PPARα agonist fenofibrate improves diabetic nephropathy in db/db mice
2006; Elsevier BV; Volume: 69; Issue: 9 Linguagem: Inglês
10.1038/sj.ki.5000209
ISSN1523-1755
AutoresC.W. Park, Y. Zhang, Xiaoyan Zhang, Jing Wu, L. Chen, Dongchul Cha, Dongming Su, Min-Huo Hwang, Xueliang Fan, L. Davis, William J. Pavan, Feng Zheng, Matthew D. Breyer, Youfei Guan,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoPeroxisome proliferator-activated receptor α (PPARα) is a member of the ligand-activated nuclear receptor superfamily, and plays an important role in lipid metabolism and glucose homeostasis. The purpose of this study is to determine whether the activation of PPARα by fenofbrate would improve diabetes and its renal complications in type II diabetes mellitus. Male C57 BLKS db/db mice and db/m controls at 8 weeks of age were divided to receive either a regular diet chow (db/db, n=8; db/m, n=6) or a diet containing fenofibrate (db/db, n=8; db/m, n=7). Mice were followed for 8 weeks. Fenofibrate treatment dramatically reduced fasting blood glucose (P<0.001) and HbA1c levels (P<0.001), and was associated with decreased food intake (P<0.01) and slightly reduced body weight. Fenofibrate also ameliorated insulin resistance (P<0.001) and reduced plasma insulin levels (P<0.05) in db/db mice. Hypertrophy of pancreatic islets was decreased and insulin content markedly increased (P<0.05) in fenofibrate-treated diabetic animals. In addition, fenofibrate treatment significantly reduced urinary albumin excretion (P<0.001). This was accompanied by dramatically reduced glomerular hypertrophy and mesangial matrix expansion. Furthermore, the addition of fenofibrate to cultured mesangial cells, which possess functional active PPARα, decreased type I collagen production. Taken together, the PPARα agonist fenofibrate dramatically improves hyperglycemia, insulin resistance, albuminuria, and glomerular lesions in db/db mice. The activation of PPARα by fenofibrate in mesangial cells may partially contribute to its renal protection. Thus, fenofibrate may serve as a therapeutic agent for type II diabetes and diabetic nephropathy. Peroxisome proliferator-activated receptor α (PPARα) is a member of the ligand-activated nuclear receptor superfamily, and plays an important role in lipid metabolism and glucose homeostasis. The purpose of this study is to determine whether the activation of PPARα by fenofbrate would improve diabetes and its renal complications in type II diabetes mellitus. Male C57 BLKS db/db mice and db/m controls at 8 weeks of age were divided to receive either a regular diet chow (db/db, n=8; db/m, n=6) or a diet containing fenofibrate (db/db, n=8; db/m, n=7). Mice were followed for 8 weeks. Fenofibrate treatment dramatically reduced fasting blood glucose (P<0.001) and HbA1c levels (P<0.001), and was associated with decreased food intake (P<0.01) and slightly reduced body weight. Fenofibrate also ameliorated insulin resistance (P<0.001) and reduced plasma insulin levels (P<0.05) in db/db mice. Hypertrophy of pancreatic islets was decreased and insulin content markedly increased (P<0.05) in fenofibrate-treated diabetic animals. In addition, fenofibrate treatment significantly reduced urinary albumin excretion (P<0.001). This was accompanied by dramatically reduced glomerular hypertrophy and mesangial matrix expansion. Furthermore, the addition of fenofibrate to cultured mesangial cells, which possess functional active PPARα, decreased type I collagen production. Taken together, the PPARα agonist fenofibrate dramatically improves hyperglycemia, insulin resistance, albuminuria, and glomerular lesions in db/db mice. The activation of PPARα by fenofibrate in mesangial cells may partially contribute to its renal protection. Thus, fenofibrate may serve as a therapeutic agent for type II diabetes and diabetic nephropathy. The worldwide prevalence of type II diabetes is rapidly increasing, and diabetic nephropathy is projected to become most common cause of end-stage renal disease and cardiovascular events in the industrialized world. Insulin resistance is a central and pathogenic feature of type II diabetes1Henry R.R. Insulin resistance: from predisposing factor to therapeutic target in type 2 diabetes.Clin Ther. 2003; 25: B47-B63Abstract Full Text PDF PubMed Scopus (49) Google Scholar, 2Olefsky J.M. Nolan J.J. Insulin resistance and non-insulin-dependent diabetes mellitus: cellular and molecular mechanisms.Am J Clin Nutr. 1995; 61: 980S-986SPubMed Google Scholar, 3Stumvoll M. Haring H. Insulin resistance and insulin sensitizers.Horm Res. 2001; 55: 3-13Crossref PubMed Scopus (50) Google Scholar contributing to the development of obesity, dyslipidemia, hypertension, and cardiovascular disease.4Sowers J.R. Frohlich E.D. 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Peroxisome proliferator-activated receptor {gamma}: implications for cardiovascular disease.Hypertension. 2004; 43: 297-305Crossref PubMed Scopus (126) Google Scholar PPARα binds to a specific peroxisome proliferators response element (PPRE) in the promoter element of target genes, forming a heterodimer with the 9-cis-retinoic acid receptor RXRα. PPARα target genes include several key enzymes actively involved in lipid metabolism. PPARα is particularly abundant in tissues exhibiting high levels of energy metabolism, including the brown fat tissue, liver, kidney and heart, and to a lesser extent in skeletal muscle.9Guan Y. Zhang Y. Davis L. Breyer M.D. Expression of peroxisome proliferator-activated receptors in urinary tract of rabbits and humans.Am J Physiol. 1997; 273: F1013-F1022PubMed Google Scholar, 10Braissant O. Foufelle F. Scotto C. et al.Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-α, -β, and -γ in the adult rat.Endocrinology. 1996; 137: 354-366Crossref PubMed Scopus (1736) Google Scholar Although its role in lipid metabolism is most firmly established, it has recently been found that PPARα may also play an important role in enhancing insulin action.11Kim H. Haluzik M. Asghar Z. et al.Peroxisome proliferator-activated receptor-alpha agonist treatment in a transgenic model of type 2 diabetes reverses the lipotoxic state and improves glucose homeostasis.Diabetes. 2003; 52: 1770-1778Crossref PubMed Scopus (145) Google Scholar, 12Koh E.H. Kim M.S. Park J.Y. et al.Peroxisome proliferator-activated receptor (PPAR)-alpha activation prevents diabetes in OLETF rats: comparison with PPAR-gamma activation.Diabetes. 2003; 52: 2331-2337Crossref PubMed Scopus (131) Google Scholar These findings suggest that PPARα ligands might provide novel therapeutic agents for the treatment of type II diabetes. Here we report the effect of fenofibrate, a specific PPARα ligand, on hyperglycemia, insulin resistance, and diabetic nephropathy in type II diabetic db/db mice. We report that PPARα activation by fenofibrate improves insulin sensitivity, glucose control, and diabetic nephropathy associated with decreased urine albumin excretion and attenuated glomerular mesangial matrix accumulation. Compared to untreated db/db mice, daily food consumption in fenofibrate-treated db/db mice was initially not different, but subsequently significantly decreased after 4 weeks of treatment (P<0.05, Table 1). After 2 months of treatment with fenofibrate, the body weight of db/db mice was slightly reduced compared with that of control db/db mice (P<0.05) (Figure 1a).Table 1Effects of fenofibrate on body weight, food intake, and lipid profiles in nondiabetic db/m and diabetic db/db micedb/dbdb/db-Fenodb/mdb/m-FenoBody weight (g) Baseline42.5±1.441.9±1.724.5±1.825.7±2.4 Final53.4±3.747.0±4.0*P<0.05,26.8±1.424.5±3.9Food intake (g) Baseline4.5±0.75.2±0.80.8±0.21.0±0.3 Final4.1±0.82.2±0.6**P<0.01 vs db/db mice and1.1±0.30.7±0.3Lipid profiles TC (mg/dl) Baseline177.1±19.5173.3±13.8111.9±32.9120.2±32.0 Final159.0±15.9273.2±35.6*P<0.05,101.9±9.4209.5±21.9# VLDL12.5±2.713.3±3.6NDND LDL13.1±3.010.3±3.0NDND HDL140.4±12.4189.6±10.1*P<0.05,NDNDTG (mg/dl) Baseline154.3±16.3165.7±16.794.3±24.8109.4±21.4 Final153.2±26.1156.1±46.799.4±7.9117.9±25.4#P<0.05 vs db/m mice.HDL, high-density lipoprotein; LDL, low-density lipoprotein; TC, total cholesterol; TG, triglyceride; VLDL, very low-density lipoprotein; ND, not done.* P<0.05,** P<0.01 vs db/db mice and# P<0.05 vs db/m mice. Open table in a new tab HDL, high-density lipoprotein; LDL, low-density lipoprotein; TC, total cholesterol; TG, triglyceride; VLDL, very low-density lipoprotein; ND, not done. Fenofibrate treatment dramatically improved glycemic control in db/db mice so that both blood sugar and HbA1c levels were reduced to levels similar to that seen in db/m mice (Figure 1b and c). Compared to untreated db/db mice, serum insulin levels in db/db mice receiving fenofibrate treatment were significantly reduced after 8 weeks' fenofibrate treatment (2.6±1.2 vs 4.5±0.9 ng/ml, P<0.05) (Figure 1d). Surprisingly, we noticed a late increase in serum insulin levels in untreated db/m mice at 16 weeks of age that was also blocked by fenofibrate treatment (Figure 1d). Similarly, db/db mice receiving fenofibrate treatment exhibited improved insulin sensitivity reflected by significantly lower HOMAIR indexes compared to that in untreated db/db animals (2.2±1.3 vs 16.6±3.9, P<0.001). Decreased HOMAIR indexes were also observed at the end of study in db/m mice treated with fenofibrate (data not shown). Untreated db/db mice exhibited marked hyperplasia and hypertrophy (Figure 2a). In contrast, fenofibrate-treated db/db mice showed dramatically reduced islet size to values within the range of that in db/m mice (P<0.01) (Figure 2b). Immunostaining study further demonstrated that insulin content as reflected by insulin-positive area per islet was significantly increased in pancreatic islets in db/db mice treated with fenofibrate treatment compared to untreated mice (P<0.05) (Figure 2c). No change in islet size and insulin immunoreactivity was found in fenofibrate treated db/m mice. Serum triglyceride levels appeared to be lower in db/db mice, but slightly increased in db/m mice following 8 weeks' treatment (Table 1). In contrast, serum total cholesterol levels increased in both db/db and db/m mice after 2 months treatment with fenofibrate (P<0.05). The lipoprotein profile showed that the major cholesterol subfraction affected by fenofibrate was comprised of increased high-density lipoprotein-cholesterol with little change in very low-density lipoprotein- or low-density lipoprotein-cholesterol levels in db/db mice (Table 1). Blood Na+, K+, Cl-, hematocrit, and creatinine levels showed no significant difference among the groups. Compared to untreated db/db mice, fenofibrate-treated db/db mice exhibited a slight increased in blood urea nitrogen (BUN) (P<0.05) (Table 2). The anion gap in treated db/db mice was significantly higher than that in untreated db/db mice (13.4±1.8 vs 10.6±2.1, P<0.02), suggesting ketosis could contribute to unmeasured anions in treated db/db mice.Table 2Influences of fenofibrate on blood chemistry in nondiabetic db/m and diabetic db/db micedb/dbdb/db-fenodb/mdb/m-fenoBUN (mg/dl)21.6±4.227.3±4.3*P<0.05 vs db/db group.25.6±4.630.0±5.0Creatinine (μg/ml)1.08±0.210.86±0.131.08±0.111.30±0.10Anion gap10.6±2.113.4±1.8*P<0.05 vs db/db group.NDNDHematocrit (%)49.0±2.348.1±2.245.2±2.747.0±3.2* P<0.05 vs db/db group. Open table in a new tab Fenofibrate treatment of db/db or db/m mice did not affect kidney weight (Table 3). In contrast, fenofibrate treatment induced dramatic hepatomegaly and reduced epididymal adipose tissue mass in both db/db and db/m mice. These changes were more prominent in db/db mice. Fenofibrate also increased cardiac weight in both db/db and db/m mice (P<0.05).Table 3Kidney, liver, heart, and epididymal fat tissue weight (per 100 g body weight) in nondiabetic db/m and diabetic db/db mice treated without or with fenofibratedb/dbdb/db-fenodb/mdb/m-fenoKidney0.48±0.040.49±0.100.54±0.090.53±0.08Liver5.19±0.1610.69±0.81**P<0.001 vs db/db mice;4.57±0.2112.9±2.34P<0.001 vs db/m mice.Heart0.28±0.050.35±0.08*P<0.05 and0.50±0.04**P<0.001 vs db/db mice;0.54±0.09#P<0.05 andEpididymal5.48±0.404.83±0.40*P<0.05 and0.69±0.16**P<0.001 vs db/db mice;0.56±0.28#P<0.05 and* P<0.05 and** P<0.001 vs db/db mice;# P<0.05 and## P<0.001 vs db/m mice. Open table in a new tab Untreated db/db mice consumed more water (data not shown) and exhibited greater urine volume than control db/m mice (Figure 3a). Following fenofibrate treatment for 2 weeks, water intake (data not shown) and urine output rapidly decreased to levels seen in db/m mice (P<0.001, Figure 3a). Untreated db/db mice exhibited a persistent increase in urine albumin excretion. However, after treatment with fenofibrate for 2 weeks, urine albumin excretion decreased to levels comparable to that in db/m mice. Albuminuria in db/db mice was persistently reduced throughout the treatment period (P<0.01, Figure 3b). Fenofibrate treatment in db/m mice did not exhibit any change in water intake (data not shown), urine output (Figure 3a), or albuminuria (Figure 3b). Marked glomerular mesangial expansion in db/db mice was improved by fenofibrate treatment (Figure 4a and b). Glomerulometric determinations further showed significantly decreased glomerular surface area in fenofibrate-treated db/db mice (P=0.03) as well as a tendency for reduced mesangial area (P=0.07, Figure 4b). PPARα mRNA was detected in freshly isolated glomeruli, cultured MCT (a murine renal proximal tubule cell line) cells, as well as mesangial cells from db/db mice, as assessed by reverse transcriptase-polymerase chain reaction (Figure 5a and b). PPARα mRNA levels were increased by 1.4-fold in the kidneys of db/db mice compared to db/m mice (P<0.05, n=3), as assessed by real-time polymerase chain reaction analysis. PPARα protein expression was also evident by Western blot in two db/db mice mesangial cell lines (Figure 5c). PPRE3X luciferase reporter analysis demonstrated that the fenofibrate significantly increased luciferase activity in db/db mouse mesangial cells (Figure 6a). Cells cultured under high glucose exhibited more type I collagen production than in cells cultured with normal glucose. Treatment with fenofibrate (10 μM) significantly suppressed collagen I production stimulated by high glucose (Figure 6b). The present studies demonstrate that the PPARα agonist fenofibrate improves insulin resistance, glucose control, and adiposity in a mouse model of type II diabetic db/db mice. Fenofibrate treatment also reduces 24-h urinary albumin excretion and improves renal histopathologic changes, including reduced glomerular hypertrophy and mesangial matrix expansion in db/db mice. These beneficial renal effects of fenofibrate appear to be associated both with its insulin-sensitizing effect as well as a direct action on cultured glomerular mesangial cells. These findings suggest that PPARα may represent a potential therapeutic target in treating type II diabetes and its renal complications. Accumulating evidence suggests that PPARα activators may improve insulin resistance in type 2 diabetic animals12Koh E.H. Kim M.S. 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Park J.Y. et al.Peroxisome proliferator-activated receptor (PPAR)-alpha activation prevents diabetes in OLETF rats: comparison with PPAR-gamma activation.Diabetes. 2003; 52: 2331-2337Crossref PubMed Scopus (131) Google Scholar showed that fenofibrate treatment prevents the development of diabetes in OLETF rats by reducing adiposity, improving peripheral insulin action, and exerting beneficial effects on pancreatic β-cells. Here we report that activation of PPARα by fenofibrate also improved glycemic control in db/db mice by attenuating insulin resistance, reducing pancreatic islet hypertrophy, enhancing islet insulin expression, and increasing high-density lipoprotein-cholesterol. Taken together, these studies are consistent with the possibility that PPARα activators may provide a novel therapeutic approach for treating insulin resistance and type II diabetes. The db/db mouse is characterized by a G-to-T point mutation of the leptin receptor gene, leading to abnormal receptor splicing and defective signaling of leptin.19Sharma K. McCue P. Dunn S.R. Diabetic kidney disease in the db/db mouse.Am J Physiol Renal Physiol. 2003; 284: F1138-F1144Crossref PubMed Scopus (334) Google Scholar, 20Chen H. Charlat O. Tartaglia L.A. et al.Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice.Cell. 1996; 84: 491-495Abstract Full Text Full Text PDF PubMed Scopus (1859) Google Scholar The present studies provide confirmation that the PPARα agonist fenofibrate decreases food intake by a mechanism independent of leptin action. This finding is in sharp contrast to thiazolidinedione PPARγ activators, which increase body weight and adipose tissue mass.21Spiegelman B.M. Flier J.S. 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Both indirect metabolic effects and direct renal effect seem likely. Improved glucose control and reduced hyperinsulinemia associated with fenofibrate treatment may contribute to reduced albuminuria and improved renal glomerular lesions in db/db mice.33Michel O. Heudes D. Lamarre I. et al.Reduction of insulin and triglycerides delays glomerulosclerosis in obese Zucker rats.Kidney Int. 1997; 52: 1532-1542Abstract Full Text PDF PubMed Scopus (33) Google Scholar, 34Buckingham R.E. Al-Barazanji K.A. Toseland C.D.N. et al.Peroxisome proliferator-activated receptor-γ agonist, rosiglitazone, protects against nephropathy and pancreatic islet abnormalities in Zucker fatty rats.Diabetes. 1998; 47: 1326-1334Crossref PubMed Scopus (201) Google Scholar In addition, direct renal actions appear to be involved in beneficial renal effect of fenofibrate in diabetic nephropathy. This possibility is supported by the fact that PPARα activator fenofibrate suppressed exaggerated type I collagen production in high-glucose treated mesangial cells. Therefore, direct renal action may also play an important role in mediating renoprotective effect of fenofibrate in diabetic nephropathy. In summary, the present studies show the PPARα agonist fenofibrate markedly improves hyperglycemia and insulin resistance in db/db mice without inducing weight gain or adiposity. Treatment with fenofibrate also results in marked renoprotective effect in these animals. Our studies suggest that PPARα could serve as an important therapeutic target for treating type II diabetes and diabetic nephropathy as well. Six-week-old male C57BLKS/J db/db and db/m mice were purchased from Jackson Labs and housed under a standard condition. Fenofibrate (0.2%, w/w, Sigma, St Louis, MO, USA) was mixed into the standard chow diet, and provided to db/db mice (n=8) and age- and gender-matched db/m mice (n=7) for 2 months starting at age of 8 weeks. Control db/db mice (n=8) and control db/m mice (n=6) received normal mouse chow for 8 weeks. In total, 250–300 mg/kg/day of fenofibrate were administered in treated db/db and db/m group. Blood was collected following an overnight fast. Blood glucose and HbA1c levels were measured using HemoCue B-Glucose kit (HemoCue AB, Angelholm, Sweden) and DCA 2000+ HbA1c kit (Bayer, Elkhart, IN, USA), respectively. Plasma insulin levels were measured using radioimmunoassay kit (Linco Reasearch, St Charles, MO, USA). Blood BUN and hematocrit and serum creatinine were measured using iStat-Kit (HESKA, Fort Collins, MO, USA) and HPLC, respectively.35Dunn S.R. Qi Z. Bottinger E.P. et al.Utility of endogenous creatinine clearance as a measure of renal function in mice.Kidney Int. 2004; 65: 1959-1967Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar Serum lipid profile was measured using GPO-Trinder kit (Sigma, St Louis, MO, USA) and FPLC. HOMAIR index was calculated as follows: Fasting glucose (mmol/l) × fasting insulin (mU/l)/22.5. A 24-h urine collection was obtained using metabolic cages. Urine albumin and creatinine concentrations were measured by an immunoassay and the Jaffe alkaline picrate reaction (DCA 2000+Analyzer, Bayer, Elkhart, IN, USA). Kidney and pancreas samples were fixed in 4% formaldehyde. Histology was assessed following HE or PAS staining. Pancreatic samples were also stained with insulin antibody (1:100, Zymed). The surface area of staining of islet was quantified using morphometric software.36Zheng F. Plati A.R. Potier M. et al.Resistance to glomerulosclerosis in B6 mice disappears after menopause.Am J Pathol. 2003; 162: 1339-1348Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar To examine the effect of fenofibrate on glomerular volume and matrix area, glomerulometry analysis was utilized using PAS-stained kidney sections as previously reported.37Wen M. Segerer S. Dantas M. et al.Renal injury in apolipoprotein E-deficient mice.Lab Invest. 2002; 82: 999-1006Crossref PubMed Scopus (101) Google Scholar Murine mesangial cells from a db/db mouse were cultured with some modifications as previously reported38Zheng F. Fornoni A. Elliot S.J. et al.Upregulation of type I collagen by TGF- in mesangial cells is blocked by PPAR activation.Am J Physiol Renal Physiol. 2002; 282: F639-F648Crossref PubMed Scopus (21) Google Scholar and characterized by positive staining for α-smooth muscle actin and negative staining for vWF and cytokeratin. Total RNA was extracted from glomeruli of male db/db and db/m mice, mesangial cells isolated from wild-type and PPARα null mice and MCT cells, a mouse proximal tubule cell line39Kuncio G.S. Alvarez R. Li S. et al.Transforming growth factor-beta modulation of the alpha 1(IV) collagen gene in murine proximal tubular cells.Am J Physiol. 1996; 271: F120-F125PubMed Google Scholar using Tri Reagent (MRC, Cincinnati, OH, USA). Expression of PPARα was determined by reverse transcriptase-polymerase chain reaction using a specific set of primers: 5′-CGT TCC AGC CCT TCC TCA GTC AGC-3′ (sense) and 5′-GAC ATC CCG ACA GAC AGG CAC TTG-3′ (antisense). In addition, real-time polymerase chain reaction was utilized to assess the mRNA levels of PPARα mRNA in male db/m and db/db mice (8-week-old, n=3). Samples containing equal amounts of protein (100 μg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and incubated with a rabbit anti-PPARα antibody (Santa Cruz). The specificity of the antibody used was further confirmed by an immunoprecipitation study using an additional polyclonal antibody from Sigma (Cat# P0869). Mesangial cells were transfected with PPREx3 TK-Luc38Zheng F. Fornoni A. Elliot S.J. et al.Upregulation of type I collagen by TGF- in mesangial cells is blocked by PPAR activation.Am J Physiol Renal Physiol. 2002; 282: F639-F648Crossref PubMed Scopus (21) Google Scholar, 40Guan Y. Zhang Y. Schneider A. et al.Peroxisome proliferator-activated receptor-gamma activity is associated with renal microvasculature.Am J Physiol Renal Physiol. 2001; 281: F1036-F1046Crossref PubMed Scopus (72) Google Scholar (Qiagen Inc., Valencia, CA, USA). After incubation for 24 h, the transfection mixture was replaced with complete media containing either vehicle or fenofibrate (10 μM). After 24 h, cells were harvested in 1 × luciferase lysis buffer (Dual Luciferase Kit, Promega) and relative light units were determined using a luminometer (Mono light 2010, Analytical Luminescence Laboratory, San Diego, CA, USA). Mesangial cells (2 × 104) were seeded in each well of a 24-well plate. After washing cells three times with 1 × phosphate-buffered saline, normal (5 mM) or high glucose (30 mM) medium containing 0.1% fetal bovine serum was added to the cells in the presence or absence of 10 μm fenofibrate for 72 h. Media and cell lysate were collected for determination of type I collagen production by enzyme linked immunosorbent assay as previously described.38Zheng F. Fornoni A. Elliot S.J. et al.Upregulation of type I collagen by TGF- in mesangial cells is blocked by PPAR activation.Am J Physiol Renal Physiol. 2002; 282: F639-F648Crossref PubMed Scopus (21) Google Scholar Final values were normalized for cell numbers. The data expressed as means±s.d. Significance of difference between two groups was evaluated using Student's t-test. For multiple comparisons, one-way analysis of variance was used to evaluate differences among groups. A P-value of <0.05 was considered statistically significant. We thank Drs Kumar Sharma and Steve Dunn and the AMDCC U01 for help in measuring serum creatinine. These studies were supported by the National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) DK065074-02 (to YG), DK64118 (to FZ) and P01-DK 38226 (to MDB), and a funding from the Genzyme Renal Innovations Program and the NNSF of China (30271521 and 104001) (to YG).
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