Heme oxygenase-1 (HO-1) is an anti-oxidant enzyme normally upregulated in response to oxidant injury. Here we determined the role of HO-1 in podocyte apoptosis in glomeruli of streptozotocin-treated rats and in immortalized mouse podocytes cultured in media containing normal or high glucose. HO-1 expression, its activity, the ratio of Bax/Bcl-2 protein, and active caspase-3 fragments were all significantly higher in isolated glomeruli of diabetic rats and in high glucose-treated podocytes. These increases were inhibited by zinc protoporphyrin treatment of the rats or by HO-1 siRNA treatment of the podocytes in culture. The number of apoptotic cells was also significantly increased in the glomeruli of diabetic rats and in high glucose-treated podocytes. Inhibition of HO-1 accentuated the increase in apoptotic cells both in vivo and in vitro. Our findings suggest that HO-1 expression protects against podocyte apoptosis under diabetic conditions.

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(-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.

) 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 glucoseinduced 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), NGϩ24.4 mM mannitol (NGϩM), or 30 mM glucose (HG) with or without mutant MCP-1 (mMCP-1), CCR2 small interfering (si)RNA, or CCR2 inhibitor (RS102895). To examine the relationship between MCP-1 and transforming growth factor (TGF)-␤1, MCs were also treated with TGF-␤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-␤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-␤1 levels were significantly attenuated by mMCP-1, CCR2 siRNA, and RS102895 (P Ͻ 0.05). MCP-1 directly increased ECM protein expression, and this increase was inhibited by an anti-TGF-␤1 antibody. In addition, TGF-␤1-induced ECM protein expression was significantly abrogated by the inhibition of the MCP-1/CCR2 system (P Ͻ 0.05). These results suggest that an interaction between the MCP-1/CCR2 system and TGF-␤1 may contribute to ECM accumulation in DN. diabetic nephropathy; monocyte chemoattractant protein-1; transforming growth factor-␤1; extracellular matrix MONOCYTES/MACROPHAGES ARE the principle inflammatory cells found in the diabetic kidney (6, 9, 29). These cells are extravasculated from the bloodstream through a process mediated by chemokines secreted from resident glomerular cells. Chemokines are a family of chemotactic cytokines that induce the migration of various cell types, and to date Ͼ40 chemokines have been identified (31). Among them, monocyte chemoattractant protein (MCP)-1 is the most extensively studied chemokine. In the kidney, MCP-1 is expressed in mesangial cells (MCs) and tubular epithelial cells (22,26) and is known to be involved in the pathogenesis of various renal diseases, including diabetic nephropathy. Previous studies have demonstrated that plasma MCP-1 levels are increased in type 1 diabetes with microalbuminuria (4) and that urinary levels of MCP-1 are also increased in accordance with the extent of albuminuria (1,20). In addition, it has been reported that glomerular MCP-1 expression is increased in experimental diabetic rats and that this increase is associated with the number of infiltrated monocytes in the glomeruli (5, 6). Moreover, an angio...