kb fragment in pJ2M was also cloned in antisense orientation.

    A reporter plasmid containing a 586-bp nested deletion (from position 1772 to 1186) within this 2.5-kb DNA region (termed pJ2Mdel) was generated using PCR with pJ2M as template and primers F12/R12 (Table 1) complementary to the gap ends. Following DpnI treatment, the PCR-generated pGL3-basic construct was ligated, and the sequence was verified. Empty pGL3 vector containing no insert was used as a negative control. pGL3-control containing the SV40 enhancer/promoter was used as a positive control. Promoter activity was estimated from the ratio of hPCLN-1-driven luciferase activity in pGL3-basic vector to promoterless pGL3-basic vector.

    For reporter gene activity driven by distal hPCLN-1 promoter fragments, the DNA region from position 1738 to 1493 as well as a series of five deletion fragments 5'-truncated in increments of 35 bp were PCR generated with human genomic DNA and primers F13, F14, F15, F16, F17, F18, with R13. All six fragments were cloned into the pGL3-promoter vector, which carries an SV40 minimal promoter upstream to the luciferase reporter gene. In these experiments, promoter activity was determined from the ratio of hPCLN-1 promoter-driven luciferase activity in the pGL3-promoter vector to that in the empty pGL3-promoter vector.

    Point mutations in the 2.5-kb insert-containing plasmid, pJ2M, and the 210-bp insert-containing plasmid, pJ210 (see RESULTS), were generated using PCR with the mutated primers F19 and R14 (Table 1).

    In all transfection experiments, plasmid pCH110 (Pharmacia, Uppsala, Sweden), containing the LacZ gene driven by the CMV promoter, was used to normalize for transfection efficiency. DNA for transfections was purified using Nucleobond AX (Macherey Nagel, Duren, Germany).

    Transient transfections and reporter gene assays. OK, HEK293, and MDCT cells were plated (5 x 104/dish) in 24-well dishes in serum-containing medium. Cotransfections were performed using Fugene 6 (1.2 μl/well, Roche) with 0.3 μg reporter plasmid and 0.3 μg pCH110. NIH3T3 cells (4 x 105/dish) were seeded in 6-well plates and incubated overnight at 37°C. Cells were transfected 24 h later using Polyfect (10 μl/well, Qiagen) with 0.75 μg reporter plasmid and 0.75 μg pCH110. All plates were incubated at 37°C for 48 h. For enzymatic assays, cells were washed with PBS (150 mM NaCl, 15 mM sodium phosphate, pH 7.3) and lysed by incubating in 200 μl/well M-Per (Pierce, Cheshire, UK) for 5 min at 37°C. Lysed cells were centrifuged, and the supernatant was aliquoted (50 μl/well) into 96-well plates. Fifty microliters of Luciferase Assay Reagent (Promega) were automatically added, and the light intensity of the reaction was immediately read in a luminometer (Lucy, Anthos, Austria) for a period of 10 s. Luciferase activity was normalized to -galactosidase activity, which was measured in identical cell lysates. One hundred sixty microliters of ONPG substrate (Sigma, St. Louis, MO) were added to 30 μl of cell lysate in each well of a 96-well plate. The reaction mixture was incubated for 30 min at 37°C, or until yellow color developed. -Galactosidase measurements were performed by a luminometer with a 405-nm filter. Measurements of luciferase and -galactosidase were performed in duplicate.

    In some experiments, 48 h after transfection, the medium was replaced with fresh medium containing 0 (low), 0.7 (normal), or 1.5 mM (high) Mg2+ (Biological Industries). In other experiments, the fresh medium contained 1,25(OH)2 vitamin D (from a stock solution of 104 M in ethanol, Sigma) at a final concentration of 5 x 107 M. Control experiments were carried out with 0.5% ethanol. Cells were exposed to experimental media for 24 h.

    EMSA. Nuclear extracts were prepared from cells using the method of Dignam (9). Briefly, confluent cells were grown on 100-mm plates, washed in 3 ml of PBS (150 mM NaCl, 15 mM sodium phosphate, pH 7.3) supplemented with protease inhibitor mix (Complete, Roche), scraped, and pelleted. The pellet was resuspended in ice-cold lysis buffer (in mM: 10 HEPES, pH 7.9, 1.5 MgCl2, 420 NaCl, 0.2 EDTA, and 1 DTT as well as protease inhibitor mix) and incubated at 4°C for 20 min. Following centrifugation, the supernatant, containing the nuclear extract, was diluted 1:2 with (in mM) 20 HEPES (pH 7.9), 100 KCl, 0.2 EDTA, and 1 DTT as well as 20% glycerol and protease inhibitors. Protein concentration was measured using Bradford reagent (Sigma) at 595 nm with bovine serum albumin as the standard.

    Double-stranded oligonucleotides corresponding to promoter sequences of interest were end-labeled with [-32P]ATP (DuPont-New England Nuclear) using T4 polynucleotide kinase (NEB, Beverly, MA). Binding reactions contained (in mM) 10 Tris?HCl (pH 8.0), 250 KCl, 0.5 EDTA, and 0.2 DTT as well as 0.1% Triton-X 100, 12.5% glycerol (vol/vol), 1 μg poly-dIdC-labeled probe (5 x 104 counts/min), and 50 μg of nuclear extracts. In some reactions, a 50-fold molar excess of unlabeled double-stranded oligonucleotide was added for specific competition. Following a 30-min incubation period on ice, complexes were resolved on 4% nondenaturing polyacrylamide gels in 1x TBE buffer. The gels were dried and autoradiographed.

    RESULTS

    Cloning the 5'-flanking region of hPCLN-1. A DNA fragment stretching over 7.5 kb in the 5'-flanking region of hPCLN-1, reaching 9 bp from the gene’s translation start site, was isolated. Computer analysis of this fragment disclosed a variety of putative transcription factor binding sites, among them recognition sites for several transcription factors known to be tissue restricted and expressed in the kidney. These included hepatocyte nuclear factor 5 (HNF-5), GATA factors, and a peroxisone proliferator-activated receptor (PPAR) binding site. The hPCLN-1 promoter also contained binding sites for transcription factors involved in signal transduction, such as activator protein (AP)-1 and AP-3 as well as a putative TATA box.

    The 5'-flanking regions of PCLN-1 in mouse and rat genomic DNA were located using NCBI-BLAST with the respective cDNA sequences. Comparison of the 5'-flanking region of hPCLN-1 to that in mouse (accession no. NW-000107, region: 23187252 ... 23287252) and rat (accession no. AC106700) revealed very little overall sequence identity within 2.4 kb 5' to the translation site. However, the 51-bp region from position 392 to 341, relative to the hPCLN-1 translation start site, was 79 and 81% identical to the mouse and rat PCLN-1 5'-flanking region sequences, respectively.

    Comparison of the hPCLN-1 5'-flanking region to that of a variety of genes encoding various ion channels and transporters, known to be specifically expressed in the kidney, such as the voltage-gated Cl channels ClC-K1 and ClC-K2, aquaporin-2, the K+ channel ROMK, Tamn-Horsfall protein, and the thiazide-sensitive NaCl cotransporter, did not reveal areas of sequence homology.

    Kidney cell-specific expression of hPCLN-1. RT-PCR followed by Southern blot analysis with nested probes (see MATERIALS AND METHODS) detected endogenous PCLN-1 mRNA in HEK293 and OK cells using an hPCLN-1 probe and in HEK293 and MDCT cells using an mPCLN-1 probe (Fig. 2A). Paracellin-1 mRNA was not detected in NIH3T3 cells.

    Western blot analysis with paracellin-1-specific antibody detected paracellin-1 protein expression in HEK293 and OK but not in NIH3T3 cells (Fig. 2B). Addition of 100-fold molar excess of paracellin-1 peptide to the antibody, 24 h before incubation with the blot, competed out the paracellin-1 band (data not shown).

    Kidney cell-specific activity of the hPCLN-1 promoter. To verify that the 5'-flanking region of PCLN-1 contained a functional cell-specific promoter, reporter gene assays were conducted. The 7.5-kb hPCLN-1 5' flanking region, as well as three 5'-deletion products, were cloned upstream to a luciferase reporter gene in promoterless pGL3-basic vectors in sense orientation.

    The resulting plasmids (Fig. 1), designated pJ12 (7,514-bp insert), pJ2L (4,687-bp insert), pJ2M (2,554-bp insert) and pJ2.7 (733-bp insert), were transfected into the PCLN-1-expressing cell lines HEK293, OK, and MDCT. Reporter gene activities were compared with those in transfected NIH3T3 cells, which do not express PCLN-1. As shown in Fig. 3, all three PCLN-1-expressing renal cell lines displayed a similar pattern of luciferase activity when transfected with pJ12, pJ2L, pJ2M, and pJ2.7. Luciferase activity was maximally induced by pJ2M (2.5 kb) and gradually diminished, as the insert size was either increased or decreased. Specifically, the lowest luciferase activity resulted from transfection of the 7.5-kb insert-containing plasmid (pJ12). Truncation of the 5'-flanking region from 7,514 to 4,687 bp (pJ2L) did not significantly increase luciferase activity. A further decrease in size from 4,687 to 2554 bp (pJ2M) caused a significant increase in luciferase activity in all three renal cell lines. When the 7.5-kb fragment in the luciferase vector was further shortened to 0.733 kb (pJ2.7), luciferase activity in all four renal cell lines was markedly reduced. Transfection of each of the four constructs into the control cell line NIH3T3 showed negligible induction of luciferase activity.

    Taken together, these results indicated that the 2.5-kb hPCLN-1 promoter fragment cloned in pJ2M contained an active promoter. The lack of stimulation in NIH3T3 cells suggested that the activity of the hPCLN-1 promoter was kidney cell specific. The results also suggested that kidney-specific expression of the hPCLN-1 promoter was due, at least in part, to tissue-specific transcriptional regulation.

    Although the induction pattern was similar in all three PCLN-1-expressing renal cell lines, its magnitude was very different between cells. The highest levels of induction appeared in OK cells and the lowest in MDCT cells. HEK293 cells displayed intermediate levels of induction. Based on these findings, OK cells were selected as experimental cells in our next set of experiments.

    Deletion analysis of the hPCLN-1 promoter. To further explore the 5'-flanking region of hPCLN-1, a second set of deletion constructs was tested (Fig. 1), two between pJ2L and pJ2M (designated pJ2/3.9 and pJ2/3.3) and two between pJ2M and pJ2.7 (designated pJ2/1.9 and pJ/1.4). All eight constructs were transiently transfected into OK and NIH3T3 cells. As evident from Fig. 4, the highest level of luciferase activity resulted from transfection of the 2.5-kb insert-containing plasmid (pJ2M). Increasing the size of this fragment by 763 bp (pJ2/3.3) caused a 50% reduction in luciferase activity, which remained unchanged when the 2.5-kb fragment was lengthened by 1,432 bp (pJ2/3.9). When the 2.5-kb fragment was lengthened by 2,133 (pJ2L) or 4,960 bp (pJ12), luciferase activity gradually diminished by 60 and 90%, respectively, compared with pJ2M. Decreasing the size of the 2.5-kb fragment by 572 (pJ2/1.9) and 1,096 bp (pJ2/1.4) brought about a 30 and 77% reduction in luciferase activity, respectively. A further truncation of the insert from 1,458 to 733 bp left luciferase activity at 23% that of pJ2M. No significant differences in luciferase activity between the various fragments tested were demonstrated in control NIH3T3 cells (data not shown).

    To further explore the 2.5-kb 5'-flanking region of hPCLN-1, which displayed the highest promoter activity, an orientation study was performed in OK cells. As shown in Fig. 5, when the 2.5-kb promoter fragment in pGL3 vector was reversed (pJ235M), luciferase activity was 80% lower than activity induced by the same sequence in sense orientation (pJ2M).

    Taken together, these results suggested that a positive regulatory element, most likely located between positions 1982 and 1458, and a negative regulatory element likely positioned between positions 3317 and 2554, are involved in hPCLN-1 promoter activity in renal cells.

    Binding of nuclear proteins to the hPCLN-1 promoter. To investigate the location and nature of the presumed positive regulatory element between positions 1982 and 1458, binding of nuclear proteins to this region was examined. The 525-bp DNA fragment was divided into 15 sequential double-stranded oligonucleotides, which were 5' end-labeled with 32P and incubated with nuclear proteins. DNA-protein complexes were resolved from unbound DNA by nondenaturing gel electrophoresis. Binding patterns were compared between nuclear extracts from OK cells and those from control NIH3T3 cells. As shown in Fig. 6, incubation of seven sequential DNA fragments located in the proximal part of the 525-bp sequence (between positions 1770 and 1491) with nuclear extracts from OK cells produced several retarded bands ( in Fig. 6) that were absent when the DNA was incubated with nuclear extracts from NIH3T3 cells. Binding was specific, since addition of a 50-fold molar excess of unlabeled oligonucleotide abolished DNA-protein complexes. DNA binding was absent in lanes without the nuclear extract. When oligonucleotides originating from the distal part of the 500-bp region (from position 1982 to 1771) were incubated with nuclear proteins from OK and NIH3T3 cells, the protein-DNA binding pattern was similar in the two cell lines (data not shown).

    Taken together, these results suggested that OK cells, the renal cell line OK, but not nonrenal NIH3T3 cells, contain nuclear proteins that bind specifically to the hPCLN-1 promoter in the 280-bp region between positions 1770 and 1491.

    Deletion analysis of the DNA region containing the proposed positive regulatory element of hPCLN-1. To investigate the functional importance of the 280-bp hPCLN-1 promoter region implicated in transcriptional activation and nuclear protein binding, a deletion of the region between positions 1772 and 1186 within the pJ2M reporter vector was created. When the resulting plasmid, designated pJ2Mdel, was transfected into OK cells, luciferase activity was reduced by 60% relative to pJ2M (Fig. 7A).

    To further define the positive regulatory element within the 280-bp promoter sequence of functional importance, deletion analysis of this segment was carried out. The hPCLN-1 promoter sequence from positon 1738 to 1493 was 5'-truncated in increments of 35 bp. These progressively shorter DNA fragments were cloned upstream to a luciferase reporter gene in SV40 minimal promoter-containing vectors, pGL3-promoter, and transfected into OK cells. Figure 7B illustrates that luciferase activity was highest with the construct containing the hPCLN-1 promoter sequence from position 1703 to 1493. Truncation of the promoter to position 1668 caused little change in reporter activity. However, when the promoter was truncated to position 1633, reporter activity diminished by 50%. Two more sequential 35-bp deletions caused only minor reductions in luciferase activity.

    Taken together, these experiments suggest that the 70-bp DNA sequence between positions 1703 and 1633, which binds nuclear proteins from renal cells, contains a positive regulatory element involved in transcriptional activation. Computer analysis of this segment, revealed a single PPRE at position 1655, which is known to bind the transcription factor PPAR (11, 15, 20).

    Most PPREs described so far consist of a direct repeat of two hexamer half-sites separated by several nucleotides (14). The first half-site is highly conserved (AGGTCA) whereas the second is not. The sequence of the PPRE identified in the hPCLN-1 promoter contains one half-site, which is a perfect consensus sequence, but no clearly recognizable second half-site. This sequence, however, has been shown to enable transcription factor binding and activation (3).

    Several in vivo (37, 44) and in vitro (7, 30) studies have demonstrated that Mg2+ restriction increases Mg2+ transport in the renal tubule whereas Mg2+ load decreases it (22, 31). To explore the molecular mechanisms of this modulation, we examined the effect of changes in ambient Mg2+ concentration on the activity of the hPCLN-1 promoter. For this purpose, OK cells were transiently transfected with the 2.5-kb hPCLN-1 promoter fragment (pJ2M) driving maximal luciferase activity. Following transfection, the cells were exposed for 24 h to media containing 0 (low), 0.7 (normal), or 1.5 mM (high) Mg2+. As demonstrated in Fig. 8, Mg2+ restriction caused a 52% increase, and Mg2+ load a 72% decrease, in luciferase activity in OK cells compared with conditions of normal Mg2+ concentration. These findings suggest that ambient Mg2+ concentration may modulate Mg2+ transport at the transcriptional level.

    Effect of 1,25(OH)2 vitamin D on the PPRE-containing DNA region in the hPCLN-1 promoter. Since the vitamin D receptor (VDR) and PPAR both belong to the nuclear receptor superfamily of transcription factors and thus share several biological characteristics (6, 15, 26), we examined the effect of 1,25(OH)2 vitamin D on the hPCLN-1 promoter sequence containing the PPRE half-site. We first examined the action of 1,25(OH)2 vitamin D on the 2.5-kb hPCLN-1 promoter fragment (pJ2M). For this purpose, OK cells, transiently transfected with this fragment were exposed to 5 x 107 M 1,25(OH)2 vitamin D for 1620 h, which caused promoter activity to decrease by 56% (Fig. 9A). Experimental conditions were selected based on preliminary experiments with varying 1,25(OH)2 vitamin D concentrations (106 to 107M) (data not shown).

    We next examined whether the effect of 1,25(OH)2 vitamin D on the promoter was PPRE dependent. For this purpose, OK cells transfected with pGL3-promoter vector containing a 210-bp hPCLN-1 promoter fragment (pJ210) corresponding to the hPCLN-1 promoter sequence between positions 1703 and 1493 harboring the PPRE site, or a 70-bp 5' deletion product (pJ140) of this same promoter sequence, between positions 1633 and 1493, lacking the PPRE, were exposed to 1,25(OH)2 vitamin D under similar conditions. 1,25(OH)2 vitamin D treatment reduced the activity of the PPRE-containing promoter fragment by 52% compared with control non-1,25(OH)2 vitamin D-treated cells (Fig. 9B). This effect of 1,25(OH)2 vitamin D was markedly diminished in cells transfected with the PPRE-lacking fragment. Moreover, the actual difference in promoter activity between the PPRE-containing and PPRE-lacking fragments decreased fivefold following 1,25(OH)2 vitamin D treatment (Fig. 9B).

    To further establish the role of PPRE in hPCLN-1 promoter activity, three bases of the PPRE half-site were point-mutated in both pJ2M and pJ210. OK cells transfected with these constructs were exposed to 1,25(OH)2 vitamin D as above. As shown in Fig. 10A, the decrease in pJ2M activity following 1,25(OH)2 vitamin D treatment was greatly diminished in cells transfected with the mutated promoter. When a similar experiment was carried out with mutated pJ210 (Fig. 10B), the 1,25(OH)2 vitamin D-induced reduction in luciferase activity seen in cells transfected with wild-type, PPRE-containing pJ210 was completely abolished once the PPRE site was mutated.

    These experiments suggest that 1,25(OH)2 vitamin D modulates hPCLN-1 activity by a mechanism that appears to involve the PPRE half-site in the gene promoter.

    DISCUSSION

    In this study, we describe the transcriptional analysis of the promoter of the human paracellin-1 gene. We demonstrate that a 70-bp region between positions 1633 and 1703 may play a key role in the activity of the hPCLN-1 promoter (Figs. 6 and 7). Furthermore, we provide evidence that an interplay between a positive regulatory element located within this 70-bp region, and a more distally located negative regulatory element on the 5'-flanking region of the paracellin-1 gene, may determine renal cell-specific expression of this gene (Fig. 4). In addition, we demonstrate that ambient Mg2+ concentration affects hPCLN-1 promoter activity (Fig. 8). Finally, we show that 1,25(OH)2 vitamin D modulates hPCLN-1 promoter activity via this 70-bp, PPRE-containing, hPCLN-1 promoter fragment (Figs. 9 and 10), thereby suggesting that paracellin-1 gene expression is subject to PPAR/PPRE-mediated transcriptional regulation by this hormone.

    Reabsorption of solutes across the tubular epithelial layer depends on two separate routes: a transcellular pathway and a paracellular pathway (1, 47). The paracellular pathway is regulated by tight junctions, which form a barrier to the diffusion of solutes across epithelial cells and function as a boundary between the apical and basolateral membranes maintaining epithelial cell polarity (21). Paracellin-1, which appears to regulate the paracellular transport of Mg2+ in the TAL, is the first tight junction protein reported to be involved in ion resorption (38). Paracellin-1, or claudin 16, (38) belongs to the claudin family of proteins that participate in the formation of tight junction strands in various tissues (27) and are thought to have a major role in regulating the magnitude and nature of paracellular permeability (1). The disease mutations found in paracellin-1 in familial hypomagnesemia-hypercalciuria syndrome result in an increased resistance to the electrical seal of tight junctions, thereby decreasing epithelial ionic permeability and selectively impeding magnesium and calcium reabsorption (38, 47). Recently, it has been reported that deletion of the paracellin-1 gene is responsible for renal tubular dysplasia in cattle (29). This finding suggests that paracellin-1, in addition to its function in ion resorption, may play an important role in the normal development and organization of the renal tubule.

    hPCLN-1 mRNA was found to be expressed exclusively in the kidney (38, 42). Although cis-acting regulatory elements involved in regulating gene transcription may be dispersed throughout the gene locus, often the proximal promoter region contains elements sufficient for high levels of tissue-specific gene transcription. These elements may function as binding sites for tissue-restricted proteins that arbitrate transcriptional activation in expressing cells. In an effort to identify cis-acting elements involved in regulating kidney-specific expression of hPCLN-1, we focused our study on the 5'-flanking region of this gene. We isolated a 7.5-kb genomic sequence corresponding to the 5'-flanking region of hPCLN-1 and, using reporter gene assays, showed that the proximal 2.5-kb region contained cis-acting, positive and negative, regulatory elements that play a role in renal epithelial-specific expression of hPCLN-1. This promoter displayed high activity in PCLN-1-expressing renal cell lines but not in the fibroblast cell line NIH3T3. In the rabbit (38) and rat (42) kidney, PCLN-1 expression has been shown to be restricted to the TAL and the DCT. Several renal cell lines were used in this study. These included HEK293 and MDCT cells, as well as OK cells of proximal tubular origin. These renal cells were shown to express paracellin-1 mRNA (Fig. 2A) and protein (Fig. 2B) and to display hPCLN-1 promoter-driven luciferase activity (Fig. 3) as opposed to mouse embryonic fibroblast cells (NIH3T3), which demonstrated no expression/activity, indicating that the PCLN-1 promoter was kidney specific.

    In our study, OK cells displayed the highest level of hPCLN-1 promoter-driven reporter gene activity (Fig. 3). Hence, these cells served as the recipient cells in most of our transfection experiments. It is not entirely clear why OK cells of proximal tubular origin express paracellin-1, which was found only in the TAL and the DCT of the rabbit and the rat. Several considerations could provide an explanation for this finding. First, the nephron segment-specific expression of the paracellin-1 gene has been examined in the kidney of these two rodents only. It is possible that the expression of the paracellin-1 gene in other animals and species, including humans, extends to the tight junction of more proximal nephron segments. Second, although several properties of OK cells are consistent with a proximal tubular site of origin, this cell line was originally derived from the whole kidney and may possess characteristics of more distal regions of the nephron (10, 19). Finally, in the immature rat, Mg2+ reabsorption occurs predominantly via the paracellular pathway in the proximal tubule rather than in the loop of Henle (23, 30). It is possible that the OK cell line used in our study contains cells with characteristics of the immature proximal tubule, including expression and cell-specific activity of genes, such as PCLN-1, participating in Mg2+ reabsorption in the immature proximal tubule tight junction. Taken together, we believe that the expression of paracellin-1 mRNA and protein in OK cells, coupled with the marked increase in reporter gene activity driven by the hPCLN-1 promoter in this cell line, has made OK cells suitable as experimental cells in our study.

    Deletion analysis of reporter gene constructs containing hPCLN-1 promoter DNA suggested that hPCLN-1 transcription may be regulated by a proximal positive regulatory element positioned between 1982 and 1458 and a more distally located negative regulatory element between positions 3317 and 2554 (Fig. 4). EMSA studies of the presumed positive regulatory element-bearing region indicated that OK cells contain nuclear proteins that bind specifically to this functionally important region of the hPCLN-1 promoter (Fig. 6). NIH3T3 nuclear proteins did not bind to this DNA region. These proteins are potentially involved in tissue-specific expression of hPCLN-1. When this protein-binding DNA region was deleted from the 2.5-kb PCLN-1 promoter, a 60% reduction in promoter activity occurred. Detailed analysis revealed a 70-bp sequence within this DNA region, which seems to be responsible, at least in part, for modulating kidney-specific hPCLN-1 transcription (Fig. 7). Computer analysis of this segment disclosed a single PPAR binding site (PPRE).

    Tubular Mg2+ reclamation is modulated by a variety of hormonal and nonhormonal factors (8, 16, 30). Mg2+ restriction and Mg2+ load influence both transcellular Mg2+ transport in the DCT and paracellular Mg2+ flux in the TAL (7, 22, 31, 37, 44). Micropuncture experiments in the rat nephron have demonstrated that a low-Mg2+ diet leads to urinary retention of Mg2+ due to increased Mg2+ reabsorption in the loop of Henle (37, 44). Culturing MDCT cells in Mg2+-free medium increased their Mg2+ transport rate (7, 30). Pretreatment of MDCT cells with actinomycin D, an inhibitor of transcription, resulted in a significant decrease in this adaptive response, suggesting that the adaptive regulation of Mg2+ depletion may involve gene transcription (30). As opposed to the effect of Mg2+ deprivation, Mg2+ infusion (22) and acute elevation of Mg2+ concentration at the contraluminal membrane of the TAL (31) in rats inhibited Mg2+ resorption in this tubule segment. The molecular mechanisms underlying the adaptive response of tubular Mg2+ transport to changes in Mg2+ levels have not been investigated. In this study, we show that ambient Mg2+ concentration affects hPCLN-1 promoter activity in OK cells. Specifically, Mg2+ restriction increases hPCLN-1 promoter activity, whereas Mg2+ load reduces the activity of this promoter. These findings are in concert with the modulatory effects of Mg2+ restriction and load on Mg2+ transport observed at the tubular and cellular levels (7, 22, 31, 37, 44) and demonstrate, for the first time, that changes in Mg2+ availability may influence Mg2+ transport at the transcriptional level. The exact molecular mechanisms whereby this Mg2+ level-induced effect is achieved remain unknown. The possible involvement of the cell membrane-bound Ca2+/Mg2+-sensing receptor, or the potential role of a hypothetical Mg2+ response element residing on the hPCLN-1 promoter in the Mg2+-induced effect on transcription of this gene, remains to be explored.

    Paracellular Mg2+ transport in the TAL is known to be regulated by several hormones. These include parathyroid hormone, arginine vasopressin, aldosterone, insulin, and 1,25(OH)2 vitamin D, among others (30). Using microperfusion studies of isolated mouse TAL segments, it has been shown that most of these hormonal responses are mediated by changing the transepithelial voltage or by altering the permeability of the paracellular pathway (24, 30, 45). However, the exact molecular mechanisms of this hormone-induced effect on Mg2+ transport have not been investigated. 1,25(OH)2 vitamin D has been shown to increase Mg2+ uptake by MDCT cells (32). The effect of this hormone on paracellular Mg2+ transport has not been explored. Several hormones, including 1,25(OH)2 vitamin D (6), exert many of the biological actions by receptor-mediated effects on gene transcription. In this study, we show that 1,25(OH)2 vitamin D decreases hPCLN-1 promoter-driven luciferase activity (Figs. 9 and 10). OK cells are known to harbor vitamin D receptors (18) and, as discussed above, appear to express the paracellin-1 gene as well as possess the machinery necessary for its activity. Hence, this cell line may serve as an excellent model with which to explore the effect of this hormone on the paracellin-1 gene. Taken together, our findings provide the first direct evidence that paracellular, paracellin-1-mediated Mg2+ transport may be regulated at the transcriptional level by 1,25(OH)2 vitamin D. However, the physiological significance of the transcription-inhibiting effect of 1,25(OH)2 vitamin D shown in our study and its role in overall magnesium transport in the renal tubule remain to be clarified.

    The effect of 1,25(OH)2 vitamin D on the hPCLN-1 promoter was demonstrated in our study to involve the PPRE half-site located between positions 1633 and 1703 within this promoter (Figs. 9 and 10). The PPARs comprise a group of transcription factors that belong to the nuclear receptor superfamily to which the VDR, the thyroid hormone receptor, and the all-trans-retinoic acid receptor also belong (11, 15, 20). PPARs are ligand-regulated transcription factors that control gene expression by binding to specific response elements (PPREs) within promoters, having formed heterodimers with the retinoid X receptor (11, 15, 20). Three PPAR isoforms, PPAR, PPAR, and PPAR, have been identified (11, 15) and are expressed in several tissues including the kidney (11, 48). PPARs participate in a variety of biological processes common to various cell types, including lipid metabolism, glucose homeostasis, inflammation, cell proliferation and differentiation, apoptosis, and early development (4, 11). However, despite their abundant expression in various segments of the renal tubule (11, 48), very little is known about specific tubular transport processes that are controlled by the PPARs. Noteworthy is a recent study demonstrating enhanced renal tubular cell surface expression of the epithelial sodium channel in response to PPAR activation (12).

    In our study, we focused on 1,25(OH)2 vitamin D, known to modulate Mg2+ transport on one hand (30, 32) and to interact with the PPAR system on the other. The DNA binding site of the VDR is 46% homologous to the DNA binding site of PPAR, and both receptors are known to heterodimerize with the retinoid X receptor before binding to their specific DNA motifs (6, 15, 26). However, recent studies suggest that there is considerable flexibility in the binding sites recognized by the VDR, including the existence of some single half-sites (17). Furthermore, the outcome of the interaction between the VDR and its binding site may be an increase (5) or a decrease (25) in gene transcription. Of note is a recent study demonstrating that the VDR represses transcriptional activity of PPAR in COS1 cells (34). In our study, we provide evidence for PPRE-dependent transcriptional regulation of hPCLN-1 by 1,25(OH)2 vitamin D, and we show here, for the first time, that the PPAR/PPRE axis may play a specific role in the hormonal regulation of Mg2+ transport in the renal tubule.

    In conclusion, an interplay between a positive regulatory element and a more distally located negative regulatory element on the 5'-flanking region of the paracellin-1 gene may determine renal epithelial-specific activity of this gene. The 70-bp, PPRE-containing, DNA region between positions 1633 and 1703 may play a key role in the activity of the PCLN-1 promoter. Ambient Mg2+ concentration and 1,25(OH)2 vitamin D may modulate paracellular, paracellin-1-driven, Mg2+ transport at the transcriptional level. 1,25(OH)2 vitamin D exerts its activity on the hPCLN-1 promoter likely via the PPRE site.

    Future studies utilizing transgenic animals may verify whether the activity of this 70-bp DNA fragment, examined in the cell culture model, demonstrates tubular epithelial specifity in the intact organism, where the transgene is exposed to a more physiologically relevant environment (i.e., hormones, proteins, etc.). Once established, it is possible that these transgenic animals will serve as an excellent model with which to study the transcriptional effects of a variety of physiological as well as pathophysiological factors on the paracellin-1 gene, and thus on tubular Mg2+ reabsorption.

    GRANTS

    This work was supported by the Ruth and Allen Ziegler Fund for Pediatric Research and the Kronovet Fund for Medical Research, Technion-Israel Institute of Technology.

    ACKNOWLEDGMENTS

    We thank Dr. Karl Skorecki, Dr. Maty Tzukerman, and Dr. Sara Selig for helpful suggestions and Ayal Kelmachter and Adva Hermoni for technical assistance. We thank Judi Fichman and Hagar Shafrir for expert secretarial assistance.

    FOOTNOTES

    The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

    E. Efrati and Julia Arsentiev-Rozenfeld contributred equally to this work.

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