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...

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 ϭ 16) or streptozotocin intraperitoneally (DM, N ϭ 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 Ϫ6 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 HGstimulated MC by 70 and 91%, respectively (P Ͻ 0.05). Under diabetic conditions, Bcl-2 protein expression was decreased, whereas cleaved caspase-3 protein expression was increased (P Ͻ 0.05), and these changes were inhibited by FR167653 treatment. Apoptotic cells were also significantly increased in DM glomeruli and in HG-stimulated MC (P Ͻ 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 MOLECULAR AND CELLULAR mechanisms responsible for diabetic nephropathy remain incompletely resolved. While studies indicate involvement of hyperglycemia via the stimulation of growth factor-induced cellular hypertrophy (48), increased production of extracellular matrix (ECM) protein (2), and decreased production of matrix-degrading proteinases (20), the underlying signal transduction mechanisms mediating these processes have been less well explored.Numerous studies reveal protein kinase C (PKC) activation in diabetic glomeruli (5) and in mesangial cells cultured under high-glucose conditions (42). PKC propagates the physiological responses of receptor-ligand interactions via an array of downstream signals, such as mitogen-activated protein kinases (MAPKs). p38 MAPK, a member of the MAPK family, is known to be activated in response to stress signals, such as proinflammatory cytokines (28, 34), ultraviolet irradiation (34), osmolality changes (28, 34), and oxidative stress (4, 28), leading to apoptosis (43), prostanoid production (22), and other cellular dysfunctions (8). Since hyperosmolality and oxidant stress c...

Epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) has a role in the process of peritoneal fibrosis (PF), a serious complication in peritoneal dialysis (PD) patients. Even though monocyte chemoattractant protein-1 (MCP-1) was demonstrated to directly increase extracellular matrix (ECM) synthesis, the role of the MCP-1/CCR2 system in PD-related EMT and ECM synthesis in cultured human PMCs (HPMCs) and in an animal model of PD has never been elucidated. In vitro, HPMCs were exposed to 5.6 mM glucose (NG), NG þ MCP-1 (10 ng/ml) (NG þ MCP-1), or 100 mM glucose (HG) with or without CCR2 inhibitor (RS102895) (CCR2i) or a dominant-negative mutant MCP-1-expressing lentivirus (LV-mMCP-1). In vivo, PD catheters were inserted into 60 Sprague-Dawley rats, and saline (Control, C) (N ¼ 30) or 4.25% PD solution (PD) (N ¼ 30) was infused for 4 weeks. Twenty rats from each group were treated with empty LV or LVmMCP-1 intraperitoneally. Snail, E-cadherin, a-smooth muscle actin (a-SMA), and fibronectin protein expression in HPMCs and the peritoneum was evaluated by western blot analysis. Compared with NG cells, Snail, a-SMA, and fibronectin expression was significantly increased, while E-cadherin expression was significantly decreased in HPMCs exposed to HG and NG þ MCP-1, and these changes were significantly abrogated by CCR2i (Po0.05). In addition, MCP-1-induced EMT was significantly attenuated by anti-TGF-b1 antibody. In PD rats, Snail and fibronectin expression was significantly increased in the peritoneum, whereas the ratios of E-cadherin/a-SMA protein expression were significantly decreased (Po0.05). The thickness of the peritoneum and the intensity of Masson's trichrome staining in the peritoneum were also significantly higher in PD rats than in C rats (Po0.05). These changes in PD rats were significantly abrogated by LV-mMCP-1. These findings suggest that the MCP-1/CCR2 system is directly involved in PD-related EMT and ECM synthesis and that this is mediated, at least in part, via TGF-b1.