Gamma Linolenic Acid Exerts Anti-Inflammatory and Anti-Fibrotic Effects in Diabetic Nephropathy

PURPOSE: This study was undertaken to investigate the effects of gamma linolenic acid (GLA) on inflammation and extracellular matrix (ECM) synthesis in mesangial and tubular epithelial cells under diabetic conditions. MATERIALS AND METHODS: Sprague-Dawley rats were intraperitoneally injected with either a diluent [n=16, control (C)] or streptozotocin [n=16, diabetes (DM)], and eight rats each from the control and diabetic groups were treated with evening primrose oil by gavage for three months. Rat mesangial cells and NRK-52E cells were exposed to medium containing 5.6 mM glucose and 30 mM glucose (HG), with or without GLA (10 or 100 microM). Intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), and fibronectin (FN) mRNA and protein expression levels were evaluated. RESULTS: Twenty-four-hour urinary albumin excretion was significantly increased in DM compared to C rats, and GLA treatment significantly reduced albuminuria in DM rats. ICAM-1, MCP-1, FN mRNA and protein expression levels were significantly higher in DM than in C kidneys, and these increases were significantly abrogated by GLA treatment. In vitro, GLA significantly inhibited increases in MCP-1 mRNA expression and protein levels under high glucose conditions in HG-stimulated mesangial and tubular epithelial cells (p<0.05, respectively). ICAM-1 and FN expression showed a similar pattern to the expression of MCP-1. CONCLUSION: GLA attenuates not only inflammation by inhibiting enhanced MCP-1 and ICAM-1 expression, but also ECM accumulation in diabetic nephropathy.

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.

The Uses of Urinary betaig-h3 in Differential Diagnosis of Isolated Microscopic Hematuria / 대한신장학회지

PURPOSE: This study was undertaken to elucidate the usefulness of urinary betaig-h3 concentrations in differential diagnosis of isolated microscopic hematuria patients. METHODS: Seventy-seven patients, in whom renal biopsy was performed due to microscopic hematuria without proteinuria, were enrolled. The patients were divided into two groups, IgAN group (patients with IgA nephropathy, N=37) and NM group (patients with normal or minor change on renal biopsy, N=40), and the clinical characteristics and laboratory findings were compared between the two groups. TGF-beta and betaig-h3 concentrations in urine were determined by ELISA and were compared between the two groups. To establish the optimal cut-off value of betaig-h3/creatinine (Cr) ratio for the diagnosis of IgA nephropathy, a receiver operating characteristic curve was constructed and the sensitivity and specificity were calculated. RESULTS: A comparative analysis revealed no significant differences in age and sex ratio between the two groups. There were no differences in serum IgG, IgA, IgM, C3, and C4 levels between the two groups. The urinary betaig-h3/Cr ratio was significantly higher in the IgAN group compared to the NM group (6.632.6 vs. 4.462.6 ng/mg, p0.05). A cut-off betaig-h3/Cr ratio 4.5 has a sensitivity of 85.0% and a specificity of 77.8%. CONCLUSION: The urinary betaig-h3/Cr ratio was a good predictor for the diagnosis of IgA nephropathy. Therefore, renal biopsy should be considered in isolated microscopic hematuria patients with high urinary betaig-h3/Cr ratio.

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.