| Background:Diabetic nephropathy is one of the serious microvascular complications of diabetes; once there are massive proteinuria, kidney function will irreversibly decline and eventually develop to end-stage renal disease. As the pathogenesis is complex, there is no ideal treatment until now. Previous studies were focused on the harm of high blood glucose on diabetes and its complications; however, in recent years it is thought that the disorders of fatty acid metabolism are the primary pathophysiology of diabetes and its complications. Elevated free fatty acids in the blood, which could accumulate in non-adipose tissue and cause damage, also known as lipotoxicity, are both one of the possible causes of the onset of diabetes, and key factors in promoting and aggravating disease progression. In diabetic nephropathy, it has been proven lipotoxicity is an important pathological mechanism of progressive tubulointerstitial injury and is also correlated with the prognosis of kidneys. Electron transport flavoprotein (ETF) is an electron acceptor for at least 12 kinds of mitochondrial dehydrogenase in fatty acid β oxidation, which is composed by a and P subunits and located in mitochondria matrix. Our previous studies have found ETFβ point mutations in conserved sequences by renal cortex proteome in diabetic nephropathy, suggesting that it may be involved in diabetic nephropathy by regulating lipid metabolism.Objective:In this study, different rat models of diabetic nephropathy were established to observe the expression and distribution of ETFP and its role in diabetic kidney damage and its mechanisms; overexpression and knockdown technology were used to explore the function of ETFβ in palmitate-induced tubular epithelial cells apoptosis. Evaluation the role of ETFβ in diabetic nephropathy by in vivo and in vitro experiments could provide new ideas for further insight into the pathogenesis of diabetic nephropathy, and new targets for drug candidates.Methods:In vivo experiments are divided into two parts, the first part of which is to explore changes of ETFβ expression in rat cortical kidneys in different diabetic nephropathy models. The model established by intraperitoneal injection of streptozotocin and high-fat diet induced mild renal damage, and severe kidney injury model was made by injection of streptozotocin and unilateral nephrectomy in rats. In addition, two stages of kidney injury progression were observed in spontaneous type 2 diabetic OLETF rats model respectively, at 36 weeks of mild renal impairment, and 56 weeks of age with severe renal impairment. Expression and distribution of ETFβ in renal cortex of normal and model rats were detected by Western blot and immunohistochemistry; PAS staining was done from fixed kidney tissue to eventually analyze the relationship between ETFβ expression changes and diabetic nephropathy. Furthermore, the mechanism of renal injury was analyzed in a rat model of OLETF rats. Apoptosis was detected at two time points,36 and 56 weeks of age respectively, by TUNEL assay, and then protein changes in mitochondrial apoptosis pathway were examined through western blot analysis: cytochrome C, BAX, Bcl-2 and Cleaved caspase-3 expression, in addition to the detection of renal tissue NOX4 expression.In vitro experiments are to explore the effects of ETFβ on fatty acid-induced renal tubular epithelial cell apoptosis and its mechanism. For the first part, we observed the effects of ETFβat low expression on fatty acid-induced apoptosis by knocking down the gene. Firstly, siRNA Oligos targeting at ETFβ gene sequence were designed and screened for higher efficiency of knocking down; and then were transfected into rat proximal tubular epithelial cell line NRK 52E for 24h, followed by adding in the medium to a final concentration of 0.5 mM PA for another 24 h. At last, cells were harvested to detect of ROS generation, BAX and Bcl-2 protein expression, Cleaved caspase-3 expression, mitochondrial membrane potential and apoptosis rate by flow cytometry, and apoptosis by TUNEL assay. Apoptosis and related apoptosis pathways induced by fatty acid or knock down of ETFβcombined with fatty acids stimulate were compared to analyze the influence of ETFβ on lipotoxic apoptosis. On the other hand, by constructing ETFβ recombinant plasmid and transfecting into cells with high expression, mitochondrial apoptosis pathway was tested to observe the change brought by ETFβoverexpression.Results:1. the relationship between ETFβ and diabetic nephropathyETFβexpression was detected in two different rat model of diabetic nephropathy induced by streptozotocin injection. In diabetic nephropathy model induced by high fat diet combined with the streptozotocin injection, the maximum amount of proteinuria was 41.6 mg/24h, and renal tubular damage was mild. In another diabetic nephropathy model induced by unilateral nephrectomy combined with streptozotocin injection, proteinuria were over 300 mg/24h, and renal injury was more serious than the last model, with tubular epithelial cell degeneration, including glass drops and vacuolar degeneration, some tubular dilation or atrophy within protein or protein-like substance casts, interstitial lymphocytes and mononuclear cell infiltration and fibrosis. Decreased ETFβ expression was only seen in the latter severe renal impairment model.In a model of spontaneous type 2 diabetic OLETF rats, there was small amount of proteinuria and mild renal impairment in 36-week-old OLETF rats, whereas it progressed to massive proteinuria at 56 weeks of age, with furtherly aggravated renal pathological damage and a wide range of tubular atrophy and interstitial fibrosis. Also, decreased ETFβ expression occurred only in 56-week-old rats.2. the mechanism of renal tubular apoptosis in severe diabetic kidney injury of ratsApoptotic cells tested by TUNEL were few in kidney tubules at 36 weeks of age, while at 56 weeks there were excessive apoptosis in renal tubular epithelial cells, in which the expression of Cleaved caspase-3 increased only at 56 weeks in the renal cortex, and further study showed that the BAX and of cytoplasma cytochrome C expression were elevated, and meanwhile Bcl-2 expression was decreased. In addition, increased expression of NOX4 was found in renal tissue at 56 weeks. 3. the protection role of ETFβ on reducing renal tubular epithelial cell apoptosis induced by fatty acids and its mechanismThe effect of ETFβ knock down on NRK 52E apoptosis induced by fatty acids was studied.Three pairs of siRNA sequences targeting at ETFβ gene was designed and transfected into NRK 52E cells, one of which showed 90% knockdown rate by western blot, and was used in the following further research. We found increased ROS generation, decreased mitochondrial membrane potential, increased expression of BAX, and decreased Bcl-2, increased Cleaved caspase-3, and a lot of apoptotic cells in NRK 52E cells cultured in 0.5 mM PA for 24h. After ETFβ gene was knocked out, the above change by PA stimulation in mitochondrial apoptosis pathway was furtherly aggravated. ETFβ low expression promotes mitochondrial apoptosis induced by PA.On the other hand, the effect of ETFβ overexpression on apoptosis of NRK 52E induced by fatty acids was studied. After ETFP plasmid sequence was tested as correct cDNA by restrict enzyme digestion and sequencing, the correctly constructed recombinant plasmid was transfected into NRK 52E cells and the fusion protein was successfully expressed in the cells. Compared with PA stimulation control group, high expression of ETFP made mitochondrial membrane potential rise again, the amount of ROS and BAX expression be reduced, Bcl-2 expression be increased, cleaved caspas3 expression reduce, and finally apoptosis rate decline. These results confirmed the overexpressed genes of ETFP alleviate fatty acid-induced apoptosis in renal tubular epithelial cells. Conclusion:The expression of ETFP in kidney cortex was significantly decreased with the progression of renal injury in diabetic nephropathy rats, which suggests a relationship between ETFβ and diabetic nephropathy. Excessive apoptosis of renal tubular epithelial cells was observed, and discovery of increased expression of cytoplasmic cytochrome C and other indicators shows the mitochondrial apoptosis pathway was involved in. Fatty acid can cause decreased expression of ETFP in renal tubular cell line NRK 52E, accompanied by a significant increase in activation of the mitochondrial apoptosis pathway and apoptosis rate. Knock down of ETFβ under palmitate stimulation can significantly increase apoptosis rate; whereas ETFβ overexpression can partially reverse palmitate -induced mitochondrial apoptosis. Thus, we speculate decreased ETFβ expression may be involved in renal tubular epithelial cells apoptosis and the progression of diabetic nephropathy; while ETFβ elevation may alleviate the progression of diabetic nephropathy. |
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