Ras Dysfunction In High Glucose-induced Renal Tubular Epithelial Cell Transdifferentiation Effects And Mechanisms

Diabetic nephropathy is one of the major complications of diabetes, and is also a leading cause of end-stage renal disease, ESRD. It had been demonstrated that in type I diabetes,40% patients had a concurrent renal injury, while 15% in typeⅡ. Some of them will finally develop into renal failure. The early signs of DN are glomerular hypertrophy, extracellular matrix accumulation, thickened glomerular and tubular basement membranes. At the end stage of DN, glomerular sclerosis and diffuse interstitial fibrosis occur. The mechanism of DN has not been completely clear, and multiple factors are considered contribute to the development of DN. Researches demonstrated that the renal interstitial fibrosis is a key pathological change in all kinds of chronic kidney diseases, including diabetic nephropathy. Therefore, revealing the mechanism of the formation of renal interstitial fibrosis will be useful in developing new strategy in implement prevention or treatment on DN, and slow down the chronic renal injury.Extracellular matrix accumulation and degradation are all contributed to the renal interstitial fibrosis. The proliferation of kidney fibroblast cells and epithelial mesenchymal transition (EMT), are two key contributors of extra ECM production. Available evidences indicate that under hyperglycemic condition, renal tubular epithelial cells undergo phenotypic transition into myofibroblasts. The transitioned cell is characterized by the expression of a-smooth muscle actin, enhanced contractility and migratory ability, production of cytokines, increased proliferation, and more importantly, enhanced capacity to produce interstitial matrix, which contributes to renal fibrosis. Studies have demonstrated that many facotors, including TGF-β, are closely related to the development of EMT.Renin-angiotensin system (RAS) is an endocrine peptide system known for its involvement in blood pressure control and body salt homeostasis. In the updated concept, RAS contains two axes:the angiotensin converting enzyme (ACE)-angiotensinⅡ(AngⅡ)-AngⅡtype 1 (AT1) receptor axis and the angiotensin-converting enzyme-related carboxypeptidase (ACE2)-angiotensin (1-7) [Ang-(1-7)]-Mas receptor axis. Recently, accumulated evidences indicate that ACE2-Ang-(1-7)-Mas system is acting as counter-regulator for the ACE-AngⅡ-AT1 receptor axis. In the present study, we aimed to observe the effect of high glucose on RAS, the role of RAS in high glucose-induced EMT, and the mechanisms involved in EMT.PartⅠ:Changes in the main components of RAS under hyperglycemia condition and the role in high glucose-induced EMT.Rat kidney proximal tubular epithelia cell line (NRK-52E) was used in the present study. Changes of EMT makers, namely a-SMA, E-cadherin and Vimentin, were evaluated using fluorescence immunocytochemistry, protein levels of E-cadherin and Vimentin were detected by western blotting, mRNA levels of AGT, ACE, AT1 receptor were determined by real-time polymerase chain reaction (Real-time PCR) and the concentration of angiotensinⅡ(AngⅡ) and transforming growth factor (TGF-β1) in the culture medium were measured by enzyme immunoassay (ELISA). The results illustrated that NRK-52E cells incubated in NG (5.5 mmol/L D-glucose) exhibited a typical cobblestone-like shape, while exposure to HG (30 mmol/L D-glucose) for 48 or 72 hrs resulted in phenotypic conversion from primary epithelial cells into myofibroblast-like cells. Immunofluorescence staining result showed a significant increase in a-SMA signal and a decrease in E-cadherin. The changes in EMT marker proteins were confirmed by western blot. Treatment of NRK-52E cells with high glucose for 24 or 48 hrs induced significant increases in AGT, ACE, AT1 mRNA levels, and Ang II concentration in culture medium. In summary, high glucose induced epithelial mesenchymal transition, meanwhile, the ACE-AngⅡ-AT1 receptor axis in cultured renal tubular cells was activated by high glucose.To investigate whether the ACE-AngⅡ-AT1 receptor axis was involved in high glucose-induced EMT, selective AT1 receptor antagonist was used to block the effect of AngⅡ. Fluorescence immunostaining showed that exposure of NRK-52E cells to HG resulted in a significant decrease in E-cadherin and increase in vimentin signal. Pre-treatment with 10-5 mol/L AT1 receptor antagonist Losartan obviously attenuated the effect. Meanwhile, Western blot result also showed that epithelial marker E-cadherin protein level in cells exposed to HG was lower than that of NG.. Pre-treatment with Losartan reversed the decrease in E-cadherin in a concerntration-dependant manner. In contrast, the mesenchymal maker, Vimentin protein level was increased significantly by HG treatment. Pre-treatment with Losartan reversed the increase in Vimentin. Losartan alone had no significant effect on E-cadherin and Vimentin in NG-cultured cells. These effect demonstrated the activation in RAS by HG participates in HG induced EMT.TGF-β1 has been demonstrated to be involved in EMT both in vivo and vitro. In the experiment, ELISA result showed that TGF-β1 concentration significantly increased in the cell supernatant exposed to HG for 48 hrs. Pre-treatment with Losartan (10-5 mol/L) inhibited TGF-β1 secretion caused by HG, but did not return to normal. Losartan alone had no effect on TGF-β1 secretion. These results suggest that stimulating TGF-β1 might be a way that activated RAS participates in HG induced EMT.Fibronetin, a major component of extracellular matrix protein secretion by transitioned myofibroblast cells, was measured by Western blot. Fibronetin showed a significant increase in HG group. Pretreatment of cells with Losartan (10-5-10-7mol/L) diminished HG-induced increase in fibronectin synthesis in a dose-dependent manner. Losartan (10-5mol/L) alone had no effect on fibronectin synthesis. Moreover, MMP-9 synthesis was also significant increased after exposed to HG. Pre-treatment with Losartan (10-5mol/L) blocked the increase in MMP-9 induced by HG. These results suggest that the increased ECM production induced by HG can be attenuated by AT1 receptor blockade.PartⅡ:In the above study, we demonstrated that the activation of local RAS participates in high glucose-induced EMT. Blocking AT1 receptor suppressed but not completely restored high glucose-induced EMT. The purpose of this study was to indentify the changes in ACE2-Ang(1-7)-Mas receptor axis after HG exposure and whether ACE2-Ang(1-7)-Mas receptor axis was also involved in HG-induced EMT and its molecular mechanism.1. Effect of HG on ACE2 and Mas receptor level in NRK-52E cells.Real-time PCR analysis showed ACE2 mRNA level decreased significantly after exposed the cells to HG for 24 or 48 hrs when compared with that in NG. Mas receptor mRNA level also decreased after HG exposure for 48 hrs.2. Effect of ACE2-Ang(1-7)-Mas receptor axis on high glucose-induced EMT.Fluorescent immunocytochemistry result showed that exposure of the cell to HG resulted in a significant increase in a-SMA signal. Pre-treatment with Ang-(1-7) obviously attenuated the increase in a-SMA signal. Western Blot result showed that epithelial marker E-cadherin protein level decreased significantly after exposed the cell to HG for 48 hrs. Pre-treatment of the cell with Ang-(1-7) reversed the decrease in E-cadherin in a concentration-dependant manner. In contrast, mesenchymal maker vimentin protein level was increased significantly after HG exposure. Pre-treatment with Ang-(1-7) reversed the increase in vimentin. However, even high dose of Ang-(1-7) (10-5 mol/L) did not abolish the vimentin increase completely.3. MAPK signaling pathways, ERK and p38 intracellular, but not JNK, mediate the anti effect of Ang-(1-7) on HG-induced EMT.Western blot result showed that ERK phosphorylation level in cultured NRK-52E cells increased in a time-dependent manner after HG exposure. ERK phophorylation level has already increased after exposed the cell to HG for 15 min, and kept increasing in the succedent 60 min. The phosphorylation of JNK and p38 showed the similar changing pattern after HG expo sure. Pre-treatment of the cell with Ang-(1-7) prevented HG-induced increase in ERK phosphorylation, the effect of Ang-(1-7) was blocked by Mas receptor antagonist A-779. Ang-(1-7) did not show obvious effect on ERK phosphorylation in NG-cultured cells. Same pattern was observed on p38 that Ang-(1-7) blocked HG-induced p38 phosporylation increase and showed no obvious effect on NG-cultured cells. Mas receptor antagonist A-119 blocked the effect of Ang-(1-7) on reversing HG-induced increase in p38 phosphorylation. In contrast, Ang-(1-7) did not show obvious effect on HG-induced increase in JNK phosphorylaiton, also on NG-cultured cells.4. Ang-(1-7) inhibited the increasing of TGF-β1 production in tubular epithelial cells induced after HG exposure.Real-time PCR result showed that TGF-β1 mRNA level was up-regulated significantly after exposed the cell to HG for 24 hrs. Pre-treatment of the cell with Ang-(1-7) reversed HG-induced increase in TGF-β1 mRNA level. Consistent with the mRNA changes, ELISA result showed that TGF-β1 concentration in the cultured media increased significantly after exposed the cell to HG for 48 hrs. Ang-(1-7) attenuated HG-induced increase in TGF-β1, but not abolished the increase completely. The TGF-β1 concentration in HG-treated group was still significantly higher than control value after Ang-(1-7) treatment. Mas receptor antagonist A-779 blocked the effect of Ang-(1-7). Neither Ang-(1-7) nor A-779 showed obvious effect on TGF-β1 production in NG-cultured cells. MAPK pathway activation after HG exposure was involved in TGF-β1 over production. ERK signaling pathway blocker PD98059 significantly attenuated the HG-induced increase in media TGF-β1 concentration, but did not return it to normal. Similar results were observed after applications of p38 signaling pathway blocker SB203580 or JNK signaling pathway blocker SP600125. Combination of PD98059, SB203580 and SP600125 completely blocked HG-induced increase in TGF-p 1 secretion.5. Ang-(1-7) inhibited the increasing of extracellular matrix synthesis induced by HG Western blot analysis showed fibronectin was increased significantly after HG exposure. Pre-treatment of the cells with Ang-(1-7) attenuated HG-induced increases in fibronectin synthesis in a concentration-dependent manner. However, Ang-(1-7), even at high concentration (10-5 mol/L), did not abolish HG-induced increase in fibronectin synthesis.PartⅢOur above study demonstrated that under hyperglycemic condition, renal tubular epithelia cells undergo EMT, and both ACE-AngⅡ-AT1 axis and ACE2-Ang(1-7)-Mas receptor axis of the local RAS were involved in high glucose-induced EMT. However, the molecular mechanism that governs this process is still not very clear. MicroRNAs (miRNAs) are a family of short non-coding RNAs on average 22 nucleotides long, and are found in all eukaryotic cells. As post-transcriptional regulators, miRNAs bind to complementary sequences on target mRNAs and result in translational repression or gene silencing. The human genome may encode over 1000 miRNAs, which may target about 60% of mammalian genes. Aberrant expression of miRNAs has been implicated in numerous pathological conditions, including DN. The aim of our study is to investigate the miRNAs expression changing profile in high glucose models and to identify the differentially expressed miRNAs and its target mRNAs.1. Differential expressed miRNAs profile in NRK-52E cells with or without HG treatment.Microarrays were used to screen the differential expressed miRNAs. Among the differential expressed miRNAs, the miR-181a was elevated more than 50% in NRK-52E cells after HG treatment. Furthermore, Mas receptor, the Ang-(1-7) receptor in RAS was predicted as a potential target gene of miR-181a.2. Validation of the microarray data by Real-time PCR.The differential expression of miR-181a between NG and HG-induced NRK-52E cells was validated by Real-time PCR. Consistent with the microarray analysis, Real-time PCR showed that miR-181a level was significantly increased when cells were treated with HG for 24 hrs. Moreover, we examined the level of miR-181a in STZ-induced diabetic rat kidney. The result showed that miR-181a level up-regulated in diabetic rats when compared with control animals. Taken together, these results confirm that miR-181a is significantly up-regulated under hyperglycemia condition.3. Identification of miR-181a regulates Mas receptor expression in NRK-52E.Specific miR-181a knockdown probe was transfected into NRK-52E cells. The transfection of miR-181a knockdown probe (100pM) decreased miR-181a level significantly both NG and HG groups, while transfection of the scramble probe did not show obvious effect. Meanwhile, knockdown of miR-181a significantly elevated Mas mRNA levels in both NG and HG cultured cells. These data suggest that miR-181a directly regulate Mas receptor expression in NRK-52E cell.In summary, the present study demonstrated that:1. Under hyperglycemic condition, tubular epithelial cells undergo phenotypic transiton into myofibroblasts. The transitioned cell obtained enhanced capacity in interstitial matrix production, which may contribute to renal fibrosis in vivo. Activation of RAS ACE-AngⅡ-AT1 axis was involved in high glucose-induced EMT. Blocking AT1 receptor suppressed, but not completely halted high glucose-induced EMT.2. Under hyperglycemic condition, ACE2-Ang-(1-7)-Mas receptor axis, which counterbalance ACE-AngⅡ-AT1 pathway, is suppressed. The depression in ACE2-Ang-(1-7)-Mas receptor in NRK-52E cells also contribute to glucose-induced EMT. Activation of Mas receptor blunted HG-induced EMT via eliminating HG-induced ERK1/2 and p38 MAPK phosphorylation.3. MiR-181 a level is significantly up-regulated under hyperglycemic condition both in vivo and in vitro. Up-regulated miR-181a level contributed HG-induced EMT via suppressing Mas receptor expression.