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Antibody Treatment Shields Fetus From Zika in Mice

By Dennis Thompson

HealthDay Reporter

MONDAY, Nov. 7, 2016 (HealthDay News) -- An antibody derived from the blood of Zika-infected people may have the potential to protect developing fetuses from the ravages of the virus, a new study with mice suggests.

The antibody, called ZIKV-117, protected fetal mice from a Zika infection in their pregnant mothers, said co-senior researcher Dr. James Crowe, director of the Vanderbilt Vaccine Center in Nashville.

"The antibody treatment will clear the virus in the mother, but also protect the fetus, which is very important," Crowe said.

The antibody is only a short-term antiviral treatment, but the researchers said it demonstrates the potential of a Zika vaccine to provide people long-lasting immunity against the virus.

"No study so far has shown in any model that you could actually treat pregnant animals that are infected with Zika and protect the fetus," said co-senior researcher Dr. Michael Diamond, a professor of medicine and infectious diseases with Washington University in St. Louis. "This is the first study to do this, and it suggests that vaccines should work as well because vaccines induce antibody responses."

The researchers hope to proceed to human clinical trials for the antibody therapy within a year, Crowe said. They are currently making plans to test the antibody treatment in monkeys.

Zika virus produces a relatively mild infection in adults, with only one in every five people showing any symptoms at all, according to the U.S. National Institutes of Health.

Instead, Zika poses the biggest health threat to the fetus. That's because the virus causes severe birth defects including microcephaly, where babies are born with too-small skulls and underdeveloped brains.

More than 2,000 children have been born with microcephaly or birth defects of the central nervous system in Brazil, the nexus of the Zika outbreak in South America, according to the World Health Organization.

To find a treatment for Zika, Crowe and his colleagues isolated antibody-producing immune cells from the blood of three people who previously had been infected with the virus.

From those immune system cells, the researchers obtained and then screened 29 different anti-Zika antibodies.

日期:2016年11月9日 - 来自[Health News]栏目

This Treatment May Be Option for Plantar Fasciitis

By Alan Mozes

HealthDay Reporter

SUNDAY, March 1, 2015 (HealthDay News) -- An ultrasound technique is showing early promise as a quick and minimally invasive treatment for the common and painful foot condition known as plantar fasciitis.

The finding is based on a short-term study involving just 65 patients, the researchers noted.

"While the long-term outcome studies are in progress, the results we have seen to date are very promising," said study lead author Dr. Rahul Razdan, an interventional radiologist with Advanced Medical Imaging in Lincoln, Neb.

The American Orthopaedic Foot and Ankle Society describes plantar fasciitis as essentially an "overuse injury" resulting from inflammation of a band of tissue in the sole of the foot that links the heel bone to the base of the toes.

According to Razdan, standard treatment includes painkillers, cortisone shots, icing, heating, massage, silicone arch supports, and physical therapy centered on the benefits of controlled stretching. For some patients, invasive surgery is another option.

Dr. Raymond Monto is an orthopedic surgeon at Nantucket Cottage Hospital in Nantucket, Mass. He said that about 85 percent of patients will recover from plantar fasciitis with sufficient rest and standard treatment. However, the remaining 15 percent are so-called "problem patients" for whom typical treatments fail to provide relief.

"So when you're looking at chronic cases of disabling morning pain lasting three or four months or more, then absolutely it is warranted to explore new treatment options," said Monto, "because the treatments we currently have are just not that great for these kind of stubborn cases."

Razdan said the new ultrasound therapy is an entirely "novel approach" that uses ultrasonic energy to cut and remove damaged, pain-generating tissue while sparing healthy foot tissue.

In the study, Razdan's team tested the procedure on 65 patients who sought care at an interventional radiology clinic in 2013 and 2014.

All had chronic plantar fasciitis, and all had failed to respond to standard treatments.

During the ultrasound therapy, doctors guided a hollow needle tip into an area of "problem" tissue by means of ultrasound guidance. Once in position, the tip targeted a combination of high frequency/low amplitude sound to the damaged foot region. That broke up the pain-generating tissue, which was then extracted out of the foot.

日期:2015年3月3日 - 来自[Health News]栏目


By Amy Norton

HealthDay Reporter

WEDNESDAY, June 4, 2014 (HealthDay News) -- Severe skin infections are often treated with IV antibiotics for days. But two new drugs -- given once a week, or just once -- could offer an alternative, researchers report.

The findings come from two independent studies published June 5 in the New England Journal of Medicine. In one, researchers found that a single-dose IV antibiotic called oritavancin worked as well as standard antibiotic treatment among 954 patients with serious skin infections.

In the other trial, involving more than 1,300 patients, researchers tested an IV antibiotic called dalbavancin, which was given once a week for two weeks. Again, it was as effective as the standard first-choice treatment.

That standard is the long-used antibiotic vancomycin, which has to be infused twice a day, for up to 10 days. And that can mean an extended stay in the hospital.

One infectious disease expert said the new drugs could "revolutionize" the treatment of serious skin infections, by avoiding prolonged treatment.

"They're easy to use," said Dr. Henry Chambers, a professor of medicine at the University of California, San Francisco. "And easy is better than hard, all things being equal."

Dr. G. Ralph Corey, the lead researcher on the oritavancin trial, agreed.

"It's simple and it works," said Corey, a professor of medicine and infectious diseases at Duke University in Durham, N.C.

Some patients might be able to receive an infusion of the drug and then go home, according to Corey. "With others you may need to watch them for 12 to 24 hours, to see how they do," he said.

"Oritavancin kills [bacteria] very rapidly, so clinicians can note a response within 24 hours," Corey said.

Oritavancin is not yet on the market, but dalbavancin is. The U.S. Food and Drug Administration approved dalbavancin on May 23, under the brand name Dalvance. So far, it is cleared only for treating bacterial infections of the skin and underlying tissue.

Many of those infections are caused by methicillin-resistant Staphylococcus aureus bacteria. That strain, often called MRSA, is considered a "superbug" because it is resistant to many existing antibiotics. Vancomycin has long been the go-to treatment for MRSA infections.

Both of the new antibiotics worked against MRSA skin infections in their respective studies. But whether they are effective for infections of other body tissue "remains to be seen," said Chambers, who wrote an editorial that accompanied the studies.

Staph bacteria, including MRSA, can cause serious infections of the blood, bones and organs such as the heart and lungs.

Corey said the new drugs should "absolutely" be tested against those types of infections, too.

The oritavancin trial included 954 patients with wound infections (from surgery or accidents) or other skin infections that were inflamed, swollen and causing systemic symptoms like fever.

日期:2014年6月5日 - 来自[Health News]栏目

New Treatment for Aggressive Breast Cancer Shows Some Promise

By Dennis Thompson

HealthDay Reporter

WEDNESDAY, Dec. 11, 2013 (HealthDay News) -- Women with aggressive breast cancer who receive combination targeted therapy with chemotherapy prior to surgery have a slightly improved chance of staying cancer-free, researchers say.

However, the improvement was not statistically significant and the jury is still out on combination treatment, said lead researcher Dr. Martine Piccart-Gebhart, chair of the Breast International Group, in Brussels.

"I don't think that tomorrow we should switch to a new standard of care," she said.

Piccart-Gebhart presented her findings Wednesday at the 2013 San Antonio Breast Cancer Symposium, alongside other research that investigated ways to improve treatment for women with HER2-positive breast cancer. This aggressive form of cancer is linked to a genetic irregularity.

Other researchers reported the following:

  • The targeted drug trastuzumab (Herceptin) worked better in HER2-positive breast cancer tumors containing high levels of immune cells.
  • A combination of the chemotherapy drugs docetaxel and carboplatin with Herceptin appeared to be the best postsurgery treatment option.

Overall, the studies were good news for women with HER2-positive breast cancer, which used to be one of the most fatal forms of the disease. Researchers reported long-term survival rates higher than 90 percent for women treated using the targeted therapy drugs.

"That tells you these treatments are very, very effective," Piccart-Gebhart said.

Piccart-Gebhart's combo targeted therapy trial is evaluating whether the HER2-targeted drugs Herceptin and lapatinib (Tykerb) work better when combined on top of standard chemotherapy.

The trial involved 455 patients with HER2-positive breast cancer with tumors larger than 2 centimeters.

The women were given chemotherapy prior to surgery along with either Herceptin, Tykerb, or a combination of the two targeted drugs. They also were treated after surgery with whichever targeted therapy they had been receiving.

Piccart-Gebhart reported that 84 percent of the patients who received the combination targeted therapy between 2008 and 2010 have remained cancer-free, compared with 76 percent who only received Herceptin.

"It's too early today to say this dual treatment saves more lives. We can't say that on the basis of this trial," she noted.

The drawbacks of this combination therapy are cost and side effects, Piccart-Gebhart said. Targeted therapies cost tens of thousands of dollars, and combining the two drugs increases toxic side effects such as diarrhea and rash.

"There is a price to pay in terms of side effects," she said. "There will be a price to pay in terms of drug costs."

This study was supported by funds from GlaxoSmithKline. Piccart-Gebhart has received honoraria from Roche, and her institution has received research funding from GlaxoSmithKline.

The second study involved 156 patients who received chemotherapy and Herceptin before surgery. However, this study focused on the levels of immune cells called lymphocytes that had infiltrated the breast tumors.

日期:2013年12月16日 - 来自[Health News]栏目

Renal 20-HETE inhibition attenuates changes in renal hemodynamics induced by L-NAME treatment in pregnant rats

【关键词】  cytochrome

    Department of Physiology and Center for Biotechnology and Genomic Medicine, Medical College of Georgia, Augusta, Georgia
    Department of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas
    Department of Physiology, Medical College of Georgia, Augusta, Georgia, and Renal Department of Memorial Hospital, Sun Yat-Sen University, Guangdong Province, People’s Republic of China


    We previously reported that inhibition of nitric oxide (NO) synthesis by N-nitro-L-arginine methyl ester (L-NAME) during late pregnancy leads to increased production of renal vascular 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P-450 (CYP) 4A-derived vasoconstrictor, in pregnant rats. However, the effect of upregulation of vascular 20-HETE production on renal function after NO inhibition is not known. To test the hypothesis that increased gestational vascular 20-HETE synthesis after NO inhibition is involved in mediating blood pressure and renal functional changes, we first determined the IC50 value of the effect of nitroprusside (SNP), a NO donor, on renal 20-HETE production in cortical microsomes. We then divided pregnant rats and age-matched virgin rats into a vehicle control group, an L-NAME treatment group (0.25 mg/ml in drinking water), and a group treated with L-NAME plus N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS; CYP4A-selective inhibitor, 10 mg?kg1?day1 iv). After 4 days of treatment, we measured blood pressure, renal blood flow (RBF), renal vascular resistance (RVR), and glomerular filtration rate (GFR) in each group. The addition of SNP (IC50 = 22 μM) decreased renal cortical 20-HETE production. In pregnant rats, L-NAME treatment led to significantly higher mean arterial pressure (MAP) and RVR, and lower RBF and GFR. Combined treatment with DDMS and L-NAME significantly attenuated the increases in MAP and RVR and the decrease in GFR, but not the reduction in RBF induced by L-NAME treatment. L-NAME and L-NAME plus DDMS had no significant impact on renal hemodynamics in virgin rats. In addition, chronic treatment with DDMS selectively inhibited cortical 20-HETE production without a significant effect on CYP4A expression in L-NAME-treated pregnant rats. In conclusion, NO effectively inhibits renal cortical microsomal 20-HETE production in female rats. In pregnant rats, the augmentation of renal 20-HETE production after NO inhibition is associated with increased MAP and RVR, whereas decreased GFR is negated by treatment of a selective and competitive CYP4A inhibitor. These results demonstrate that the interaction between renal 20-HETE and NO is important in the regulation of renal function and blood pressure in pregnant rats.

    pregnancy; cytochrome P-450; arachidonic acid; eicosanoid; kidney; nitric oxide; hypertension

    THE MAJOR CYTOCHROME P-450 (CYP)-derived eicosanoid in rat kidney, 20-hydroxyeicosatetraenoic acid (20-HETE), is formed primarily by CYP4A isoforms. The expression of CYP4A isoforms and production of 20-HETE in the small arterial vessels is well documented (12, 30, 34). In the microvessels, 20-HETE depolarizes vascular smooth muscle by inhibiting Ca2+-activated K+ channels and enhances the conductance of L-type Ca2+ channels (11, 40), thereby sensitizing vascular smooth muscle to constrictor stimuli and causing vasoconstriction (39). In the rat kidney, 20-HETE inhibits ion transport in the different segments of nephron. For example, in the renal tubules, 20-HETE inhibits Na+-K+-ATPase activity in a concentration-dependent manner (0.1 nM-1 μM) through phosphorylation of the -subunit of Na+-K+-ATPase by protein kinase C (25, 28). Escalante et al. (10) demonstrated the inhibitory effect of 20-HETE on sodium transport in the thick ascending limb of the loop of Henle (TALH). Interestingly, Wang and Lu (37), using a patch-clamp technique, demonstrated that 20-HETE blocks the 70-pS K+ channel in the apical membrane of TALH cells, thus limiting the amount of K+ available for transport via Na+-K+-2Cl cotransporter and reducing the positive potential in the lumen, which is the main driving force for passive reabsorption of cations in the TALH. Because of these biological activities, 20-HETE has been linked to regulation of renal function and blood pressure in many animal models of hypertension (15, 20, 31).

    Preeclampsia, which affects 510% of pregnancies in the U.S., is characterized by increased arterial blood pressure, generalized vasoconstriction, increased systemic resistance, widespread vascular endothelial damage, decreased fetal growth, and proteinuria (18). The exact mechanisms that mediate preeclampsia are still unknown. Several reports have suggested that nitric oxide (NO) may play an important role in its development (5, 19). Moreover, different investigators have demonstrated that chronic inhibition of NO synthesis in pregnant rats results in signs similar to those of preeclampsia (23, 38). Several studies have demonstrated that NO inhibits 20-HETE synthesis and interferes with 20-HETE vasoconstrictor activity in vivo (13, 27, 32). We previously demonstrated that NO binds differently to CYP4A1 and CYP4A3, the major renal CYP isoforms in female rats, and inhibits their catalytic activity (36). Moreover, the inhibition of NO production by N-nitro-L-arginine methyl ester (L-NAME) during late pregnancy in rats causes the augmentation of 20-HETE synthesis in renal microvessels (36). However, it is not known whether the upregulation of renal microvessel 20-HETE production affects renal function in L-NAME-treated pregnant rats.

    In the present study, we examined the effect of N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), a selective CYP4A inhibitor, on various renal functional parameters, including mean arterial pressure (MAP), renal blood flow (RBF), renal vascular resistance (RVR), and glomerular filtration rate (GFR) in pregnant rats treated with L-NAME during days 14-17 of gestation. Age-matched virgin rats served as controls. This study provides valuable information regarding the interaction between NO and 20-HETE in the regulation of renal function and blood pressure in pregnant rats.


    Materials. We obtained [1-14C]arachidonic acid (56 mCi/mmol) from DuPont-New England Nuclear (Boston, MA). All HPLC solvents and chemicals for buffer were obtained from Sigma (Milwaukee, WI). We purchased 20-HETE, epoxyeicosatrienoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs) standard from Cayman Chemicals (Ann Arbor, MI).

    Animals. All animals were purchased from Charles River Laboratories (Wilmington, MA). We conducted experiments in pregnant (timed pregnancy) and age-matched female Sprague-Dawley rats. All rats were maintained on a 12:12-h light-dark cycle and were housed two rats to a cage. All animal protocols were approved by the Institutional Animal Care and Use Committee and were in accordance with the protocols for animal use outlined in the Guide for the Care and Use of Laboratory Animals.

    Protocol to evaluate the effect of sodium nitroprusside on renal microsomal 20-HETE production. We examined the stability of the NO donor sodium nitroprusside (SNP) with NO-sensitive litmus paper using Griess reagent [0.5 g of sulfanilamide plus 20 mg of N-(1-naphthyl)ethylenediamine dihydrochloride] dissolved in 10 ml of methanol (24). We preincubated SNP (1556 μM, final concentration) with renal cortical microsomes (150 μg) isolated from female rats at 37°C for 20 min, then added [1-14C]arachidonic acid (0.4 μCi, 7 nmol), NADPH (1 mM), buffer (10 mM MgCl2 and 100 mM KH2PO4, pH 7.2) to the incubation mixtures in a final volume of 0.15 ml. Incubation was then carried out at 37°C for an additional 30 min. The reaction was terminated by acidification to pH 3.54.0 with 2 M formic acid. We extracted metabolites with ethyl acetate and determined 20-HETE production in the renal microsomes by HPLC as previously described (35). We also determined the IC50 value as previously described (33).

    Protocol to evaluate the effect of DDMS on renal function in L-NAME-treated pregnant and virgin rats. We divided the pregnant rats (day 14 of gestation) and age-matched virgin rats into three groups (6 rats per group): 1) a vehicle-treated group (45% 2-hydroxypropyl--cyclodextrin), 2) an L-NAME treatment group; (0.25 mg/ml in drinking water), and 3) a group treated with L-NAME (0.25 mg/ml in drinking water) plus N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS; 10 mg?kg1?day1 iv). Each group was treated for 4 days. The dosages of L-NAME and DDMS were based on our previous studies and a literature search (4, 36). After treatment, we used these pregnant rats and age-matched female rats for a renal functional study. On day 18 of pregnancy, after finishing the functional study, we immediately removed their kidneys for renal cortical homogenate preparation to be used to assess renal 20-HETE production and CYP4A expression.

    Renal functional measurements. We performed the renal functional study in pregnant rats and age-matched control female rats from the three treatment groups. Each rat was weighed before surgery. We anesthetized the rats with 2% isoflurane delivered by an anesthesia apparatus. Then, we placed one polyethylene cannula in the trachea (PE-205) to allow free breathing, one in the bladder (PE-240) to collect urine, one in the femoral artery (PE-50) for measuring and recording MAP with a pressure transducer, and one (PE-50) in the femoral vein for the infusion of agents. We then began infusing saline (3 ml/h iv) and a dose of 0.5 ml of FITC inulin (8 mg/ml in PBS, Sigma) was administered over 2 min as a priming dose. We performed a left laparotomy and placed a Transonic flow probe (Transonic System, Ithaca, NY) over the left renal artery to measure RBF. During the experiments, the rats’ body temperature was maintained at 37°C by a temperature controller (Cole Palmer Instrument) connected to a heating mat and a rectal temperature probe. After a volume of saline containing 6.2% BSA equal to 1.25% body wt had been infused, the intravenous infusion was switched to saline without BSA, but still with FITC inulin at 4 mg/ml. At least 45 min were allowed for equilibration following surgery before the beginning of the 30-min urine collections. Arterial blood (0.4 ml) was drawn from the femoral artery in the middle of each 30-min clearance period for measurement of GFR. An equal volume of normal saline was infused for volume replacement. MAP, RBF, and RVR were obtained from a computerized data collection and analysis system (EMKA Technologies, Falls Church, VA). We determined the concentration of FITC-inulin in plasma and urine using a GENios Plus fluorescent plate reader (Tecan, Research Triangle Park, NC) at 485-nm excitation and 538-nm emission. We used the concentration of FITC inulin in the plasma and urine to calculate GFR as described previously (29).

    Isolation of renal cortical homogenates. After the renal functional study, we immediately isolated the kidneys from treated and control rats. Renal cortex from each rat was homogenized in 1 ml of 10 mmol/l potassium buffer (pH 7.7) containing 250 mmol/l sucrose, 1 mmol/l EDTA, 0.1 mmol/l phenylmethylsulfonyl fluoride (PMSF), and 7.5 μl/ml protease inhibitor cocktail (Sigma). We centrifuged the homogenates for 15 min at 3,000 g and for 30 min at 11,000 g and collected supernatants and stored them at 80°C. The protocol for homogenate preparation was adapted from that of Hoagland et al. (14).

    Arachidonic acid metabolism in renal cortical homogenates. We incubated renal cortical homogenates (1 mg) isolated from treated and control with [1-14C]arachidonic acid (0.2 μCi, 3.5 nmol) and NADPH (1 mmol/l, final concentration) in 1 ml potassium phosphate buffer (100 mmol/l, pH 7.4) containing 10 mmol/l MgCl2 for 15 min at 37°C. The reaction was terminated by acidification to pH 3.54.0 with 2 mol/l formic acid, after which arachidonic acid metabolites were extracted with ethyl acetate. We evaporated the ethyl acetate under nitrogen, resuspended the metabolites in 50 μl methanol, and injected them into the HPLC column. We performed reverse-phase HPLC on a 5-μm ODS-Hypersil column, 4.6 x 200 mm (Hewlett Packard, Palo Alto, CA) using a linear gradient of acetonitrile:water:acetic acid ranging from 50:50:0.1 to 100:0:0.1 at a flow rate of 1 ml/min for 30 min. The elution profile of the radioactive products was monitored by a flow detector (In/us System, Tampa, FL). We confirmed the identity of 20-HETE and DHETs with authentic standards. The activity of 20-HETE formation was estimated based on the specific activity of the added [1-14C]arachidonic acid and was expressed as pmoles per milligram of protein per minute as described previously (14, 35).

    Western blot analysis. We separated renal cortical homogenates (10 μg) from treated and control rats by electrophoresis on a 10 x 20-cm, 8% SDS-polyacrylamide gel at 25 mA/gel at 4°C for 1820 h. The proteins were transferred electrophoretically to an enhanced chemiluminescence (ECL) membrane. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) containing 10 mmol/l Tris?HCl, 0.1% Tween 20, and 150 mmol/l NaCl for 90 min, then washed three times with TBS. We incubated the membranes for 10 h with goat anti-rat CYP4A1 (1:2,000; Gentest, Woburn, MA) at room temperature, washed them several times with TBS, and incubated them again for 1 h with a 1:5,000 dilution of horseradish peroxidase-coupled rabbit anti-goat secondary antibody for CYP4A1. We developed the immunoblots using an ECL detection kit (Amersham, Arlington Heights, IL). To normalize the expression of CYP isoforms, we incubated renal cortical homogenates (10 μg) from treated and control rats with a 1:5,000 dilution of mouse anti-chicken -actin antibodies (Sigma, St. Louis, MO) for 10 h. The secondary antibody was horseradish peroxidase-coupled rabbit anti-mouse antibody (1:5,000). Immunoreactive -actin was detected as described earlier. We scanned the ECL films of Western blot analyses and performed densitometric analysis with Scion Image software using a gray color scale as a standard (SPSS 10.0).

    Statistical analysis. Data are expressed as means ± SE. All data were analyzed by SPSS 10.0 computer software (SPSS, Chicago, IL). We used one-way ANOVA or Student’s unpaired two-tailed test for statistical analysis. Statistical significance was set at P < 0.05.


    Effect of SNP on renal cortical microsomal 20-HETE production. To examine the stability of NO donor SNP, we incubated SNP (0.01 to 1 mM) at 37°C for varying times, then tested a drop of solution from each concentration with NO litmus paper prepared using filter paper that had been incubated with Griese reagent (24). As shown in Fig. 1A, SNP released NO (an orange spot shown by arrow bar) within 1020 min of incubation. The litmus paper proved to be a convenient and useful method for qualitatively checking NO production and indicated that SNP stably released NO within 20 min at 37°C. To examine whether NO has an inhibitory effect on 20-HETE production in renal cortical microsomes, we incubated renal cortical microsomes isolated from female rats with SNP (1556 μM) at 37°C for 20 min, then incubated them with arachidonic acid and NADHP and determined 20-HETE production by HPLC. As shown in Fig. 1B, the addition of SNP inhibited 20-HETE production in renal cortical microsomes in a concentration-dependent manner, with an IC50 of 22 μM.

    Effects of DDMS on renal function of L-NAME-treated pregnant and control rats. The preceding study showed upregulation of renal vascular 20-HETE production after chronic treatment of pregnant rats with L-NAME (36). To assess the contribution of elevated 20-HETE production to renal hemodynamics after withdrawal of NO, we examined renal functional parameters in pregnant and age-matched virgin female rats treated with vehicle, L-NAME, or L-NAME plus DDMS. As shown in Fig. 2, MAP was significantly decreased on day 18 of gestation compared with that in control nonpregnant rats. L-NAME treatment from days 14 to 17 of gestation caused a significant elevation of blood pressure, from 97 ± 3 to 134 ± 4 mmHg. This elevation of MAP was attenuated by combined treatment with L-NAME and DDMS. RBF values were slightly increased in pregnant rats compared with virgin rats (6.1 ± 0.4 vs. 5.1 ± 0.4 ml/min). L-NAME treatment of pregnant rats significantly decreased RBF from 6.1 ± 0.4 to 3 ± 0.3 ml/min, whereas L-NAME plus DDMS treatment did not significantly alter RBF in L-NAME-treated pregnant rats. RVR values in pregnant rats were significantly lower than those in nonpregnant control rats (15.3 ± 1.7 vs. 21.3 ± 1.6 mmHg?ml1?min1, P < 0.05). L-NAME treatment of pregnant rats also caused a significant increase in RVR, from 15.3 ± 1.7 to 45.3 ± 4.0 mmHg?ml1?min1. Moreover, the elevation of RVR in response to L-NAME was attenuated by treatment with L-NAME plus DDMS. As shown in Fig. 3, the GFR values in pregnant rats were significantly higher than those in nonpregnant control rats (1.6 ± 0.1 vs. 1.1 ± 0.04 ml/min, P < 0.05). L-NAME treatment markedly decreased GFR in pregnant rats to the levels in nonpregnant rats. This reduction of GFR by L-NAME treatment was attenuated by treatment with L-NAME plus DDMS (Fig. 3). In contrast, the MAP, RBF, RVR, and GFR in L-NAME-treated virgin rats did not significantly differ from control rats (Figs. 2 and 3). Similarly, combined treatment with L-NAME and DDMS had no impact on the changes in MAP, RBF, RVR, and GFR in virgin rats (Figs. 2 and 3).

    Effects of DDMS on renal cortical 20-HETE production of L-NAME-treated rats. To study the selectivity of the effect of DDMS on renal 20-HETE synthesis, we used HPLC to examine renal cortical arachidonic acid metabolism in pregnant rats treated with L-NAME and L-NAME plus DDMS. As shown in Fig. 4A, incubation of renal cortical homogenates isolated from L-NAME treatment with [14C]arachidonic acid and NADPH produced DHETs and 20-HETE. After treatment with DDMS plus L-NAME, the conversion of arachidonic acid to 20-HETE (-hydroxylase activity) decreased by 44%. DHETs production was not affected compared with L-NAME treatment (Fig. 4A). The -hydroxylase activity in the L-NAME treatment group was 49 ± 4 pmol?min1?mg protein1. Treatment with DDMS plus L-NAME caused a 44% reduction in this activity to 26 ± 3 pmol?min1?mg protein1 (n = 4, P < 0.05). The selective inhibition of 20-HETE by DDMS treatment was also observed in L-NAME plus DDMS-treated virgin rats (data not shown). To examine the effect of DDMS treatment on CYP4A expression, we conducted Western blot analysis for CYP4A in renal cortical homogenates isolated from pregnant rats treated with L-NAME and DDMS plus L-NAME. A representative Western blot of CYP4A isoforms after two treatments is shown in Fig. 4B. Renal cortical homogenate CYP4A protein levels were similar in rats treated with L-NAME and DDMS plus L-NAME.


    Normal pregnancy in rats is associated with increases in GFR and RBF and reductions in RVR and MAP (1, 17). Many different investigators have suggested that NO plays an important role in the regulation of renal function and blood pressure in pregnant rats (18, 19). NO causes the relaxation of smooth muscle in blood vessels, and the mechanisms for the action of NO on blood pressure regulation during pregnancy may be attributable to the upregulation of NO synthesis in blood vessels. It is also possible that pregnancy-induced changes in hormonal background contribute to the upregulation of NO production. Supporting this hypothesis, a prior study by Chen et al. (7) showed that estrogen stimulates NO synthesis through endothelial NO synthase (eNOS). Another study by Binko and Majewski (6) showed that estrogen increases the expression of inducible NO synthase (iNOS) in the vascular smooth muscle of blood vessels. In addition, Conrad et al. (8) demonstrated that the binding of NO to hemoglobin is detected only in the blood of pregnant, not nonpregnant, rats and Alexander et al. (1) demonstrated that increased NO production in pregnant rats is associated with upregulation of neuronal NO synthase (nNOS) and iNOS in the kidneys. These findings support the notion that upregulation of the NO system may be important in regulating vascular tone and blood pressure during pregnancy. To investigate the role of NO on the regulation of functional changes during pregnancy, Kassab et al. (16) used L-NAME, a competitive inhibitor of NO synthase, to study the consequence of NO inhibition on blood pressure and systemic and renal hemodynamic changes in pregnant rats. In this study, L-NAME-induced hypertension in pregnant rats is associated with increased RVR and decreased RBF (16). However, the mechanisms whereby L-NAME causes these changes in renal hemodynamics are still not very clear.

    One possibility is that chronic blockade of NO by L-NAME in pregnant rats results in the amplification of renal vasoconstrictor factors that cause the changes in renal function. In a previous study, we demonstrated a marked increase in renal vascular 20-HETE production after L-NAME treatment of pregnant rats (36). Because 20-HETE is a vasoconstrictor of renal arterioles, we hypothesize that this increased renal vascular 20-HETE synthesis after chronic NO inhibition may contribute to the alteration of renal function in L-NAME-treated pregnant rats. The essential findings of the present study, that selective inhibition of renal 20-HETE production in pregnant rats attenuates the changes in renal hemodynamics caused by L-NAME treatment, are in accord with that hypothesis. This conclusion is based on the observation that L-NAME-induced renal functional changes such as increased MAP, increased RVR, and decreased GFR are attenuated by treatment with DDMS, a selective inhibitor of 20-HETE production. Although we observed attenuation of the changes of renal hemodynamics by DDMS treatment in L-NAME-pregnant rats, this treatment cannot totally reverse the changes in renal hemodynamics, returning them back to the levels in control pregnant rats (Figs. 2 and 3), perhaps because of increases in the sensitivity of vasoconstriction systems other than 20-HETE after NO blockade. Molnar and Hertelendy (22) demonstrated that in rats at late pregnancy, acute treatment with L-NAME enhances the sensitivity of the pressor responses to angiotensin II, norepinephrine, and arginine vasopressin (22). Edwards et al. (9) showed that L-NAME treatment during mid to late pregnancy causes elevation in the level of plasma endothelian-1. However, further investigation is needed to determine whether these vasoconstriction systems are involved in the regulation of renal hemodynamics after chronic NO blockade.

    Besides its action on vascular relaxation, NO inhibits CYP enzyme systems (21, 31). The mechanisms for the inhibition of CYP enzymes by NO are thought to be reversible binding between NO and the heme moiety of CYP isoforms and irreversible modification of CYP isoforms by NO (21). In this regard, Sun et al. (32) showed that the addition of NO donors to recombinant CYP4A2 leads to the binding of NO to the heme moiety of CYP4A2 (an increases in the visible light absorbance at 440 nm). Minamiyama et al. (21) showed that NO can irreversibly modify the cysteine residues of CYP proteins. More recently, we demonstrated that NO binds to the heme moiety of CYP4A1 and CYP4A3 isoforms, the major enzymes for renal 20-HETE production in female rats, and that the NO donor SNP inhibits both CYP4A1 and CYP4A3-catalyzed 20-HETE production (36). These results are consistent with the results shown in Fig. 1, that SNP constantly releases NO and that SNP inhibits renal 20-HETE production in female rats. These results and those from our previous study (36) provide solid biochemical evidence of the interaction between NO and CYP4A enzymes and this interaction results in the inhibition of 20-HETE production in the kidneys.

    Several investigators demonstrated the functional implications of interaction between NO and 20-HETE in vivo. For example, Magdalena et al. (2) found that acute administration of DDMS attenuated the NO-mediated vasodilatory response in rat renal arterioles and that a reduction in MAP and RVR was mediated by NO donor. Another report by the same group demonstrated that inhibition of 20-HETE production by DDMS treatment contributes to the cerebral vasodilation response to NO in rats (3). In addition, Oyekan and McGiff (26) showed that acute inhibition of 20-HETE by 12,12-dibromododec-enoic acid, another selective inhibitor of 20-HETE production (33), attenuated acute L-NAME-induced reductions in RBF and GFR, as well as increases in MAP and RVR in male rats. More recently, Hercule et al. (13) demonstrated that the treatment of antisense oligodeoxynucleotides for CYP4A isoforms attenuates both the reduction in RBF and the increase in RVR induced by L-NAME treatment. Because we did not observe the significant renal hemodynamic changes in L-NAME and L-NAME plus DDMS-treated virgin rats (Figs. 2 and 3), the interaction between NO and CYP4A isoforms may be a unique and important mechanism in the regulation of renal function during pregnancy. In pregnant rats, the overproduction of NO in the kidneys (1) can lead to binding of NO to CYP4A isoforms in renal arterioles and mask the vasoconstrictive effect of 20-HETE during normal pregnancy. When chronic L-NAME treatment blocks NO production, NO no longer keeps the 20-HETE system under control resulting in the elevation of renal vascular 20-HETE production (36) and the amplification of renal vasoconstriction mechanisms that cause increased RVR and MAP (Fig. 2). More importantly, we showed that the increases in RVR and MAP induced in L-NAME-treated pregnant rats were attenuated by the inhibition of renal 20-HETE synthesis (Fig. 2). Taken together, the present study solidly demonstrates that the interaction between NO and 20-HETE has many functional implications with respect to the regulation of blood pressure and renal function in pregnant rats.

    It is well recognized that 20-HETE is formed endogenously in various tissues and exerts potent biological effects on cellular functions. Studies of its role on cellular levels are impeded by the difficulty in selectively targeting its synthesis and effects. This difficulty arises from that fact that the commonly used CYP inhibitors such as 1-aminobenzotriazole and 1-octadecynoic acid do not selectively inhibit 20-HETE production (31). We previously characterized several selective inhibitors of 20-HETE production in renal microsomes (33). Among them, DDMS was found to be highly specific for 20-HETE production, with an IC50 value about 2 μM, whereas its IC50 value for EETs formation is 60 μM. In the present study, we used DDMS to block the arachidonic acid -hydroxylase pathway and found that DDMS specifically blocked 20-HETE production without significantly affecting DHETs formation in pregnant rats (Fig. 4A). In addition, chronic DDMS treatment did not affect the renal expression of CYP4A isoforms in L-NAME-treated pregnant rats (Fig. 4B), which suggests that DDMS is a competitive inhibitor. Similar results for the competitive inhibition of 20-HETE by chronic DDMS treatment have been reported in the literature (4). Thus these data demonstrate that the selective inhibition of 20-HETE production by DDMS in vivo will be a very useful tool for elucidating the effect of 20-HETE in mediating physiological functions.

    In summary, we presented evidence that chronic inhibition of NO production by L-NAME amplifies a vasoconstrictor system operating through 20-HETE that contributes to the changes in renal hemodynamics following suppression of NO production in pregnant rats. This conclusion is supported by the chronic effect of DDMS, a selective inhibitor of 20-HETE production, which inhibits the renal response to L-NAME-treated pregnant rats. This study demonstrates that activation of the renal 20-HETE pathway after removal of NO in the kidneys during pregnancy affects the regulation of renal function and blood pressure and that interaction between NO and 20-HETE may have important functional implications during pregnancy.


    This study was supported by National Institutes of Health (NIH) Grant R01-HL-70887 to M.-H. Wang and by NIH Grant DK-38226 and a grant from Robert A. Welch Foundation to J. R. Falck.


    The authors thank Dr. D. M. Pollock for providing the protocol for measuring GFR. The authors also thank J. Cole for editorial assistance.


    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.

    H. Huang and Y. Zhou contributed equally to the development of this research study.


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日期:2013年9月26日 - 来自[2005年第288卷第11期]栏目

Insulin treatment enhances AT1 receptor function in OK cells

【关键词】  sodium

    Heart and Kidney Institute, College of Pharmacy, University of Houston, Houston, Texas


    Increased renal sodium retention is considered a major risk factor contributing to hypertension associated with chronic hyperinsulinemia and obesity. However, the molecular mechanism involved is not understood. The present study investigates the effect of insulin treatment on AT1 receptor expression and ANG II-induced stimulation of Na/H exchanger (NHE) and Na-K-ATPase (NKA) in opossum kidney (OK) cells, a proximal tubule cell line. The presence of the AT1 receptors in OK cells was confirmed by the specific binding of 125I-sar-ANG II and by detecting 43-kDa protein on Western blot analysis with AT1 receptor antibody and blocking peptide as well as by expression of AT1 receptor mRNA as determined by RT-PCR. Insulin treatment (100 nM for 24 h) caused an increase in 125I-sar-ANG II binding, AT1 receptor protein content, and mRNA levels. The whole cell lysate and membrane showed similar insulin-induced increase in the AT1 receptor protein expression, which was blocked by genistein (100 nM), a tyrosine kinase inhibitor, and cycloheximide (1.5 μg/ml), a protein synthesis inhibitor. Determination of ethyl isopropyl amiloride-sensitive 22Na+ uptake, a measure of the NHE activity, revealed that ANG II (1100 pM)-induced stimulation of NHE in insulin-treated cells was significantly greater than in the control cells. Similarly, ANG II (1100 pM)-induced stimulation of ouabain-sensitive 86Rb+ uptake, a measure of NKA activity in insulin-treated cells, was significantly greater than in the control cells. ANG II stimulation of both the transporters was blocked by AT1 receptor antagonist losartan, suggesting the involvement of AT1 receptors. Thus chronic insulin treatment causes upregulation of AT1 receptors, which evoked ANG II-induced stimulation of NHE and NKA. We propose that insulin-induced increase in the renal AT1 receptor function serves as a mechanism responsible for the increased renal sodium reabsorption and thus may contribute to development of hypertension in conditions associated with hyperinsulinemia.

    angiotensin II; Na-K-ATPase; Na/H exchanger

    ANGIOTENSIN II (ANG II) is an important regulator of renal and cardiovascular functions. ANG II binds mainly to two types of ANG II receptors, namely AT1 and AT2, which on activation initiate a cascade of signaling leading to the cellular response (10). The AT1 receptors promote cell growth and proliferation and produce vasoconstriction and antinatriuresis, (12, 21, 31, 34, 38), whereas AT2 receptors are reported to produce responses opposite to those produced by the AT1 receptors (11). Within the kidney, systemic and locally produced ANG II, via the activation of tubular AT1 receptors, serves as a potent hormone that participates in the reabsorption of the filtered sodium from the lumen and thus helps maintain sodium homeostasis and regulates blood pressure (13, 28). At molecular level, ANG II activates AT1 receptors coupled with Gi proteins and modulates multiple second messenger systems such as lowering of cellular cAMP and activation of phospholipase A2 in proximal tubule epithelial cells (26, 39). The modulation of these second messenger levels by ANG II leads to the stimulation of tubular sodium transporters, namely, Na/H exchanger, Na-K-ATPase, and Na/HCO3 cotransporters, thereby increases tubular sodium reabsorption (7, 8, 15). Numerous studies suggest that an abnormal AT1 receptor function, either caused by excessive availability of ANG II or increased AT1 receptor signaling per se, contributes to the shift in pressure natriuresis and the development of hypertension (9, 17, 25, 28, 36, 37).

    It is well established that obesity is a major cause of insulin resistance leading to hyperinsulinemia and essential hypertension (19). Although there is a correlation between hyperinsulinemia and enhanced renal sodium reabsorption, there is evidence suggesting that chronic hyperinsulinemia is not a major factor responsible for the increases in renal sodium reabsorption and blood pressure (20). In vivo studies suggest that the renal AT1 receptor function in obese Zucker rats is increased, which contributes to a greater reabsorption of the filtered sodium and to the development of hypertension in these animals (1, 34). Recently, we provided in vitro evidence that the AT1 receptor numbers are increased in the brush-border membranes (BBM) and ANG II produces a greater stimulation of Na/H exchanger (NHE) in proximal tubules of obese compared with lean Zucker rats (6). We hypothesized that hyperinsulinemia, a characteristic of obese Zucker rats, may be responsible for the increase in the AT1 receptor expression and the enhanced ANG II-mediated stimulation of the sodium transporters. Therefore, to study the effect of insulin on AT1 receptor expression and their influence on the stimulation of sodium transporters, we have utilized proximal tubule cell line derived from the opossum kidney (OK cells), which maintains cellular response to ANG II (24, 36). The OK cells were chronically treated with insulin, followed by measuring AT1 receptor ligand binding and protein and mRNA expression and the ANG II-mediated stimulation of NHE and NKA activities in the treated cell.


    Cell culture media and serum were purchased from GIBCO-BRL. ANG II, cycloheximide, genistein, and insulin were purchased from Sigma (RBI). 86RbCl, 22NaCl, and 125I-sar-angiotensin were purchased from New England Nuclear Life Sciences. Antibodies NHE31-A for NHE3 and AT1 (N-10, SC-1173) for AT1 receptors were purchased from Alpha Diagnostic (San Antonio, TX) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. All other chemicals of highest purity available were purchased from Sigma.

    Cell culture and drug treatments. OK cells were purchased (American Type Culture Collection, Manassas, VA) and grown in 24-well plastic cell culture plates or 100-mm culture dishes in DMEM culture media supplemented with serum (10%) under constant flow of 5% carbon dioxide mixed with air in a cell culture incubator set at 37°C, as suggested in the manufacturer's manual and reported by us (27). For consistency, cell passages 510 were utilized in this study. At 8090% confluency, the OK cells were washed with serum-free medium and further incubated in serum-free medium with vehicle (control cells) or insulin, 100 nM (treated cells) for 24 h. Also, to investigate the effect of tyrosine kinase in insulin action, OK cells were incubated with insulin in the presence of genistein (100 nM). Furthermore, cells were also chronically coincubated with insulin and cycloheximide to investigate the role of de novo protein synthesis in insulin action. After 24 h of incubation, the cells were washed three times with serum-free media and stabilized for 3 h in same media to ascertain that insulin is absent from the medium and does not have acute effect per se on sodium transporters. Effects of ANG II on the activities of NKA and NHE were measured in the intact cells, while the quantification of AT1 receptor expression was performed in the cell membranes and the cell extracts, as described below.

    Quantification of AT1 receptors. OK cell membranes were prepared, as we described earlier (27). Briefly, OK cells were lysed with 10 mM tris[hydroxymethyl]aminomethane (Tris) buffer (pH 7.4) containing a cocktail of protease inhibitors (Complete 1 697 498, Roche Diagnostic) and 1 mM phenylmethylsulfonylfluoride (PMSF) followed by homogenization and sonication. The cell lysates were centrifuged at 200 g for 5 min; resulting supernatant was further centrifuged at 37,000 g for 20 min, to obtain membrane pellets. The pellets were suspended in Tris buffer. In another set of experiments, the cell lysates, without any centrifugation, were used for measuring total contents of AT1 receptor protein in the cell. Protein in the membrane samples and the cell lysates was determined by using a bovine serum albumin (BCA) kit (Pierce) with BSA as standard.

    125I-sar-ANG II binding. To the OK cell membranes, 125I-sar-ANG II binding was performed essentially as described earlier (23). The membranes (50 μg protein) were incubated with 30 pM of 125I-sar-ANG II at 30°C for 20 min in a shaking water bath. The assay was terminated by rapid filtration on GF/C filters under vacuum. The radioactivity on the filters was counted in a LKB gamma counter. Nonspecific binding was determined by performing the binding assay in the presence of 1 μM unlabeled ANG II.

    Western immunoblotting. The AT1 receptors and NHE3 were detected and quantified by Western blotting, as we have described earlier (23). Basolateral membranes were used a positive control for AT1. Briefly, the sample proteins were solubilized in Laemmeli buffer. Protein samples of the membranes (515 μg) and the lysates (1025 μg) of the control and the treated cells and the proximal tubular membranes (5 μg) were resolved on 10% SDS-polyacrylamide gel electrophoresis and transferred onto Immobilon P membrane (blot). The AT1 receptors and NHE3 were detected by using affinity-purified polyclonal anti-peptide AT1 receptor and anti-NHE3 antibodies, respectively, HRP-linked anti-rabbit IgG and enhanced chemiluminescence. Signals were recorded as bands on Kodak X-ray films. Approximate molecular mass and the density of the protein bands were determined using molecular mass markers and densitometric analysis (Kodak Imaging Station), respectively. The density of the bands was compared between samples from the control and insulin-treated OK cells.

    RT-PCR for AT1. Total RNA was isolated from the OK cells and rat renal cortex by the RNeasy mini kit (QIAGEN, Valencia, CA) and 1μg of RNA was used for cDNA synthesis and amplification for AT1 receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, used as an internal control) using Advantage cDNA PCR Kit (BD Biosciences, Clonetech, Palo Alto, CA). The PCR for AT1 cDNA was performed utilizing sense (5'-CCA AAG TCA CCT GCA TCA TC-3') and antisense primers (5'-CAC AAT CGC CAT AAT TAT CCT A-3'). The primers used for AT1 cDNA were designed from the cDNA sequences common to rat AT1A and AT1B receptors (3, 33). These primers correspond to regions where no sequence divergence exists between AT1A and AT1B, and amplify a 305-bp cDNA fragment from position 723 to 1028 in the AT1A sequence and from position 630935 in the AT1B sequence (3, 33). The GAPDH cDNA was amplified using the sense primer (5'- TAC TCC TTG GAG GCC ATG TA-3') and the antisense primer (5'-CGT GGA GTC TAC TGG CGT CT-3') (Ref. 3), which yielded a 723-bp product. The PCR products, were resolved on 1.5% agarose gel, stained with ethidium bromide, and subjected to densitometry of the bands using FluorChem 8800 (Alpha Innotech Imaging System).

    Effect of ANG II on NHE and NKA activity in intact OK cells. Activity of the NHE was measured by 22Na+ uptake while the activity of NKA was measured by 86Rb+ uptake as well as by adenosine 5'-triphosphate (ATP) hydrolysis methods, as described earlier (4, 14, 16) with slight modifications. 1) 22Na+ uptake: Briefly, stabilized OK cells were rinsed three times with sodium-free buffer consisting of (in mM) 120 tetramethylammonium chloride (TMA-Cl), 5 KCl, 1 CaCl2, 1 MgCl2 {pH 6.40 ± 0.01 and 295 ± 1 mosmol/KgH2O with 18 Tris base, 28 N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), and 18 2-(N-morpholino)ethanesulfonic acid}. The assay was performed in the presence of a 1 mM ouabain and 100 μM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) to minimize Na+ exit from the cell. The cells were incubated for 710 min in Na+-free buffer containing ANG II (1100 pM). In AT1 receptor antagonist assays, 1 μM losartan was added 5 min prior to ANG II. The uptake was initiated by addition of 22Na+ (10 μCi/ml) in a buffer containing (in mM) 40 NaCl, 110 TMA-Cl, 5 KCl, 1 CaCl2, 1 MgCl2 (pH 7.40 and 295 ± mosmol/kgH2O with 18 mM Tris base and 28 mM HEPES). The cells were maintained at 37°C for 3 min and the reaction was terminated by washing the wells four times with 1 ml ice-cold isosmotic LiCl-HEPES buffer (pH 7.4). Cells were lysed with 3% sodium dodecyl sulfate (SDS) [0.5 ml/well] and radioactivity was counted directly in cell lysate by gamma-counter. A portion of the lysates was used to determine the protein contents, and the values for each sample were corrected per milligram protein. The uptake was also measured in the presence of ethyl isopropyl ameloride (EIPA, 10 μM), a NHE inhibitor. The NHE activity was determined as the difference of 22Na+ uptake in the absence and presence of EIPA. The effect of ANG II in control and insulin-treated cells was calculated over respective basal 22Na+ uptake. 2)86Rb+ uptake: The control and insulin-treated cells after stabilization for 3 h in the medium free of serum and insulin, as described above, were incubated with vehicle or ANG II (1100 pM) for 10 min at 37°C. Similar to 22Na+ uptake, AT1 specificty was determined by preincubating the cells with 1μM losartan (AT1 antagonist). The 86Rb+ uptake was initiated by the addition of 10 μl of 86Rb+ in DMEM to obtain final concentration of 3 μCi/ml 86Rb+. After 10 min of incubation at 37°C, the uptake was stopped by aspirating the media and rinsing the wells four times with 1.0 ml/well ice-cold phosphate-buffered saline. Preliminary results show that 86Rb+ uptake is linear for at least 15 min. Similar to the above protocol, the cells were lysed by SDS, radioactivity was counted, and protein contents of the lysates were determined. The NKA activity was determined as the difference between 86Rb+ uptake in the absence (total activity) and presence (ouabain-insensitive) of ouabain (1 mM). Ouabain inhibited 70% of the total uptake. 3) ATP hydrolysis: cells were incubated with or without 10 pM ANG II for 10 min at 37°C. Cell suspension (0.05 mg/ml) was used to assay 1mM ouabain-sensitive NKA activity by end-point phosphate hydrolysis of 4 mM ATP as described earlier (4). The inorganic phosphate (Pi) released was determined calorimetrically.

    Statistical analysis. Differences between means were evaluated using the unpaired t-test or analysis of variance with Newman-Keuls multiple test, as appropriate. P < 0.05 was considered statistically significant.


    Quantification of AT1 receptors. Western blot analysis with the AT1 receptor antibody revealed the presence of 43-kDa protein band in OK cell membranes and in the rat proximal tubular membranes. The blocking peptide-treated AT1 receptor antibody did not label any band, suggesting the presence of AT1 receptor-specific band detected with the antibody (Fig. 1A). Furthermore, these cells also express a moderate levels of AT1 receptor mRNA. The RT-PCR product in OK cells was of similar size (300 bp), although smaller in intensity compared with rat renal cortex (Fig. 1B), thus suggesting that AT1 message level in OK cells is not markedly lower than proximal tubules. Of the note, the intensity of AT1 protein band in OK cells is smaller than proximal tubules.

    As illustrated in (Fig. 1B), the amplification of cDNA for AT1 receptors, synthesized from OK cells and the rat kidney, shows a single band that corresponds to the size of 300 bp on a 100-bp DNA ladder. Since the expected size of our PCR product is 305 bp, we believe that the bands obtained are the ones for the AT1 receptor. Since kidney is known to express AT1 receptors in abundance, we used the mRNA from the rat kidney as a control for the AT1 receptor expression studies. The presence of the bands at the same position in the cDNA of OK cells samples suggesting that OK cells also express the AT1 receptors.

    A comparison of the membrane AT1 receptor density revealed that treatment of OK cells with insulin caused more than twofold increase in the AT1 receptor expression compared with the control cells (Fig. 2A). Insulin treatment also caused a significant increase in AT1 receptor message levels (Fig. 2B). 125I-sar-ANG II binding to OK cell membranes revealed specific binding as 30 and 40% of the total binding in control and insulin-treated samples, respectively. The specific binding was approximately twofold higher in the membranes of insulin-treated (1.76 ± 0.03 fmol/mg protein) compared with control cells (0.94 ± 0.09 fmol/mg protein; Fig. 2C). A similar increase in the AT1 receptor expression was observed in the lysates of cells treated with insulin compared with the control cells. When the cells were incubated with insulin in the presence of genistein (10 nM), a tyrosine kinase inhibitor, the upregulatory effect of insulin on AT1 receptor was completely abolished, while genistein alone had no effect on the AT1 receptor protein expression (Fig. 3). This suggested the role of tyrosine kinase, a characteristic of insulin receptors, in the insulin-mediated upregulation of AT1 receptors. Furthermore, when the cells were incubated with insulin in presence of cycloheximide, the insulin-induced increase in the AT1 receptor protein was abolished, suggesting the role of protein synthesis in insulin action on AT1 receptor increase (Fig. 4).

    Effects of ANG II on NHE and NKA activity. ANG II (1100 pM) stimulated 22Na uptake over basal in both the control and insulin-treated cells. However, the stimulatory effect of ANG II was significantly greater in the insulin-treated compared with the control cells (Fig. 5A). The basal 22Na+ uptake was similar in the control and insulin-treated cells (basal: control, 4 ± 0.1; insulin-treated, 4.35 ± 0.15 nmol 22Na+?mg protein1?min1). To test whether ANG II-induced stimulation of NHE3 was AT1 specific, 22Na+ uptake was carried out in presence of losartan, an AT1 antagonist. As shown in Fig. 4A, 1 μM losartan blocked ANG II-induced stimulation of NHE3 both in control and insulin-treated cells. Losartan per se did not affect the 22Na+ uptake in absence of ANG II. To determine whether ANG II causes any changes in NHE3 protein expression, NHE3 surface antigen was measured in both control and insulin-treated cells. As shown in Fig. 5B, exposure to ANG II alone for 10 min did not affect NHE3 protein contents on the membranes. ANG II (1100 pM) stimulated 86Rb uptake over basal in both the control and the treated cells, being greater stimulation in the treated compared with the control cells (Fig. 6A). Basal 86Rb uptake in the control and insulin-treated cells was similar (basal: control, 15.2 ± 0.3; insulin-treated, 15.9 ± 0.4 nmol 86Rb+?mg protein1?min1). As shown in Fig. 5A, losartan blocked the ANG II effect both in control and insulin- treated cells, suggesting AT1-specific effects. Because OK cells form monolayers by attaching to surface through basolateral membranes, this may impose limitations when pump activity is measured in intact cell. To address this concern, the NKA activity was also measured in whole cell lysate. Cells were scraped from culture dishes to recover maximum basolateral side. As shown Fig. 6B, ANG II (10 pM) stimulated NKA activity both in control and insulin-treated cells with maximum effect being observed in insulin-treated cells. Also, insulin per se did not change the ATPase activity of the pump. These results suggest that attached cells do not impose any limitation on the validity of 86Rb+ uptake as a measure of pump activity.


    Present study demonstrates that chronic insulin treatment of OK cells causes upregulation of the AT1 receptor numbers with an increase in protein expression of the receptors, which on activation by ANG II produces greater stimulation of NHE and NKA activity.

    Obesity is a major cause of hypertension (20). There is evidence suggesting an enhanced function of ANG II receptors in terms of an increase in the renal sodium reabsorption and their contribution to the development of hypertension in obesity (1, 18, 34). Recently, we reported that ANG II binding sites and the stimulation of NHE by ANG II were enhanced in the isolated preparations of proximal tubules of obese Zucker rats compared with the control lean Zucker rats (6). Because obesity is usually associated with insulin resistance and hyperinsulinemia, we speculated that increased level of insulin played a role in the enhanced action of ANG II on sodium transport. We tested this hypothesis by using an in vitro model of OK cells, a cell line of proximal tubule epithelial cells derived from opossum kidney. Several studies have utilized OK cells as an in vitro model to study ANG II-mediated cellular responses (24, 35). However, there are no reports that demonstrate the expression and quantification of AT1 receptors in these cell lines. Therefore, we first determined the expression of AT1 receptor mRNA and protein in OK cells, using RT-PCR and specific AT1 receptor antibody and blocking peptide. We found that OK cell express AT1 receptor mRNA and protein as expressed in the rat proximal tubular membranes and reported earlier (22, 23). A comparison of AT1 receptor expression between control and insulin-treated cells revealed that insulin treatment of OK cells caused an upregulation of the AT1 receptors in the membranes, as well as in the whole cell lysates and was blocked by cycloheximide, a protein synthesis inhibitor. This suggests that the increase in the AT1 receptor expression may be due to an increase in the receptor protein synthesis, which leads to a proportionally higher expression of the receptors on the cell membranes. This is supported by an increase in AT1 mRNA level and ligand binding to the membranes of insulin-treated cells. Insulin-mediated upregulation of the AT1 receptors was blocked by genistein, suggesting the role of tyrosine kinase. Chronic treatment with insulin has been shown to upregulate AT1 receptors in other cells lines, such as vascular smooth cells (30) and rabbit proximal tubule epithelial cells (5). Our studies are consistent with those reported in the smooth muscle cells, in that insulin upregulates the AT1 receptor at the protein synthesis level, which is sensitive to genistein. However, this is the first report showing that OK cells express detectable and quantifiable levels of AT1 receptors and hence these cells may be used as a model of epithelial cells for studying the regulation of endogenously expressed AT1 receptors.

    Having established that chronic insulin treatment increases the AT1 receptor expression in OK cell membranes, we tested whether such an increase in the AT1 receptors in response to ANG II produces a greater stimulation of sodium transporters in insulin-treated cells. We found that ANG II indeed produced a greater stimulation of both the NKA and the NHE3 activity in insulin-treated compared with the control cells. It should be noted that after stabilization of cells for 3 h in insulin-free medium, the basal activity of both the transporters was similar in the control and insulin-treated cells. This suggests that the increase in AT1 receptors, and not the transporter per se, might be responsible for the enhanced ANG II-mediated stimulation of the transporters in insulin-treated cells. Although the AT1 receptors expressed in OK cell membranes are very low, the response to picomolar concentrations of ANG II in our study are also supported by other studies (14). Earlier, we have reported an increase in the AT1 receptor number and the enhanced ANG II-mediated stimulation of NHE3 in the isolated proximal tubule of obese Zucker rats (6). Although insulin has been shown to cause mild sodium retention (2, 32), the present study suggests that the upregulation of AT1 receptors and the enhanced ANG II-mediated stimulation of sodium transporters caused by chronic hyperinsulinemia may lead to a powerful antinatriuretic response to ANG II. It is likely that insulin-induced upregulated AT1 receptors may also produce other known cellular effects associated with the AT1 receptors, such as cell growth and cell proliferation (38), and contribute to the pathophysiology associated with hyperinsulinemia and obesity.

    In brief, this is the first report, to our knowledge, showing that OK cells express detectable and quantifiable AT1 receptors. Insulin treatment of OK cells upregulates AT1 receptors, which on activation by ANG II produce greater stimulation of sodium transporters. Such an increase in AT1 receptor function serves as a mechanism responsible for the increased renal sodium reabsorption and development of hypertension in conditions associated with hyperinsulinemia.


    This study is supported by National Institutes of Health Grant DK-61578.


    The authors thank L. Pillai for providing technical assistance in maintaining OK cell cultures and performing Western blot analysis.


    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6期]栏目


【摘要】  目的 比较单独使用153Sm-EDTMP内照射治疗肿瘤多发骨转移与153Sm-EDTMP联合局部外照射治疗肿瘤多发骨转移的治疗结果,以探讨联合治疗的临床价值。方法 临床确诊为恶性肿瘤多发骨转移的住院或门诊患者59例,38例采用联合治疗的方法,21例单独使用153Sm-EDTMP内照射治疗。患者血常规检查:白细胞>3×109/L,血小板>80×109/L,肝肾功能正常。内照射治疗方法:静脉注射153Sm-EDTMP,每人1~3次,最多4~5次,每次给药剂量(mCi)=病人体重×系数(0.5~0.8),每次治疗间隔时间为6~8周,治疗1周后复查血常规,如血象降低,及时给予升血象治疗。外照射治疗方法:局部定位为痛点放疗和体积较大的骨转移灶,照射野一般不超过3个,剂量一般为3Gy/次,隔天1次,一般照射4~10次。具体部位因人而异。结果 一般治疗后2周~2个月回访患者观察疗效。153Sm-EDTMP联合外放疗38例,疼痛消失或基本消失22例(58%),疼痛减轻10例(26%),疼痛无明显改善3例(8%),疼痛加重3例(8%);单独使用153Sm-EDTMP治疗21例,疼痛消失或基本消失7例(33%),疼痛减轻10例(48%),疼痛无明显改变2例(9.5%),疼痛加重2例(9.5%)。结论153Sm-EDTMP联合局部外照射治疗疗效明显优于单独使用153Sm-EDTMP,毒副作用小,是提高患者生存质量有效、安全的治疗方法。

【关键词】  153Sm-EDTMP;外照射治疗;肿瘤多发骨转移;联合治疗;临床价值

 153Sm-EDTMP combined with exclusive inner radiation treatment on multiple bone metastatic tumors

  YANG Xiao-rong.Hubei Cancer Hospital,Wuhan 430079,China

  [Abstract] Objective The research compares 153Sm-EDTMP exclusive inner radiation treatment with 153Sm-EDTMP joint local external radiation treatment of the multiple bone metastatic tumors, in order to investigate the clinical value of combined treatment.Methods Fifty-nine outpatients and inpatients who are clinical diagnosed of malignant bone metastatic tumor, thirty-eight patients adopt combined treatment, and the others adopt 153Sm-EDTMP exclusive inner radiation treatment. Patients' blood routine examination: leukocyte>3×109/L, thrombocyte>80×109/L, the function of liver and kidney is normal. Inner radiation treatment: intravenous injection of 153Sm-EDTMP, each patient takes one to three times, four to five times at the most, each drug doses (mCi)=patient's weight×coefficient (0.5 ~ 0.8), every treatment interval is 6 ~ 8 weeks, takes blood routine examination again after the treatment, if the patient's hemogram reduces, give the treatment to raise the hemogram promptly. External radiation treatment: local orientation in pain point radiation treatment and volume of bone metastases, radiation field does not exceed three generally, dose for 3Gy/times commonly, take treatment every two days, and irradiate four to ten times, specific treatment for different parts and patients.Results Pay a return visit to the patients after treatment in two weeks until two months .Thirty-eight patients adopt 153Sm-EDTMP combined treatment, twenty-two patients' pain disappeared or disappear basically (58%), ten patients' pain ease off (26%), three patients' pain do not improve obviously (8%), three patients pain get worse before treatment(8%). Twenty-one patients adopt 153Sm-EDTMP exclusive inner radiation treatment, seven patients' pain disappeared or disappear basically (33%), ten patients' pain ease off (48%), two patients' pain do not improve obviously (9.5%), two patients' pain get worse (9.5%).Conclusion The 153Sm-EDTMP combined treatment is better than the 153Sm-EDTMP exclusive inner radiation treatment obviously, its side effect is little. It is an effectual and safe treatment method for raising the patients' quality of life.

  [Key words] 153Sm-EDTMP;external radiation treatment; the multiple bone metastatic tumors;combined treatment;clincal value


  1 资料与方法

  我院临床确诊为恶性肿瘤多发骨转移的住院或门诊患者59例,38例采用联合治疗的方法,21例单独使用153Sm-EDTMP内照射治疗。患者男30例,女29例,年龄28~78岁。原发肿瘤包括前列腺癌、乳腺癌、肺癌、鼻咽癌、胃癌、肝癌、直肠癌等。 患者血常规检查:白细胞>3×109/L,血小板>80×109/L,肝肾功能正常。内照射治疗方法:静脉注射153Sm-EDTMP,每人1~3次,最多4~5次,每次给药剂量(mCi)=病人体重×系数(0.5~0.8),每次治疗间隔时间为6~8周,治疗1周后复查血常规,如血象降低,及时给予升血象治疗[1]。外照射治疗方法:局部定位为痛点放疗和体积较大的骨转移灶,照射野一般不超过3个,剂量一般为3Gy/次,隔天1次,一般照射4~10次。具体因部位因人而异[2]。

  2 结果

  治疗后2周~2个月回访患者,观察疗效。见表1。表1 两种方法治疗结果比较由此可见,153Sm-EDTMP联合局部外照射治疗疗效明显优于单独使用153Sm-EDTMP。

  3 讨论

  许多晚期的恶性肿瘤如乳腺癌、前列腺癌以及肺癌等常伴发多发性骨转移,约有50%的患者有日益加重的剧烈疼痛,其治疗方法主要有药物镇痛(世界卫生组织三阶梯药物止痛)、外照射治疗、手术治疗、放射性核素治疗(内放疗)。三阶梯药物止痛在多数情况下可获得良好的效果,但有时因其严重的胃肠道反应等副作用而无法进行;外照射治疗对转移灶外单发者止痛疗效确切,但对于多发灶却无能为力;手术治疗可使部分病例的疼痛缓解,尤其是单侧肢体或骨盆的疼痛明显,但疗效短,且创伤大。放射性核素治疗是利用药物的趋骨性,在转移性骨肿瘤病灶内有较高的浓聚,利用其发射的β射线对肿瘤进行内照射,从而达到止痛、抑制病灶增长甚至破坏转移肿瘤病灶的目的。153Sm-EDTMP在骨内的聚集与局部骨血流和骨代谢密切相关,在肿瘤骨转移灶的聚积量是正常骨的2~25倍,可以对病灶起到极好的治疗作用而对正常骨的影响极小;153Sm-EDTMP发射的β射线可以直接作用于肿瘤细胞;它还可以降低碱性磷酸酶和前列腺素水平,减轻骨质溶解,修复骨质[3,4]。局部外照射治疗是将放射源(X线治疗机产生的普通X线,加速器产生的高能X线,还有各种加速器所产生的电子束、质子、快中子、负兀介子以及其他重粒子等)与患者身体保持一定距离进行照射,射线从患者体表穿透进人体内一定深度,达到治疗肿瘤的目的。局部外照射治疗定位为痛点放疗和体积较大的骨转移灶,可以使患者疼痛得到很好的缓解,提高患者的生存质量。153Sm-EDTMP产生的 β射线射程达不到治疗的深度,局部外放疗正好弥补了这一缺陷。153Sm-EDTMP联合局部外照射治疗不仅使全身骨转移灶得到了治疗,更使一些敏感疼痛的骨骼加强治疗。结论:153Sm-EDTMP联合局部外照射治疗疗效明显优于单独使用153Sm-EDTMP,毒副作用小,是提高患者生存质量有效、安全的治疗方法,具有较好的临床价值。

   1 卫生部医政司.核医学诊断与治疗规范.北京:科技出版社,1997:292-295.

  2 林晓丹,刘枫,王兆武.多发性骨转移瘤的综合治疗.实用肿瘤杂志,2000,15:121-122.

  3 Turner JH,Martindale AA,Sorby P,et al. 153Sm-EDTMP therapy of disseminate skeletal metastasis. Eur J Nucl Med ,1989,15:784-795.

  4 潘中允. 放射性核素治疗学. 北京:人民卫生出版社. 2006:231-239.


日期:2013年2月27日 - 来自[2010年第7卷第6期]栏目


【摘要】  目的 探讨儿童支气管哮喘药物临床治疗的顺应性。方法 选择2007年8月至2009年2月于本院儿科门急诊及病房治疗的3~12岁哮喘儿童148例,通过回顾性问卷调查方式了解这些患儿哮喘发作时使用药物治疗情况及患儿本身及其家长对于口服和吸入药物治疗方法的接受程度。结果 在所有接受调查的148例患儿中,哮喘发作时首选口服药物治疗的占84%,首选吸入药物治疗的仅占16%;对于口服和吸入两种不同治疗方法的接受程度比较,儿童愿意接受口服药物治疗的占73%,家长愿意接受口服治疗的占78%,二者比例均远高于愿意接受吸入治疗的儿童和家长的比例。结论 目前口服治疗仍是儿童支气管哮喘发作时首选治疗,且吸入药物治疗在儿童及其家长中的接受程度也远远低于口服药物治疗。

【关键词】  儿童;支气管哮喘;治疗;顺应性

Compliance of clinical treatment for bronchial asthmatic in children

  DAI Qiang,WU Lan-ying,XIA You-jia,et al.Department of Pediatrics,Chinese and Western Medicine Hospital,Shanghai 200082,China

  [Abstract] Objective To explore compliance of clinical treatment for bronchial asthmatic in children. Methods 148 asthmatic children in emergency door and in hospital from Aug.2007 to Feb.2009 were selected,understanding treatment for the children when an asthma attack with retrospective questionnaire and acceptance of oral treatment and inhalation treatment for asthmatic children and their parents.Results The proportion of preferred oral treatment was 84% when an asthma attack in whole 148 children,only 16% asthmatic children preferred inhalation treatment at first;compare acceptance of two treatments,the proportion of asthmatic children who want oral treatment was 73%,the proportion of parents who want oral treatment was 78%,both proportion was far higher than the proportion of acceptance of inhalation treatment for asthmatic children and their parents.Conclusion Oral treatment remains first choice when asthma attack for children,the acceptance of inhalation treatment is far less than oral treatment in asthmatic children and their parents.

  [Key words] children;bronchial asthma;treatment;compliance


  1 资料与方法

  1.1 一般资料 于2007年8月至2009年2月至本院儿科门急诊及住院哮喘患儿共148例,均符合全国儿科哮喘协作组制定的儿童哮喘病诊断标准[1]。年龄3~12岁,平均(6.54±2.31)岁,男87例, 平均(6.45±2.21)岁,女61例,平均(6.67±2.45)岁。其中3~6岁66例,6~9岁58例,9岁以上24例。

  1.2 方法 采用回顾性问卷调查方法了解患儿哮喘发作时使用药物治疗情况及患儿本身及其家长对于口服药物治疗和吸入治疗方法的接受程度,试验根据年龄分为三组,3~6岁组、6~9岁组和9岁以上组。调查问卷表由固定专科医生进行询问、填写。

  1.3 统计学方法 采用SPSS 11.5软件进行χ2检验和秩和检验。

  2 结果

  所有148例哮喘患儿中,哮喘发作时首选口服药物治疗者125例,其中口服速效β2受体激动剂 53 例(沙丁胺醇或特布他林),茶碱类 68 例,激素类 4 例(强的松);首选吸入治疗者23例,其中吸入速效β2受体激动剂 22 例(沙丁胺醇或特布他林),激素 1 例(布地奈德);两者之间的差异具有统计学意义(P=0.000)(表1);各年龄组内比较均有明显差异,而不同年龄组间口服及吸入治疗比较差异无统计学意义(P=0.724)。发作间歇期服药治疗的33例,其中口服顺尔宁19例;不规则吸入激素13例(丙酸倍氯米松、布地奈德);口服中药调理1例,无一例儿童于发作间歇期坚持规则吸入维持治疗。



  对于患儿家长行治疗意愿调查,愿意自己孩子口服药物治疗的115例,占78%。原因:(1)口服药物治疗简单,起效快58例,占50%;(2)吸入治疗技术要求较高,孩子很难准确掌握38例,占33%;(3)担心长时间吸入激素对孩子的生长发育有一定影响15例,占14%;(4)家长缺乏对哮喘的认识4例,占3%;愿意自己孩子行吸入治疗的33例,占22%。两者之间的差异具有统计学意义(P=0.000)(见表2)。表1 148例儿童哮喘发作时治疗情况注:*三组不同年龄段儿童口服及吸入治疗人数差异比较均有显著统计学意义(P=0.001)(组内比较);△不同年龄组口服及吸入治疗人数差异比较无明显差异(P=0.724)(组间比较);χ2=63.153,P1=0.000口服治疗使用不同药物差异比较;χ2=19.174,P2=0.000吸入治疗使用不同药物差异比较表2 哮喘治疗顺应性调查注:*仅于发作时坚持使用;三组患儿对于口服及吸入治疗顺应性组间比较Z1=0.456,P1=0.648;三组患儿家长对于口服及吸入治疗顺应性组间比较;Z2=0.020,P2=0.933


  3 讨论








    1 中华医学会儿科学分会呼吸学组《中华儿科杂志》编辑委员会.儿童哮喘诊断与防治指南.中华儿科杂志,2008,46(10):745.

  2 上海医学会儿科呼吸组.上海市0岁~14岁儿童支气管哮喘患病情况调查.临床儿科杂志,2002,30(3):146.

  3 诸葛亚玲,胡国华,姚静婵.布地奈德气雾剂治疗儿童哮喘临床观察.儿科药学杂志,2003,9(2):37.

  4 陈育智.儿童支气管哮喘防治常规(试行).中华儿科杂志,2004,42(2):100-106.

  5 崔文兰.哮喘患者激素吸入治疗依从性的探讨.中华临床医学实践杂志,2006,5(4):348.


日期:2011年6月29日 - 来自[2009年第6卷第5期]栏目
共 26 页,当前第 1 页 9 1 2 3 4 5 6 7 8 9 10 11 :



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