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Previous studies have shown that mineralocorticoid receptor (MCR) blocker reduces proteinuria in diabetic nephropathy (DN), but the role of aldosterone in podocyte injury has never been explored in DN. This study was undertaken to elucidate whether a Local aldosterone system existed in podocytes and to examine its role in podocyte apoptosis under diabetic conditions. In vitro, immortalized podocytes were exposed to 5.6 mM glucose (NG), NG + 24.4 mM mannitol, and 30 mM glucose (HG) with or without [10.sup.-7] M spironolactone (SPR). In vivo, 32 Sprague-Dawley rats were injected with diluent (C, n = 16) or streptozotocin intraperitoneally [diabetes mellitus (DM), n = 16], and 8 rats from each group were treated with SPR for 3 mo. Aldosterone synthase (CYP11B2) and MCR mRNA and protein expression were determined by real-time PCR and Western blot, respectively, and aldosterone levels by radioimmunoassay. Western blot for apoptosis-related molecules, Hoechst 33342 staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to determine apoptosis. CYP11B2 and MCR expression were significantly higher in HG-stimulated podocytes and DM glomeruli compared with NG cells and C glomeruli, respectively, along with increased aldosterone levels. Western blot analysis revealed that cleaved caspase-3 and Bax expression was significantly increased, whereas Bcl-2 expression was significantly decreased in HG-stimulated podocytes and in DM glomeruli. Apoptosis determined by Hoechst 33342 staining and TUNEL assay were also significantly increased in podocytes under diabetic conditions. These changes in the expression of apoptosis-related proteins and the increase in apoptotic cells were inhibited by SPR treatment. These findings suggest that a local aldosterone system is activated and is involved in podocyte apoptosis under diabetic conditions. diabetic nephropathy; podocytes; aldosterone; apoptosis; high glucose doi: 10.1152/ajprenal.00101.2009.

Recent studies have demonstrated that an inflammatory mechanism contributes to the pathogenesis of diabetic nephropathy (DN). It is also known that colchicine (Col) can prevent various renal injuries via its anti-inflammatory action. However, the effect of colchicine on DN has never been explored. This study was undertaken to elucidate the effect of colchicine on inflammation and extracellular matrix accumulation in DN. In vivo, 64 rats were injected with diluent (C; n = 32) or streptozotocin intraperitoneally (DM, n = 32). Sixteen rats from each group were treated with Col. In vitro, rat mesangial cells and NRK-52E cells were cultured in media with 5.6 mM glucose (NG) or 30 mM glucose (HG) with or without [10.sup.-8] M Col. Monocyte chemotactic protein-1 (MCP-1) mRNA expression was determined by real-time PCR (RTPCR), and the levels of MCP-1 in renal tissue and culture media were measured by ELISA. RT-PCR and Western blotting were also performed for intercellular adhesion molecule-1 (ICAM-1) and flbronectin (FN) mRNA and protein expression, respectively, and immunohistochemical staining (IHC) for ICAM-1, FN, and ED-1 with renal tissue. Twenty-four-hour urinary albumin excretion at 6 wk and 3 mo were significantly higher in DM compared with C rats (P < 0.05), and colchicine treatment significantly reduced albuminuria in DM rats (P < 0.05). Col significantly inhibited the increase in MCP-1 mRNA expression and protein levels under diabetic conditions both in vivo and in vitro. ICAM-1 and FN expression showed a similar pattern to the expression of MCP-1. IHC revealed that the number of ED-I(+) cells were significantly higher in DM compared with C kidney (P < 0.005), and this increase was significantly attenuated by Col treatment (P < 0.01). In conclusion, Col prevents not only inflammatory cell infiltration via inhibition of enhanced MCP-1 and ICAM-1 expression but also ECM accumulation in DN. These findings provide a new perspective on the renoprotective effects of Col in DN. inflammation; MCP-1; ICAM-1; fibronectin; afferent arteriole; contraction; losartan

Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that plays an important role in the recruitment of macrophages. Although previous studies have demonstrated the importance of MCP-1 in the pathogenesis of diabetic nephropathy (DN) in terms of inflammation, the role of MCP-1 and its receptor (C-C chemokine receptor 2; CCR2) in extracellular matrix (ECM) accumulation under diabetic conditions has been largely unexplored. This study was undertaken to investigate the functional role of the MCP-1/CCR2 system in high glucose-induced ECM (fibronectin and type IV collagen) protein expression in cultured mesangial cells (MCs). Mouse MCs were exposed to medium containing 5.6 mM glucose (NG), NG+24.4 mM mannitol (NG+M), or 30 mM glucose (HG) with or without mutant MCP-1 (mMCP-1), CCR2 small interfering (si)RNA, or CCR2 inhibitor (RS 102895). To examine the relationship between MCP-1 and transforming growth factor (TGF)-[beta]1, MCs were also treated with TGF-[beta]1 (2 ng/ml) with or without mMCP-1 or CCR2 siRNA. Transient transfection was performed with Lipofectamine 2000 for 24 h. Cell viability was determined by an MTT assay, mouse and human MCP-1 and TGF-[beta]1 levels by ELISA, and CCR2 and ECM protein expression by Western blotting. Transfections of mMCP-1 and CCR2 siRNA increased human MCP-1 levels and inhibited CCR2 expression, respectively. HG-induced ECM protein expression and TGF-[beta]1 levels were significantly attenuated by mMCP-1, CCR2 siRNA, and RS102895 (P < 0.05). MCP-1 directly increased ECM protein expression, and this increase was inhibited by an anti-TGF-[beta]1 antibody. In addition, TGF-[beta]1-induced ECM protein expression was significantly abrogated by the inhibition of the MCP-1/CCR2 system (P < 0.05). These results suggest that an interaction between the MCP-1/CCR2 system and TGF-[beta]1 may contribute to ECM accumulation in DN. diabetic nephropathy; monocyte chemoattractant protein-l; transforming growth factor-[beta]1; extracellular matrix

Previous in vitro studies suggest that the p38 MAPK pathway may be involved in the pathogenesis of diabetic nephropathy, but the consequences of the inhibition of the p38 MAPK pathway have not been well elucidated in diabetic (DM) glomeruli. This study was undertaken to investigate the effect of p38 MAPK inhibitor, FR167653, on fibronectin expression and apoptosis in DM glomeruli and in high-glucose-stimulated mesangial cells (MC). In vivo, 32 Sprague-Dawley rats were injected with diluent (control, N = 16) or streptozotocin intraperitoneally (DM, N = 16). Eight rats from each group were treated with FR167653 for 3 mo. In vitro, rat MC were exposed to medium containing 5.6 mM glucose or 30 mM glucose [high glucose (HG)] with or without [10.sup.-6] M FR167653 for 24 h. Fibronectin mRNA and protein expression were determined by real-time PCR and Western blot, respectively. Western blot for apoptosis-related molecules, terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling assay, and Hoechst 33342 staining were performed to determine apoptosis. FR167653 ameliorated the increases in fibronectin-to-GAPDH mRNA ratio and protein expression in DM glomeruli by 89 and 79% and in HG-stimulated MC by 70 and 91%, respectively (P < 0.05). Under diabetic conditions, Bcl-2 protein expression was decreased, whereas cleaved caspase-3 protein expression was increased (P < 0.05), and these changes were inhibited by FR 167653 treatment. Apoptotic cells were also significantly increased in DM glomeruli and in HG-stimulated MC (P < 0.05), and FR167653 ameliorated these increases in apoptotic cells, both in vivo and in vitro. In conclusion, these findings suggest that the inhibition of the p38 MAPK pathway has a beneficial effect on the development of diabetic nephropathy by inhibiting the increase in fibronectin expression and apoptosis. p38 mitogen-activated protein kinase; fibrosis; apoptosis; diabetic nephropathy

The lipoxygenase (LO) pathway of arachidonate metabolism and mitogen-activated protein kinases (MAPKs) can mediate cellular growth and ANG II effects in vascular smooth muscle cells. However, their role in renal mesangial cells (MC) is not very clear. ANG II treatment of rat MC significantly increased 12-LO mRNA expression and formation of the 12-LO product 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE; P < 0.03]. ANG II-induced [[sup.3]H]leucine incorporation was blocked by an LO inhibitor, cinnamyl-3,4-dihydroxy-[alpha]-cyanocinnamate (P < 0.02). 12(S)-HETE and ANG II directly induced cellular hypertrophy and fibronectin (FN) expression (P < 0.01) to a similar extent. ANG II and 12(S)-HETE led to activation of [p38.sup.MAPK] and its target transcription factor cAMP-responsive element-binding protein (CREB). ANG II-and 12(S)-HETE-induced CREB activation and [[sup.3]H]leucine incorporation were blocked by the [p38.sup.MAPK] inhibitor SB-202190. A specific molecular inhibitor of rat 12-LO mRNA, namely, a novel ribozyme, could attenuate ANG II-induced FN mRNA. Thus [p38.sup.MAPK]-dependent CREB activation may mediate ANG II- and LO product-induced FN expression and cellular growth in rat MC. ANG II effects may be mediated by the LO pathway. These results suggest a novel interaction between LO and [p38.sup.MAPK] activation in MC matrix synthesis associated with renal complications. angiotensin II; 12(S)-hydroxyeicosatetraenoic acid; hypertrophy; [p38.sup.MAPK]; cAMP-responsive element-binding protein; fibronectin; rat