The Effect of Pioglitazone on the Expression of Transforming Growth Factor (TGF)-beta and Fibronectin in Diabetic Nephropathy / 대한신장학회잡지

BACKGROUND: Peroxisome proliferator-activated receptor (PPAR)-gamma is a member of the nuclear receptor superfamily. PPAR-gamma plays an important role in numerous cellular processes including adipogenesis, insulin sensitivity, cell cycle progression, cell differentiation, inflammation, and extracellular matrix production. This study investigated the effect of a PPAR-gamma agonist on the progression of diabetic nephropathy in OLETF rats. METHODS: 30 week-old male OLETF rats were treated for 10 weeks as follows:diabetic control (DM), no treatment:pioglitazone therapy (DM+Pio). LETO rats were used as non-diabetic control (control). Body weight, blood pressure, blood sugar, creatinine, total cholesterol, triglyceride, and urinary protein excretion were measured. Histological analysis was taken with light microscope. Glomerular protein and mRNA expression of transforming growth factor (TGF)-beta1 and fibronectin were estimated by Western blot and RT-PCR. Kidney sections were stained for fibronectin by immunohistochemistry. RESULTS: Serum glucose, triglyceride and urinary protein excretion were decreased in DM+Pio rats compared to DM rats (p<0.05). PAS staining showed glomerular hypertrophy, mesangial expansion, nodular sclerosis, and glomerular basement membrane thickening in glomeruli of DM rats, but these changes were attenuated in glomeruli of pioglitazone-treated rats. Treatment with pioglitazone resulted in a significant decrease in TGF-beta1 protein and mRNA expression in diabetic glomeruli (80.6% and 78.4%, respectively). Glomerular expression of fibronectin protein and mRNA were also decreased in pioglitazone treatment group compared with DM group (93.1% and 98.6%, respectively). Immunohistochemical staining for fibronectin showed similar results. CONCLUSION: Increased TGF-beta1 and fibronectin mRNA and protein expressions in diabetic rat glomeruli were significantly ameliorated by pioglitazone treatment. These data suggest that activation of PPAR-gamma may play an important role in prevention and treatment of diabetic nephropathy.

The Effect of High Glucose on Renin-Angiotensin System (RAS) in Podocytes / 대한신장학회잡지

The renin-angiotensin system (RAS) plays an important role in the pathogenesis of diabetic nephropathy. Recently, the activation of local RAS in mesangial cells by high glucose has been reported. However, little is known about the changes of RAS in podocytes under diabetic conditions. In this study, we examined whether RAS activation was induced in high glucose- stimulated podocytes. Immortalized mouse podocytes were exposed to medium containing 5.6 mM glucose (NG), NG+24.4 mM mannitol, or 30 mM glucose(HG). mRNA and protein expression of RAS components were determined by real time-PCR and Western blot, respectively. Angiotensin I (AI) and angiotensin II (AII) concentrations, angiotensin-converting enzyme (ACE) levels, and renin activity were also determined. Angiotensinogen (AGT) mRNA expression was significantly increased in HG-stimulated podocytes. In addition, AI and AII concentrations were significantly higher in HG-treated cell lysates and in their conditioned media. However, there were no differences in renin activity and ACE levels among the groups. AII type 1 receptor (AT1R) mRNA and protein expression were also increased by 288% (p<0.01) and 170% (p< 0.05) in HG-stimulated podocytes compared to NG- treated cells. In conclusion, HG induced AGT mRNA expression, resulting in increases in AI and AII levels. These findings suggest that increased AII production along with increased AT1R expression in podocytes under diabetic conditions may well be considered as factors actively involved in the pathogenesis of diabetic nephropathy.

Angiotensin II Receptor Blocker Induced Inhibition of Cellular Hypertrophy and Differential Expression of Cyclin-dependent Kinase Inhibitors in Cultured Podocytes Stimulated by Long-term High Glucose / 대한신장학회잡지

BACKGROUND: Hypertrophy of podocytes is observed in type 2 diabetic patients. Cellular hypertrophy requires combined effects of various mitogen- induced entry into the cell cycle and subsequent cell cycle arrest at the G1/S interphase. This cell cycle arrest is mediated by various cyclin-dependent kinase inhibitors (CKIs). We investigated the effect of angiotensin II receptor blocker (ARB) treatment on podocyte hypertrophy and CKIs expression in cultured podocytes stimulated by long-term high glucose. METHODS: Immortalized mouse podocytes were cultured in media containing 5.6 mM normal glucose (NG), 30 mM high glucose (HG), or NG+angiotensin II (AII, 10(-7)M) for 7 days with or without ARB (L-158,809, 10(-6)M). Cellular hypertrophy was assessed by measurement of cellular protein/cell counts, and CKIs mRNA and protein expression were assessed by reverse-transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. RESULTS: Cellular hypertrophy was induced in podocytes exposed to HG or AII compared to NG cells and this HG-induced cellular hypertrophy was inhibited with ARB treatment by 70% (p<0.05). In addition, there were 1.5-fold and 2.0 fold increases in p27Kip1 mRNA and protein expression, respectively, in HG-stimulated podocytes compared to NG- treated cells (p<0.05). p27Kip1 mRNA and protein expression were also increased in cultured podocytes stimulated by AII by 156% and 199%, respectively (p<0.05). ARB treatment ameliorated HG-induced increase in p27Kip1 mRNA by 75% and protein expression by 70% (p<0.05). In contrast, there were no significant changes in p21Cip1 and p57Kip2 protein expression in cultured podocytes exposed to HG or AII. CONCLUSION: High glucose induced significant cellular hypertrophy and increased p27Kip1 mRNA and protein expression in cultured mouse podocytes, and these changes were effectively inhibited by ARB treatment.

Angiotensin II Receptor Blocker Induced Inhibition of Cellular Hypertrophy and Differential Expression of Cyclin-dependent Kinase Inhibitors in Cultured Podocytes Stimulated by Long-term High Glucose / 대한신장학회잡지

BACKGROUND: Hypertrophy of podocytes is observed in type 2 diabetic patients. Cellular hypertrophy requires combined effects of various mitogen- induced entry into the cell cycle and subsequent cell cycle arrest at the G1/S interphase. This cell cycle arrest is mediated by various cyclin-dependent kinase inhibitors (CKIs). We investigated the effect of angiotensin II receptor blocker (ARB) treatment on podocyte hypertrophy and CKIs expression in cultured podocytes stimulated by long-term high glucose. METHODS: Immortalized mouse podocytes were cultured in media containing 5.6 mM normal glucose (NG), 30 mM high glucose (HG), or NG+angiotensin II (AII, 10(-7)M) for 7 days with or without ARB (L-158,809, 10(-6)M). Cellular hypertrophy was assessed by measurement of cellular protein/cell counts, and CKIs mRNA and protein expression were assessed by reverse-transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. RESULTS: Cellular hypertrophy was induced in podocytes exposed to HG or AII compared to NG cells and this HG-induced cellular hypertrophy was inhibited with ARB treatment by 70% (p<0.05). In addition, there were 1.5-fold and 2.0 fold increases in p27Kip1 mRNA and protein expression, respectively, in HG-stimulated podocytes compared to NG- treated cells (p<0.05). p27Kip1 mRNA and protein expression were also increased in cultured podocytes stimulated by AII by 156% and 199%, respectively (p<0.05). ARB treatment ameliorated HG-induced increase in p27Kip1 mRNA by 75% and protein expression by 70% (p<0.05). In contrast, there were no significant changes in p21Cip1 and p57Kip2 protein expression in cultured podocytes exposed to HG or AII. CONCLUSION: High glucose induced significant cellular hypertrophy and increased p27Kip1 mRNA and protein expression in cultured mouse podocytes, and these changes were effectively inhibited by ARB treatment.

Effect of losartan on the cyclooxygenase 2 expression in the obese Zucker rat kidney / 中华肾脏病杂志

Objective To investigate the effect of angiotensinⅡ(AngⅡ) type 1 receptor blocker losartan on the cyclooxygenase 2 (COX-2) expression in metabolic syndrome (MS)kidney. Methods Seven-week-old male obese Zucker rats,a model of MS,were randomly divided into losartan treated and untreated group,and lean Zucker rats were used as controls.The obese Zucker rata of treated group received losartan for 4 months continuously.COX-2 expression was examined for all rats after 4 months.AngⅡ-stimulated mesangial cells and cortical tissue from AngⅡ-infused C57BL/6 mouse kidney by osmotic minipumps were used in this study.RNA and protein were obtained from renal cortical tissue or mesangial cells for RT-PCR and Western blot.Results Compared to the lean controls,obese Zucker rats showed a significant increase of COX-2 expression in the renal cortical tissue and these abnormalities were prevented by administration of losartan. Furthermore,the direct stimulation of AngⅡincreased COX-2 expression in mesangial cells in vitro and renal cortical tissue in vivo.Conclusions MS-induced COX-2 expression in the kidney is regulated by AngⅡ.Losartan as a non COX-2 inhibitor can protect MS kidney,at least in part,by inhibition of COX-2 activation.

Effect of 12-lipoxygenase on the AT1 receptor expression in mesangial cells / 中华肾脏病杂志

Objective To investigate the effect of 12-lipoxygenase (12-LO) on the angiotensinⅡtype 1 receptor(ATlR) expression in mesangial cells (MC).Methods p38 MAPK activation and ECM protein expression were determined using AngⅡ-stimulated MC derived from normal and 12-LO knockout mice.AT1R expression was determined using 12-LO product 12(S)- HETE-stimulated MC,MC transfected with 12-LO gene and microdissected glomeruli derived from 12-LO knockout mice.RT-PCR and Western blot were used for evaluating mRNA and protein expression respectively.Results AngⅡstimulation increased p38 MAPK activation and ECM protein expression in normal MC,but not in MC derived from 12-LO knockout mice.Time-dependent and dose-dependent experiment showed that 12 (S)- HETE increased AT1R protein' expression in MC. Similarly,12 (S)-HETE increased AT1R mRNA expression in MC compared with control MC (P＜0.01). Furthermore,AT1R expression was lower in glomeruli derived from 12-LO knockout mice relative to genetic controls (P＜0.01) and MC stably overexpressing 12-LO had greater AT1R protein and mRNA expression relative to control MC (P＜0.01).Conclusion 12-LO activation can upregulate ATIR expression in MC.

The Effect of ACE Gene Polymophism on the Antiproteinuric Effect of Angiotensin II Receptor Antagonist in Patients with Non-diabetic Chronic Renal Disease / 대한신장학회잡지

BACKGROUND: Angiotensin II, a potent vasoconstrictor, plays a key role in renal injury and in the progression of chronic renal disease of diverse causes. In every organ system, the biologic effects of angiotensin II are mediated through its interaction with specific receptors on cell membranes. Angiotensin II receptor antagonist specifically inhibits angiotensin II-mediated physiologic responses such as systemic and renal vasoconstriction, sodium reabsorption by renal proximal tubule, and stimulation of aldosterone and adrenergic hormone release by the adrenal gland. It has been reported that losartan, angiotensin II receptor antagonist, has a significant antiproteinuric effect in patients with diabetic and non-diabetic renal disease. This study was carried out to investigate the effect of angiotensin-converting enzyme (ACE) gene polymorphism on the renal response to angiotensin II receptor antagonist in non-diabetic proteinuric chronic renal patients. METHODS: Seventy patients with non-diabetic chronic renal disease with urinary protein excretion greater than 500 mg/day were enrolled in this prospective study. Subjects were given losartan 50 mg o.d. for the first 12 weeks, and then were given to 100 mg o.d. for another 12 weeks. RESULTS: Twelve weeks and twenty-four weeks later, blood pressure, urinary protein excretion, total cholesterol, and triglyceride decreased significantly compared with baseline values. There was a significant correlation between the levels of baseline urinary protein excretion and the magnitudes of the reduction of urinary protein excretion after treatment with losartan. Baseline blood pressure, BUN, serum creatinine, and urinary protein excretion were not different in the responder group (patients with more than 30% reduction of urinary protein excretion after losartan treatment) compared with the nonresponder group. Systolic blood pressure and mean arterial pressure in the responder group were significantly lower than the nonresponder group after twelve and twenty-four weeks. Urinary protein excretion in the responder group was significantly lower than the nonresponder group after twelve weeks. When the patients were divided into three groups according to ACE gene polymorphism, II, ID and DD, there were no significant differences in the blood pressure change, reduction of urinary protein excretion following losartan treatment and distributions of responder among three groups. CONCLUSION: Our results suggest that angiotensin II receptor antagonist, losartan, significantly reduced blood pressure and proteinuria in patients with non- diabetic chronic renal disease. The magnitude of antiproteinuric effect of losartan was not influenced by ACE gene polymorphism. However, further studies with large number of patients are required to confirm the issues regarding the ACE gene polymorphism and the antiproteinuric effects of angiotensin II receptor antagonist in non-diabetic chronic renal disease.

The Effect of High Glucose on p38 MAPK Pathway and Fibronectin Synthesis in Cultured Rat Mesangial Cells / 대한신장학회잡지

BACKGROUND: The p38 mitogen-activated protein kinase (MAPK) pathway is activated by several stress factors, potentially leading to cellular apoptosis and growth. Little is known about the pattern of p38 MAPK pathway activation in mesangial cells under high glucose conditions. We examined the activity and expression of p38 MAPK members, p38 MAPK, MAPK kinase 3/6 (MKK3/6), c-AMP responsive element binding protein (CREB), and MAPK phosphatase-1 (MKP-1) in cultured mesangial cells exposed to high glucose. METHODS: Mesangial cells were subcultured from rat glomeruli isolated by sieving technique. After serum restriction for 48 hours mesangial cells were exposed to 5.6 mM glucose (low glucose, LG), 5.6 mM glucose+24.4 mM mannitol (LG+M), or 30 mM glucose (high glucose, HG) for 3 minutes to 48 hours with or without SB203580. Western blot was performed to determine the activity and protein expression of p38 MAPK members. RT-PCR and ELISA were performed for fibronectin mRNA expression and fibronectin synthesis, respectively. RESULTS: p38 MAPK and CREB activities were significantly increased in mesangial cells exposed to HG compared with LG or LG+M after 10 minutes and was sustained at higher levels to 48 hours (p<0.05), but total p38 MAPK and CREB protein expressions did not differ. MKP-1 showed a similar pattern as p38 MAPK and CREB (p<0.05). MKK3/6 acitvity was significantly higher in HG cells after 3 minutes and remained at higher levels throught the study period (p<0.05). Fibronectin mRNA expression and fibronectin synthesis were significantly increased in mesangial cells exposed to HG after 48 hours (p< 0.05). SB203580 (1 micrometer) pretreatment for 1 hour significantly reduced HG-induced fibronectin mRNA expression and fibronectin synthesis by 73% and 69%, respectively (p<0.05). CONCLUSION: p38 MAPK activity was increased in mesangial cells exposed to HG in parallel with increased MKK3/6 activity, resulting in CREB activation and increased fibronectin synthesis. This activated p38 MAPK may play a role in the pathogenesis of diabetic nephropathy.

P-Cadherin is Decreased in Glucose-Stimulated Podocytes and in Experimental Diabetic Nephropathy / 대한신장학회잡지

BACKGROUND: Proteinuria is a cardinal feature of glomerular disease including diabetic nephropathy, and glomerular filtration barrier is considered as a filter restricting protein excretion in urine. We tested whether the expression of P-cadherin, a molecule known to be located at the slit diaphragm, was altered by high glucose in cultured podocytes in vitro and by diabetes in vivo. METHODS: In vitro, immortalized mouse podocytes were cultured in media with 5.6 mM glucose (NG), NG+24.4 mM mannitol (NG+M), or 30 mM glucose (HG) for 7 days at 37dgrees C. Cell lysates were used for RT-PCR and Western blot. For animal studies, twelve Sprague-Dawley rats were injected with diluent (Control, C, N=6) or streptozotocin (DM, N=6) intraperitoneally, and were sacrificed after 6 weeks. RT-PCR and Western blot for P-cadherin mRNA and protein expression, respectively, were performed with sieved glomeruli, and immunohistochemistry with renal tissue. RESULTS: HG significantly reduced P-cadherin mRNA and protein expression in cultured podocytes by 47% and 62%, respectively (p<0.05). Twenty-four hour urinary albumin excretion was significantly higher in DM (12.80+/-1.12 mg/day) compared to C rats (3.15+/-0.24 mg/day) (p<0.05). Glomerular P-cadherin mRNA expression was significantly lower in DM than that in C rats (p<0.05). P-cadherin protein expression assessed by Western blot and immunohistochemistry showed a similar pattern. CONCLUSION: Exposure of podocytes to HG in vitro and diabetes in vivo reduced P-cadherin mRNA and protein expression. These findings suggest that the decrease in P-cadherin expression is connected to the early changes of diabetic nephropathy and thus may contribute to the development of proteinuria.

Changes of ZO-1 Expression in Diabetic Rat Glomeruli and Cultured Mouse Podocyte Under High Glucose Conditions and the Effect of Angiotensin II Type 1 Receptor Blocker / 대한신장학회잡지

BACKGROUND: Diabetic nephropathy (DN) is clinically characterized by persistent proteinuria. The underlying pathologic changes responsible for the nephropathy are the loss of size selective and/or charge selective properties of the glomerular filtration barrier. Size selectivity is maintained primarily by the slit diaphragm and ZO-1 is one of the basic components of it. However, the precise role of the ZO-1 in the pathogenesis of the glomerular diseases is not fully understood. We investigated the changes of ZO-1 expression in diabetic glomeruli in vivo, and by high glucose in cultured podocyte in vitro. We also evaluated the effect of angiotensin II type 1 receptor blocker (ARB) on the ZO-1 changes induced by diabetes or high glucose. METHODS: To determine the effect of ARB on podocytes ZO-1 protein and mRNA expression, immortalized mouse podocytes were incubated with RPMI medium containing normal glucose (NG, 5.6 mM) or high glucose (HG, 30 mM) with or without ARB (10-6 M, L-158, 809). For animal studies, rats were injected with diluent (Control, C, n=18) or streptozotocin. The latter were left untreated (DM, n=18) or treated with 1 mg/kg/day ARB (DM+ARB, n=18). Six rats from each group were sacrificed monthly, and Western blot and RT?PCR were performed for ZO-1 with sieved glomeruli. Renal sections were stained for ZO-1 by immunohistochemistry. RESULTS: The ZO-1 mRNA and protein expressions in podocytes exposed to HG conditions were significantly higher than those in podocytes exposed to NG media (p<0.05). ARB treatment inhibited the HG induced increase in ZO-1 mRNA and protein expression by 73% and 64%, respectively (p<0.05). Compared to the C rats (19.8+/-3.2 mg/day), 24 hour urinary protein excretion at 3 month was significantly higher in the DM rats (90.6+/-11.3 mg/day, p< 0.05), and ARB treatment partly reversed the increase in proteinuria in DM rats (51.6+/-6.6 mg/day, p<0.05). Glomerular ZO-1 mRNA and protein expressions were also significantly increased in DM than corresponding C at all duration (p<0.05). ARB treatment for 3 months in DM rats inhibited the increase in ZO-1 mRNA and protein expression by 57.5% and 70.6%, respectively (p<0.05). ARB treatment for 3 months significantly ameliorated increased glomerular ZO-1 expression in DM rats as assessed by immunohistochemistry. CONCLUSION: In conclusion, ZO-1 mRNA and protein expressions were increased in podocytes exposed to HG and in DM glomeruli, and this increment in ZO-1 expression was ameliorated with ARB. Taken together, these data suggest that change of ZO-1 expression in podocytes is implicated in the early changes of diabetic nephropathy and may contribute to the development of proteinuria.

Inducible Nitric Oxide Synthase (iNOS) Expression Is Increased in Lipopolysaccharide (LPS)-Stimulated Diabetic Rat Glomeruli: Effect of ACE Inhibitor and Angiotensin II Receptor Blocker

Previously, we reported that high glucose enhanced cytokine-induced nitric oxide (NO) production by rat mesangial cells (MCs), and that the enhanced expression of the iNOS pathway may promote extracellular matrix accumulation by MCs. The present study was designed to examine whether the iNOS pathway is pathologically altered in experimental diabetic nephropathy, and whether therapy with angiotensin converting enzyme (ACE) inhibitor (imidapril: I) or angiotensin II type I receptor (AT1) blocker (L-158,809: L), ameliorates these changes. Male Sprague-Dawley rats were injected with diluent (control: C) or streptozotocin. At sacrifice after 4, 8 and 12 weeks, rats underwent either a 4 hour placebo or an intraperitoneal lipopolysaccharide (LPS, 2 mg/kg) challenge. Systolic blood pressure (SBP) and urinary protein excretion (UPE) increased significantly in diabetic (D) rats compared with C. The basal expression of glomerular iNOS mRNA was increased in D rats compared with that of C rats, by reverse- transcription (RT)-polymerase chain reaction (PCR), whereas there was no significant difference in the level of protein by Western blot analysis. Upon LPS stimulation, the iNOS mRNA and protein expression was significantly elevated in D rats. In D rats, this up-regulation, of LPS-stimulated iNOS expression, was equally ameliorated both by I and L in mRNA and protein levels. From immunohistochemistry (IHC), there was a negative staining for the iNOS within the glomeruli of five C rats without LPS treatment, but one of four rats, with LPS treatment, showed minimal iNOS staining in the glomeruli. In D rats, the glomerular mesangium and podocytes were positive for iNOS in each of three out of five rats with, and without, LPS treatment. In conclusion, LPS-stimulated glomerular iNOS expression was enhanced in diabetic pnephropathy, and the activation of angiotensin II may play a role in this enhancement.