| Diabetic nephropathy (DN) is currently a leading cause of end-stage renal disease (ESRD), which confers high morbidity and mortality rates in diabetic patients. In overt DN, expansion of the mesangial matrix, mesangial cells (MCs) loss and glomerular sclerosis are prominent, associated with renal dysfunction and proteinuria. Hyperglycemia-mediated reactive oxygen species (ROS) has been recognized as classical etiological factors of DN. ROS are also recognized as important mediators of cellular apoptosis. Only a limited number of studies examined the molecular events, and the mechanism of mesangial cell apoptosis under hyperglycemic condition remain poorly understood.A growing body of evidences suggests that endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of diabetes. This opens up a new avenue for understanding the pathology of ER stress and treatment of diabetes. The ER stress is mediated by activation of three ER transmembrane sensor proteins:the protein kinase-like ER kinase (PERK), the activating transcription factor6(ATF6) and the inositol requiring kinase1(IRE1). IRE1, which splices Xbpl mRNA, leads to translation of the spliced form of X-box binding protein1(XBP1S), IRE1/XBP1pathway is the most evolutionarily conserved pathway in ER stress. XBP1S has protective role in many cell biofunctions. Overexpression of XBP1S significantly reduced blood glucose levels in dieabetic mice. XBP1S plays a role in the maintenance of glucose homeostasis in type â…¡ DM, suggesting that upregulation of XBP1S is probably a new therapeutic approach for the treatment of diabetes.C/EBP homologous protein (CHOP) is activated during ER stress. It plays an important role in ER stress-induced apoptotic cell death. Expression of CHOP is mainly regulated at transcriptional level, its promoter contains ERSE motif. XBP1, as a nuclear transcriptional factor, can bind to ERSE and the CHOP promoter.Accumulating evidence suggests that ER stress and oxidative stress are closely linked events. XBP1has the role of anti-oxidative stress in certain cell types. Whether XBP1S is involved in the occurrence and development of DN deserved further study. If it indeed plays a protective role in DN, what is the role of CHOP?The aim of this study was to observe the changes of XBP1S expression under hyperglycemic condition, and to explore whether XBP1S is involved in the regulation of hyperglycemic-induced ROS overproduction and apoptosis. By these researches, we attempted to elucidate the role of XBPIS in DN and its mechanisms. Results:Part â… . The effects of XBPIS in high glucose-induced ROS overproduction in the cultured renal MCs and its mechanisms1. HG elevated ROS generation in cultured renal MCsWe first observed the effect of high glucose (HG,30mmol/L D-glucose) on ROS generation in cultured rat renal MCs in vitro. Compared with normal cultured cells (5,5mmol/L D-glucose), ROS production was elevated significantly after HG treatment for12h,24h and48h. Diphenylene-chloride iodonium (DPI,10-6mol/L), a NADPH oxidase inhibitor, inhibited the ROS elevation induced by HG. P47phox protein levels were increased after HG treatment for24h,48h or72h.2. Identification of the changes in XBP1S under HG condition.Compared with normal cultured cells, Western blot demonstrated that HG treatment decreased XBP1S level, while XBP1U protein level was not changed in the cultured renal MCs. RT-PCR and qPCR results showed that XBP1S mRNA level declined under HG condition.3. Effects of overexpression XBP1S on ROS and p47phox protein levelCompared with recombinant adenoviruses expressing the green fluorescent protein (Ad-GFP), recombinant adenoviruses expressing spliced XBP1(Ad-XBP1S) trasfection reduced HG induced ROS overproduction and p47phox protein increase in the cultured renal MCs.4. Effects of XBPIS knock-down on ROS activation in cultured rat renal MCs After XBPIS siRNA (50nM) transfection for48h, the ROS and p47phox protein level were remarkably increased in the cultured renal MCs.These results indicated that NADPH oxidase contributed to the ROS overproduction by HG treatment. XBP1S had the ability of anti-oxidative stress. The decreased XBP1S expression related to ROS overproduction under HG condition. P47phox participated in XBP1S regulating ROS production.Part â…¡. The effects of XBP1S on apoptosis induced by HG in the cultured renal MCs and its mechanisms1. HG induced apoptosis in the cultured renal MCsCompared with normal culured cells, Flow Cytometry results showed that the number of Annexin-V positive cells were elevated significantly after HG treatment for48h; Western blot results demonstrated that HG increased the ratio of Bax/Bcl2; Pro-caspase3and cleaved-caspase3protein levels were also increased after HG treatment for48h.2. Effect of overexpression of XBP1S on apoptosisCompared with Ad-GFP transfection group, Western blot results demonstrated that Ad-XBPIS transfection decreased pro-caspase3and cleaved-caspase3protein levels; Ad-XBP1S transfection decreased Bax and increased Bcl2protein level, the ratio of Bax/Bcl2was decreased after Ad-XBP1S transfection for48h.3. Effect of overexpression XBP1S on HG-induced apoptosisCompared with HG treatment group, Western blot results demonstrated that Ad-XBPIS decreased the ratio of Bax/Bcl2, pro-caspase3and cleaved-caspase3protein levels after transfection for48h.4. Effects of XBP1S knock-down on cell apoptosisCompared with negative control, Western blot results demonstrated that XBP1S siRNA (50nM) increased pro-caspase3and cleaved-caspase3protein levels after XBP1S siRNA transfection for48h.5. Effects of DPI on HG induced apoptosis Western blot results demonstrated that DPI (10-6mol/L) inhibited HG induced Bax/Bcl2, pro-caspase3and cleaved-caspase3protein levels after MCs cultured in DPI for48h.6. ROS sitmulated cell apoptosis in cultured MCs.MCs were exposed to H2O2(1*10-4uM) for4h, Western blot results demonstrated that H2O2increased pro-caspase3and cleaved-caspase3protein levels.These results indicated that HG treatment induced cell apoptosis. ROS overproduction contributed to HG-induced apoptosis. XBP1S had the ability of anti-apoptosis. Bax, Bc12and caspase3participated in XBP1S regulating MCs apoptosis.Part â…¢. The mechanism of XBP1S in anti-apoptosis1. Effects of HG on CHOP expression2. MCs were cultured in medium containing5.5or30mM D-glucose for48h, Western blot results showed that there was a decrease in CHOP protein level in the cells exposed to30mM D-glucose compared with that to5.5mM D-glucose. qPCR results showed that CHOP mRNA levels were also significantly decreased after exposed to30mM D-glucose for24h and48h.XBP1positive-regulated CHOP expression in cultured MCsChromatin immunoprecipitation results showed that XBP1bind to CHOP promoter region. Compared with the Ad-GFP transfection group, the CHOP protein level was significantly increased after transfection of Ad-XBP1S for48h.3. Effect of CHOP siRNA on cell apoptosis in cultured MCsCompared with negative control, Western blot results demonstrated that the transfection of CHOP siRNA (50nM) for48h had no obvious effect on Bax/Bc12ratio, pro-caspase3and cleaved-caspase3protein levels.4. Effect of CHOP siRNA on the necroptosis of cultured MCsCompared with negative control, Western blot results demonstrated that the transfection of CHOP siRNA (50nM) for48h increased BNIP3protein level. Flow Cytometry results showed that the PI positive cells were elevated significantly after the transfection of CHOP siRNA (50nM) for48h;5. Effect of HG and Ad-XBP1S transfection on the necroptosis of cultured MCs Compared with normal cultured cells, Flow Cytometry results showed that the PI positive necrotic cells were elevated significantly after HG treatment for48h; Western blot results demonstrated that the HG treatment increased BNIP3protein level, while the Ad-XBP1S transfection decreased HG-induced BNIP3protein level.These results indicated that XBP1had direct positive regulation on CHOP. HG treatment induced MCs apoptosis and necroptosis. XBP1S exhibited both anti-apoptosis and anti-necroptosis effect in the HG-treated MCs. CHOP mediated anti-necropstosis effect of XBP1S but not the anti-apoptosis effect of XBP1S.Part IV. Effect of XBP1S overexpression on ECM systhesis1. Effect of HG on ECM production in cultured MCs Compared with mannitol and normal cultured cells, Western blot results showed that the Collagen IV and Fibronectin protein levels in the culturing medium were elevated significantly after HG treatment for24h,48h and72h.2. Effect of XBP1S overexpression on HG-induced Collagen IV and Fibronectin synthesisCompared with HG group, Western blot results showed that the elevations in Collagen IV and Fibronectin protein levels in the culturing medium were inhibited by Ad-XBP1S transfection for48h 3. Changes in p47phox, Collagen IV and Fibronectin in diabetic rats Diabetic rats were induced by intropretoneal injection of streptozotocin (STZ,65mg/kg). Eight weeks after STZ injection, the renal cortexes were harvested. Compared with non-diabetic rats, Western blot analysis showed that the p47phox, Collagen IV and Fibronectin protein levels were significantly increased in diabetic renal cortex.4. Changes in XBP1S, CHOP, caspase3, and BNIP3and in the renal cortex of STZ-induced diabetic rats.Compared with non-diabetic rats, Western blot analysis showed that XBP1S and CHOP protein levels were suppressed while pro-casepase and cleaved-caspase3, BNIP3protein levels were elevated in diabetic renal cortex at the end of8weeks of STZ injection.These results indicated that HG induces the increases in ECM productuon in MCs. Overexpression of XBP1S decreased ECM productuon in MCs; In diabetic rats, increase in oxidative stress, cell apoptosis and necroptosis were observed, while the protein levels of XBP1S and CHOP were declined.Summary:XBP1S levels were declined under HG-treated MCs and diabetic renal cortex; XBP1S suppressed HG-induced oxidative stress, apoptosis and necroptosis in cultured MCs; XBP1binded to CHOP promotor and positively regulated the CHOP expression; CHOP mediated the suppression effects of XBP1S on cell necroptosis but not apoptosis. These results suggested that XBP1S had a protective role in the development of DN. |
