Effects Of Down-regulating Of Acetyl CoA Carboxylase2on Intracellular Lipid Accumulation And Epithelial Mesenchymal Transition In High Glucose Cultured Human Renal Proximal Tubular Epithelial Cells

ObjectiveDiabetic nephropathy(DN) is one of the most important and serious complications of diabetic melitus. DN is accounting for50%of all end-stage renal disease(ESRD) patients in the US, and has become the leading cause of ESRD worldwide. Although the mechanisms underlying DN are not fully understood yet, emerging evidences have suggested a role of disregulated lipid metabolism in kidney dysfunction. A recent genome-wide association study in Japanese patients revealed the association between a single nucleotide polymorphism in the acetyl-CoA carboxylase2(ACC2) with the susceptibility to type2diabetes mellitus associated nephropathy. The following analyses done in other ethnic groups suggest the association still holds. ACC2regulates fatty acid oxidation by catalyzing the carboxylation of acetyl-CoA to malonyl-CoA Malonyl-CoA inhibits carnitine palmitoyl transferase I (CPT-I), which is the rate limiting step in fatty acid uptake and oxidation by mitochondria in non-lipogenic tissues. The research shed new lights on the pathophysiology of DN. Tubulointerstitial fibrosis has been established as major pathological feature of diabetic nephpropathy, and epithelial-to-mesenchymal transition (EMT) is considered to be central to the process of tubulointerstitial fibrosis.In this study, we aimed to investigate the effection of ACC2down regulation on intracellular lipid accumulation and EMT in high glucose cultured human renal proximal tubular epithelial cells (HKC), to explore the potential association of ACC2, lipid disregulation, and tubulointerstitial fibrosis.MethodsThe lentivirus vector based on small hairpin RNA (shRNA) targeting ACC2was constructed and delivered to HKC cells cultured in high glucose. Then the cells were divided into5groups:normal glucose group (5.5mmol/L glucose), high glucose group (30mmol/L glucose), high osmotic pressure control group (5.5mmol/L glucose plus24.5mmol/L mannitol), ACC2-shRNA group (30mmol/L glucose plus ACC2-shRNA lentivirous), and NC-shRNA group (30mmol/L glucose plus empty lentivirous carrying the green fluorescent protein as negative control). After treated for96h:①Inteference effeciency of ACC2was detected by Western blot;②Morphological observation under microscope;③Intracellular lipid accumulation was obtained by Oil red O staining;④ACC2and p-ACC2protein level were detected by Western blot;⑤E-cad and a-SMA protein were detected by Western blot.Results①Inteference effeciency of ACC2ACC2protein level was successfully reduced by85%;②HKC cells form High glucose induced morphology change of cultured HKC cells then restored after the interference of ACC2;③Lipid accumulation obtained by Oil red O staining High glucose induced intracellular lipid accumulation of HKC cells, cellular lipid accumulation was reduced after the interference of ACC2;④ACC2and p-ACC2protein level detected by Western blot p-ACC2is the inactive form of ACC2, in the condition of high glucose protein level of p-ACC2reduced(P<0.05) and there was no significant change of ACC2, the activity of ACC2was enhanced; ⑤E-cad a-SMA protein detected by Western blot Protein level of epithelial cell marker protein E-cad was decreased, while interstitial cell marker protein a-SMA was increased(P<0.05), after interference of ACC2, the expression level of E-cad protein was increased(p<0.05) and a-SMA protein was reduced(P<0.05).Conclusion①High glucose enhance the activity of ACC2, induced morphology change and intracellular lipid accumulation of cultured HKC cells.②Interference of ACC2can ameliorate high glucose stimulated cellular lipid accumulation and EMT, then improve renal fibrosis.