The Effect of FR167653, p38 Mitogen-Activated Protein Kinase (MAPK) Inhibitor, on the Expression of Slit Diaphragm-Associated Proteins in Experimental Diabetic Nephropathy / 대한신장학회지

PURPOSE: This study was undertaken to investigate the effect of a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor, FR167653, on urinary albumin excretion and on the expression of slit diaphragm-associated proteins in diabetic rats. METHODS: Thirty-two Sprague-Dawley rats were injected with diluent [control (C), N=16] or streptozotocin intraperitoneally (DM, N=16). Eight rats from each group were treated with 5 mg/kg/day FR 167653 (C+FR, DM+FR) for 6 weeks. At the time of sacrifice, 24-hour urinary albumin excretion was determined by ELISA. Glomerular nephrin, P-cadherin, and ZO-1 mRNA and protein expression were determined by real-time PCR and Western blot, respectively, with sieved glomeruli. RESULTS: Urinary albumin excretion was significantly higher in DM compared to C rats, and this increase in albuminuria was significantly inhibited by the administration of FR167653 in DM rats. Glomerular phospho-p38 MAPK protein expression was significantly increased in DM rats compared to C rats, and FR167653 treatment significantly attenuated the increase in phospho-p38 MAPK expression in DM glomeruli. Nephrin mRNA and protein expression were higher in 6-week DM compared to C glomeruli, and these increases were significantly abrogated with FR167653 treatment in DM rats. In contrast, FR167653 had no effects on the decrease in P-cadherin expression and the increase in ZO-1 expression observed in DM glomeruli. CONCLUSION: These findings suggest that FR167653, a p38 MAPK inhibitor, reduce the amount of albuminuria in early diabetic nephropathy, and this anti-proteinuric effect seems to be related with the change of glomerular nephrin expression.

The Effect of Cyclosporine on P-cadherin Expression in Experimental Diabetic Nephropathy and Glucose-Stimulated Podocytes / 대한신장학회잡지

PURPOSE: We investigated whether Cyclosporin A (CsA) had the anti-proteinuric effect in diabetic rats and whether it was associated with the alteration of P-cadherin expression. METHODS: Sprague-Dawley rats were injected with diluent (C, N=16) or streptozotocin intraperitoneally (DM, N=16). Eight rats in each group were treated with 10% ethanol or with 1.5 mg/kg/day of CsA (C+CsA and DM+CsA) for 6 weeks. Immortalized mouse podocytes were cultured in media with 5.6 mM glucose (LG), LG+CsA (10-8 M), LG+TGF-beta1, 30 mM glucose (HG), or HG+CsA. Real time-PCR and Western blot were performed for P-cadherin and TGF-beta1 mRNA and protein expression, respectively, with sieved glomeruli and cell lysates. RESULTS: Urinary albumin excretion was significantly higher in DM compared with C rats, and CsA treatment inhibited the increase in albuminuria in DM rats. Glomerular P-cadherin mRNA and protein expression in DM were decreased compared with C rats, and these decreases were significantly inhibited by CsA. Glomerular TGF-beta1 mRNA and protein expression were higher in DM than C rats, and CsA treatment inhibited the increase in TGF-beta1 expression in DM. P-cadherin mRNA and protein expression in HG and LG+TGF-beta1 podocytes were lower than LG cells, and these HG-induced decrements were restored by CsA. CONCLUSION: CsA treatment reduces urinary albumin excretion in DM rats. P-cadherin expression is decreased under diabetic conditions, which is ameliorated by CsA. In addition, inhibition of the increase in glomerular TGF-beta1 expression under diabetic conditions by CsA seems to restore the P-cadherin expression, resulting in the decrease in albuminuria.

Differential Gene Expression According to the Size of Glomeruli in Experimental Diabetic Nephropathy / 대한신장학회지

PURPOSE: Although a few gene-profiling studies with whole renal tissue have been described in experimental diabetic nephropathy, there is only one microarray study using diabetic glomeruli. Furthermore, hypertrophic glomeruli have not been explored. The purpose of this study is to elucidate gene expression profiles of hypertrophic glomeruli in early diabetic nephropathy. METHODS: Forty-male Sprague-Dawley rats were injected with diluent (N=20) or streptozotocin intraperitoneally (DM, N=20) and were sacrificed at 6- and 12-week. Glomeruli were isolated by sieving technique. Glomeruli from 125 and 75 m sieves were classified into large (hypertrophic, DM-LG) and small glomeruli (DM-SG), respectively. After RNA extraction, hybridization was performed on the Rat cDNA 5K chip in triplicate, and slides were analyzed. The significant genes were selected using significant analysis of microarray. RESULTS: At 6-week, hierarchical clustering revealed that gene expression profiles of DM-LG were different from those of DM-SG, whereas DM-SG and C glomeruli showed similar gene expression pattern. In contrast, gene expression profiles at 12-week were similar between DM-LG and DM-SG, whereas C glomeruli showed different gene expression pattern from DM glomeruli. At 6-week, a total of 207 genes showed greater than 1.5-fold differential expression. 149 genes were upregulated, whereas 58 were downregulated in DM-LG. On the other hand, differential gene expression greater than 1.4-fold was observed in 37 genes at 12-week, upregulated in 26 and downregulated in 11. CONCLUSION: These results suggest that the gene expression profiles of DM-LG are different from DM-SG, and the gene expression patterns change with the progression 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.

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.

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.