The locally activated renin-angiotensin system is involved in albumin permeability in glomerular endothelial cells under high glucose conditions.

Background: Although the diabetic milieu per se , hemodynamic changes, oxidative stress and local growth factors such as angiotensin II (AII) are considered to be mediators in the pathogenesis of diabetic nephropathy, the underlying pathways mediating the changes in glomerular endothelial cells (GECs) are not well understood. Therefore, we investigated changes in the renin-angiotensin system (RAS) components in high glucose (HG)-stimulated GECs and the role of the local RAS in morphological and functional changes in GECs under diabetic conditions. Methods: We stimulated GECs with 5.6 mM glucose or 30 mM glucose with or without an angiotensin II type I receptor blocker (ARB) in vitro and also performed experiments with Sprague-Dawley rats injected with diluent ( n = 16) or streptozotocin [ n = 16, diabetes (DM)]. Eight rats from each group were treated with ARB for 3 months in vivo . Real-time polymerase chain reaction, western blot analysis, enzyme-linked immunosorbent assay and immunofluorescent staining using cultured GECs were performed. The permeability of GECs to macromolecules was assessed by measuring the passage of fluorescein isothiocyanate-labeled bovine serum albumin. Morphological changes were also evaluated by scanning and transmission electron microscopy. Results: There were significant increases in angiotensinogen expression in HG-stimulated GECs along with significant increases in AI and AII levels. Moreover, the expression of heparan sulfate glycosaminoglycans (HS-GAG) assessed by immunofluorescent staining was significantly lower and the permeability to albumin was significantly higher in GECs exposed to HG medium, and ARB treatment significantly abrogated these changes. Upon electron microscopy examination, the mean size of the GEC fenestrae was significantly greater in HG-stimulated GECs and DM rats, and these increases were significantly ameliorated by ARB. Conclusions: The local RAS within GECs was activated under HG conditions, and this activation may be associated with both an alteration in GEC fenestration and a decrease in HS-GAG, resulting in the development of albuminuria in diabetic nephropathy.

Double transduction of a Cre/LoxP lentiviral vector: a simple method to generate kidney cell-specific knockdown mice.

In a lentivirus-based gene delivery system, the incorporated gene is continuously expressed for a long time. In this study, we devised a simple way to knock down a specific gene in a kidney cell-specific pattern in adult mice by lentivirus-assisted transfer of short hairpin RNA (shRNA). Kidney collecting duct (CD)-specific aquaporin-3 (AQP3)-knockdown mice were generated by consecutive injection of Hoxb7-Cre-expressing lentivirus (LV-Hoxb7 Cre) and loxP-AQP3 shRNA-expressing lentivirus (LV-loxP shAQP3) in adult C57BL6/J mice. LV-Hoxb7 Cre was designed to express mCherry, while LV-loxP shAQP3 was designed with a floxed enhanced green fluorescent protein (EGFP)-tagged stop sequence, and thus EGFP would be expressed only in the absence of Cre recombination. In mice treated with LV-Hoxb7 Cre alone, mCherry protein expression, which indicates the presence of Cre recombinase, occurred only in CD cells. However, LV-loxP shAQP3 injection alone resulted in an increase in EGFP expression in all kidney cells, indicating the transcription of the floxed region. When LV-Hoxb7 Cre and LV-loxP shAQP3 were sequentially transduced, EGFP expression was attenuated while mCherry expression was sustained in CD cells, demonstrating a CD cell-specific recombination of the floxed region. AQP3 expression in mice injected with LV-Hoxb7 Cre or LV-loxP shAQP3 alone did not differ, but consecutive injection of LV-Hoxb7 Cre and LV-loxP shAQP3 significantly reduced AQP3 expression in CD cells. However, the expression levels of AQP3 were not altered in other cell types. Double transduction of Cre- and loxP-based lentivirus can easily generate kidney cell-specific knockdown mice, and this method might be applicable to other species.

Podocyte hypertrophy precedes apoptosis under experimental diabetic conditions.

Podocyte hypertrophy and apoptosis are two hallmarks of diabetic glomeruli, but the sequence in which these processes occur remains a matter of debate. Here we investigated the effects of inhibiting hypertrophy on apoptosis, and vice versa, in both podocytes and glomeruli, under diabetic conditions. Hypertrophy and apoptosis were inhibited using an epidermal growth factor receptor inhibitor (PKI 166) and a pan-caspase inhibitor (zAsp-DCB), respectively. We observed significant increases in the protein expression of p27, p21, phospho-eukaryotic elongation factor 4E-binding protein 1, and phospho-p70 S6 ribosomal protein kinase, in both cultured podocytes exposed to high-glucose (HG) medium, and streptozotocin-induced diabetes mellitus (DM) rat glomeruli. These increases were significantly inhibited by PKI 166, but not by zAsp-DCB. In addition, the amount of protein per cell, the relative cell size, and the glomerular volume were all significantly increased under diabetic conditions, and these changes were also blocked by treatment with PKI 166, but not zAsp-DCB. Increased protein expression of cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase, together with increased Bax/Bcl-2 ratios, were also observed in HG-stimulated podocytes and DM glomeruli. Treatment with either zAsp-DCB or PKI 166 resulted in a significant attenuation of these effects. Both PKI 166 and zAsp-DCB also inhibited the increase in number of apoptotic cells, as assessed by Hoechst 33342 staining and TUNEL assay. Under diabetic conditions, inhibition of podocyte hypertrophy results in attenuated apoptosis, whereas blocking apoptosis has no effect on podocyte hypertrophy, suggesting that podocyte hypertrophy precedes apoptosis.

Enhanced glycogen synthase kinase-3ß activity mediates podocyte apoptosis under diabetic conditions.

Glycogen synthase kinase-3ß (GSK-3ß) is involved in the pathogenesis of various kidney diseases. This study was undertaken to examine the changes in GSK-3ß activity in podocytes under diabetic conditions and to elucidate the functional role of GSK-3ß in podocyte apoptosis. In vivo, 32 rats were injected with either diluent (n = 16, C) or with streptozotocin intraperitoneally (n = 16, DM), and 8 rats from each group were treated with 6-bromoindirubin-3'-oxime (BIO) for 3 months. In vitro, immortalized mouse podocytes were exposed to 5.6 mM glucose or 30 mM glucose (HG) with or without 10 µM BIO. Western blot analysis and TUNEL or Hoechst 33342 staining were performed to identify apoptosis. Urinary albumin excretion was significantly higher in DM rats, and this increase was significantly abrogated in DM rats by BIO treatment. The protein expression of Tyr216-phospho-GSK-3ß was significantly increased in DM glomeruli and in cultured podocytes exposed to HG. Western blot analysis revealed that the protein expression of Bax and active fragments of caspase-3 were significantly increased, whereas phospho-Akt, ß-catenin, and Bcl-2 protein expression were significantly decreased in DM glomeruli and HG-stimulated podocytes. Apoptosis, determined by TUNEL assay and Hoechst 33342 staining, was also significantly increased in podocytes under diabetic conditions. The changes in the expression of apoptosis-related molecules and the increase in the number of apoptotic cells in DM glomeruli as well as in HG-stimulated podocytes were significantly ameliorated by BIO. These findings suggest that enhanced GSK-3ß activity within podocytes under diabetic conditions is associated with podocyte loss in diabetic nephropathy.

The monocyte chemoattractant protein-1 (MCP-1)/CCR2 system is involved in peritoneal dialysis-related epithelial-mesenchymal transition of peritoneal mesothelial cells.

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+MCP-1 (10 ng/ml) (NG+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=30) or 4.25% PD solution (PD) (N=30) was infused for 4 weeks. Twenty rats from each group were treated with empty LV or LV-mMCP-1 intraperitoneally. Snail, E-cadherin, α-smooth muscle actin (α-SMA), and fibronectin protein expression in HPMCs and the peritoneum was evaluated by western blot analysis. Compared with NG cells, Snail, α-SMA, and fibronectin expression was significantly increased, while E-cadherin expression was significantly decreased in HPMCs exposed to HG and NG+MCP-1, and these changes were significantly abrogated by CCR2i (P<0.05). In addition, MCP-1-induced EMT was significantly attenuated by anti-TGF-ß1 antibody. In PD rats, Snail and fibronectin expression was significantly increased in the peritoneum, whereas the ratios of E-cadherin/α-SMA protein expression were significantly decreased (P<0.05). 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 (P<0.05). 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-ß1.

The MCP-1/CCR2 axis in podocytes is involved in apoptosis induced by diabetic conditions.

Previous studies have demonstrated the importance of monocyte chemoattractant protein-1 (MCP-1) in the pathogenesis of diabetic nephropathy in terms of inflammation, but the direct role of the MCP-1/CCR2 system on podocyte apoptosis under diabetic conditions has never been explored. In vitro, mouse podocytes were exposed to a medium containing 30 mM glucose (HG) with or without CCR2 siRNA or CCR2 inhibitor (RS102895). Podocytes were also treated with MCP-1 or TGF-ß1 with or without anti-TGF-ß1 antibody, CCR2 siRNA, or CCR2 inhibitor. In vivo, 20 db/m and 20 db/db mice were divided into two groups, and ten mice from each group were treated with RS102895. Western blot and Hoechst 33342 or TUNEL staining were performed to identify apoptosis. HG-induced apoptosis and TGF-ß1 levels were significantly abrogated by CCR2 inhibition. In addition, treatment with MCP-1 directly induced apoptosis via CCR2. Moreover, TGF-ß1- and MCP-1-induced apoptosis were significantly ameliorated by the inhibition of CCR2 and anti-TGF-ß1 antibody, respectively. Glomerular expression of cleaved caspase-3 and apoptotic cells within glomeruli were also significantly increased in db/db mice compared to db/m mice, and these increases were significantly attenuated in db/db + RS102895 mice. These results suggest that interactions between the MCP-1/CCR2 system and TGF-ß1 may contribute to podocyte apoptosis under diabetic conditions.

Local kallikrein-kinin system is involved in podocyte apoptosis under diabetic conditions.

The kallikrein-kinin system (KKS) serves as the physiologic counterbalance to the renin-angiotensin system. This study was conducted to examine the changes in the expression of KKS components in podocytes under diabetic conditions and to elucidate the functional role of bradykinin (BK) in diabetes-associated podocyte apoptosis. Thirty-two rats were injected with either diluent (n = 16, C) or with streptozotocin intraperitoneally (n = 16, DM), and 8 rats from each group were treated with BK infusion for 6 weeks. Immortalized mouse podocytes were cultured in media containing 5.6 mmol/l glucose (NG), NG + 10(-7) mol/l AII (AII), or 30 mmol/l glucose (HG) with or without 10(-8) mol/l BK. Urinary albumin excretion was significantly higher in DM rats, and this increase was ameliorated by BK. Not only kininogen, kallikrein, and BK B1- and B2-receptor expression but also BK levels were significantly decreased in DM glomeruli and in cultured podocytes exposed to HG. The changes in the expressions of apoptosis-related molecules and the increase in the number of apoptotic cells in DM glomeruli as well as in HG- and AII-stimulated podocytes were significantly abrogated by BK. The suppressed KSS within podocytes under diabetic condition was associated with podocyte apoptosis, suggesting that BK may be beneficial in preventing podocyte loss in diabetic nephropathy.

The effect of statin on epithelial-mesenchymal transition in peritoneal mesothelial cells.

BACKGROUND: Statins have recently been highlighted for their pleiotropic actions distinct from cholesterol-lowering effects. Despite this interest, it is currently unknown whether statin therapy inhibits peritoneal dialysis (PD)-related epithelial-mesenchymal transition (EMT). METHODS: In vitro, human peritoneal mesothelial cells (HPMCs) were exposed to 5.6 mM glucose (NG) or 100 mM glucose (HG) with or without simvastatin (1 µM). In vivo, PD catheters were inserted into 32 Sprague-Dawley rats, and saline (C, n = 16) or 4.25% peritoneal dialysis fluid (PDF) (PD, n = 16) was infused for 4 weeks. Eight rats from each group were treated with 5 mg/kg/day of simvastatin intraperitoneally. Changes in the protein expression of EMT markers such as E-cadherin, α-SMA, Snail, and fibronectin in HPMCs and the peritoneum were evaluated by Western blot analysis and immunofluorescence or immunohistochemical staining. We also explored whether activation of the mevalonate pathway and its downstream small GTPases were involved in dialysis-related peritoneal EMT and could be inhibited by statin treatment. RESULTS: Compared to NG cells, E-cadherin expression was significantly decreased, while α-SMA, Snail, and fibronectin expression were significantly increased in HPMCs exposed to HG, and these changes were abrogated by simvastatin (p<0.05). In addition, the cobblestone-like appearance of normal HPMCs was converted into a fibroblast-like morphology after HG treatment, which was reversed by simvastatin. These EMT-like changes were also observed in HPMCs treated with geranyl-geranyl pyrophosphate (5 µM). HG significantly increased the protein expression of RhoA and Rac1 in the membrane fractions, and these increases were ameliorated by simvastatin (p<0.05). In PD rats, E-cadherin in the peritoneum was significantly decreased, whereas α-SMA, Snail, and fibronectin expression were significantly increased (p<0.05) compared to C rats. The thickness of the mesothelial layer in the peritoneum were also significantly greater in PD rats than in C rats (p<0.05). These changes of the peritoneum in PD rats were significantly attenuated by simvastatin. CONCLUSION: This study demonstrated that PD-related EMT was mediated via the mevalonate pathway, and statin treatment inhibited the EMT changes in HG-treated HPMCs and PDF-stimulated PD rats. These findings suggest that statins may be a promising therapeutic strategy for preservation of peritoneal membrane integrity in long-term PD patients.