Advanced Glycation End Products Activates A Fibrogenic Program In Diabetic Kidney Disease

Objective: Diabetic kidney disease(DKD),one of the most common complications in type 1 or type 2 diabetic patients,may induce renal tubular epithelial-to-mesenchymal transition(EMT),resulting eventually in renal fibrosis.Renal tubular EMT plays an important role in the development of renal fibrosis and then is known as readout for the progressive renal fibrosis.Renal fibrosis is based on many factors including advanced glycation end products(AGEs),mitochondrial dysfunction,autophagic dysfunction and activation of proinflammatory signaling,but the precise mechanisms remain unclear.Cluster of differentiation 36(CD36),a transmembrane glycoprotein,plays an important role in membrane transport of long chain fatty acids and metabolic inflammation.Accumulating evidences have suggested the pivotal role of CD36 in the pathogenesis and progression of kidney disease.AS a major substrate for palmitoylation,CD36 can be post-translationally modified by palmitoylation,which regulates CD36 biological functions.In the present study,we aimed to investigate whether AGES lead to renal tubular EMT and renal fibrosis by increasing the palmitoylation of CD36 in DKD and study its mechanisms.Methods: A total of 10 male C57BL/6J mice were randomly assigned into DKD group or a control group,5 mice in each group.Mice in DKD group were injected intraperitoneally with streptozotocin(STZ)to create DKD mouse model.N?-(carboxymethyl)-lysine(CML),one of the characteristic active AGEs,was detected in serum and kidney tissue of the mice by Enzyme-linked immunosorbent assay(ELISA).The levels of random blood glucose,blood urea nitrogen,serum creatinine,serum albumin and urinary albumin were also examined.The degree of kidney fibrosis was visualized by Haematoxylin-eosin(HE)and sirius red staining.Immunohistochemistry was performed to assess the expression of ?-SMA,E-cadherin,DHHC4 and DHHC6.Real time PCR was performed to check the gene expression levels of collagens(Collagen I,Collagen IV)in kidney tissue of the mice.HK-2 cells were treated with CML or CML plus 2-Bromopalmitate(2-BP).Stable transfection of HK-2 cells was carried out by co-transfection with empty vector,wildtype CD36 c DNA(WTOE)or the non-palmitoylated AA-SS mutant.We used RNAi technologies to down-regulate the Sel N expression in HK-2 cells.Western blot analysis was used to determine the expression levels of CD36,?-SMA,E-cadherin,vimentin,Sel N,DHHC4 and DHHC6.We used acyl-biotinyl exchange(ABE)to detect the palmitoylation of target proteins,including CD36,DHHC4 and DHHC6.Immunoprecipitation was performed to check the level of Sel N binding to DHHC6.Results: In our study,the STZ-induced diabetic mice were associated with higher blood glucose levels,low level of serum albumin,elevated blood urea nitrogen,decreased body weight and increased proteinuria.Over time,the diabetic mice showed typical symptoms of diabetes,including polydipsia,polyuria and polyphagia.Taken together,these data suggested that DKD occurred in the STZ-injected mice.Mice in the DKD group developed glomerular mesangial matrix expansion,whereas control mice did not develop glomerular mesangial matrix expansion.Collagen deposition was significantly increased in the renal tissue of diabetic mice compared with control mice.In comparison with the renal cortical tubules of control mice,the expression of ?-SMA and Sel N in the renal cortical tubules of diabetic mice was increased,while the expression of E-cadherin was significantly decreased.The expression levels of Col-I m RNA and Col-IV m RNA in the renal tissue of diabetic mice were also increased compared to control mice.Interestingly,we found that the palmitoylation of CD36 was significantly increased in the renal tissue of diabetic mice compared with control mice.We then found that the expression levels of ?-SMA and vimentin were significantly higher in CML-treated HK-2 cells,but levels of E-cadherin were decreased.2-BP,a palmitoyltransferase inhibitor,which could inhibit protein palmitoylation,had an antagonist effect on CML-induced EMT,decreasing the expression levels of ?-SMA and vimentin and increasing the expression levels of E-cadherin in CML-treated HK-2 cells.There were no changes in CD36 protein expression compared to the control,but CML increased the palmitoylation of CD36,and this response was decreased by 2BP in HK-2 cells.After treatment with CML,the expression levels of ?-SMA and vimentin were significantly higher,but levels of E-cadherin were lower in WTOE HK-2 cells compared with AA-SS HK-2 cells.After treatment with CML,DHHC6 activity is increased,but no differences were observed in DHHC4 activity,DHHC4 or DHHC6 expression levels in HK2 cells.Next,we found that the expression levels of Sel N were increased by CML and Sel N associated with DHHC6 but not with DHHC4 to form a complex.Sel N knockdown in HK-2 cells reduced CD36 palmitoylation and DHHC6 activity,then decreased the expression levels of ?-SMA and vimentin and increased the expression levels of E-cadherin in CML-treated HK-2 cells.Conclusion: AGES levels and palmitoylation of CD36 are associated with the activation of fibrogenic program in kidney of experimental diabetic mice.AGES promote renal tubular EMT by increasing CD36 palmitoylation.AGES up-regulate the activity of DHHC6 through DHHC6/Sel N complex formation,increasing the palmitoylation of CD36 in renal tubular epithelium.