The Mechanisms Of CD36 In SAA-induced Renal Amyloidosis

Objective: Renal AA amyloidosis is a disease caused by a variety of factors,which is caused by the deposition of insoluble amyloid protein in the cell and outside,resulting in the damage of the tissue structure and function.Nephrotic syndrome is the main clinical manifestation,which can lead to renal failure and death.Better control of inflammation can significantly improve the prognosis,but the pathogenesis of this disease is unclear.There was no effective treatment for the disease.CD36 is one of the scavenger receptor in the B family,named fatty acid translocase(FAT),too.is an important membrane protein that connects the lipid metabolism and inflammation.Recent research demonstrated that CD36 is a novel serum amyloid A(SAA)receptor mediating SAA proinflammatory activity.The aim of this study was to investigate whether CD36 is involved in renal damage induced by AA amyloidosis and the underlying mechanism.Methods: In vivo,we eight-weeks old male wild type(WT)and CD36-/-mice were randomly treated with either 0.5ml 10% casein subcutaneous injection(Casein(+)group)or normal saline(Control group)every other day.After 15 weeks,mice were killed and the serum,urine and kidney tissues were collected.The biochemical indexes of blood were detected.SAA was determined by enzyme-linked immunosorbent assay(ELISA).The expression of CD36,LC3 and AMPK was detected by q-PCR,Western blot and immunohistochemistry.Congo red and Potassium Permanganate staining was used to detect mouse renal amyloidosis.The pathological changes were observed by TEM,H&E and MASSON staining.Autophagy was detected by electron microscoy.The co-localization of two proteins was visualized by confocal immunofluorescence microscopy.Chip experiment was used to detect protein and gene promoter interaction.In vitro,human mesangial cells(HMCs)and human renal proximal tubular cells(HK2)were used.CD36 siRNA HMCs transient cell line were established by using small interfering RNA(siRNA)method.The HMCs and HK2 cells were treated by SAA at 1ug/ml in the absence or presence of the autophagy inhibitor Wortman(200 nmol/L)treatment of HK2 cells and HMCs cells(NCi+SAA group,CD36i+SAA group,NCi+SAA+wortman group,CD36i+SAA+Wortman group).Detection of AA deposition in cells was detected by immunofluorescence.Inflammatory and fibrotic gene expression in HK2 cells and HMCs cells were examined by Q-PCR.The AMPK inhibitor Compound C(10 umol/L)were used to HK2 and HMCs cells,which were divided into four groups: NCi+SAA group,CD36i+SAA group,NCi+SAA+Compound C group,and CD36i+SAA+Compound C group.Autophagy was detected by autophagy kit,and the expression of LC3 was detected by RT-PCR.The effect of AMPK suppression on HK2 and HMCs under SAA were detected by autophagy and LC3 expression.Results: In vivo,casein subcutaneous injection could increase the serum content of SAA in WT mice and AA amyloidosis as visualized by positive Congo red staining in the kidney setion from WT mice.The expression of CD36 was up-regulated in casein subcutaneous injection mice detected by immunohistochemistry and Western blot.Using Congo red staining under light microscope and polarized light,we found that CD36 deficiency could improve renal amyloidosis induced by casein.Potassium Permanganate after Congo red staining were negative,suggesting that the type of renal amyloid induced by casein was AA amyloidosis.In addition,we also found the obvious amyloid deposition in the kidney from WT casein(+)group,while the CD36-/-Casein(+)group was negative.The mice in WT Casein(+)group showed hypoproteinemia,hyperlipidemia,increased urine volume and proteinuria,which could be improved by CD36-/-.H&E,MASSON staining,Western blot,Q-PCR results showed that renal fibrosis and inflammation of mice in CD36-/-Casein(+)group was significantly lower than WT Casein(+)group.The transmission electron microscope demonstrated that autophagosome was increased in renal tubule from CD36-/-Casein(+)group;AA deposition in kidney tissue from CD36-/-Casein(+)group was reduced,but the expression of LC3 was up-regulated.The colocalization of LC3 and AA was observed in the kidney.The expression of p-AMPK in the mice kidney tissues from WT(+)group was significantly lower than that in CD36-/-Casein(+)group.The expression of LC3 in kidney tissue from CD36-/-Casein(+)group was higher than that in Casein(+)WT group.In cell culture experiments,knock down CD36 expression in cells increased autophagy significantly,but decreased AA accumulation,inflammation,and renal fibrosis index after the treatment with SAA.Wortman,an autophagy inhibitor abolished above changes induced by SAA.In addition,the expression of autophagy marker were decreased significantly after treatment with AMPK inhibitor Compound C in all groups,and there was no significant difference among these groups.Conclusion: 1.Casein induces CD36 expression and the occurrence of renal AA amyloidosis in WT mice.2.the deficiency of CD36 can significantly improve the deposition of amyloid fibrils and renal damage in renal tissue.3.the deletion of CD36 may regulate the production of autophagy and eliminate excessive SAA to protect kidney function.4.deletion of CD36 may play an important roles in the activation of AMPK and up regulation of LC3 promoter activity.