The Effects Of CD36 Palmitoylation On Kidney Injury In Mice Fed On A High-fat Diet

Objective: FAT/CD36 has a close relationship with metabolic inflammation.The expression of CD36 in kidney is related to the pathologic degree of kidney injury,a study about pathogenesis of diabetic nephropathy showed that the expression of CD36 can mediate apoptosis and degeneration of the proximal renal tubular epithelial cells.Palmitoylation is a post-translational lipid modification.,CD36 can also be palmitoylated,Our previous study found that increase of CD36 palmitoylation can promote the inflammation of liver and adipose tissues,abnormal lipid accumulation and cell apoptosis.However,there were no discoveries whether the palmitoylation of CD36 was associated with kidney injury.Here we will explore whether the palmitoylation of CD36 is associated with kidney injury,and then we will further investigate its molecular mechanism.Methods: In the first part,C57BL/6J mice received a high-fat diet were randomly assigned to three groups,the first group(NC)were infected with the empty vector by tail intravenous injection,the second group were injected of lentivirus of WTOE,the third group were infected with lentivirus of AASS by tail vein injection.Then urine samples of 24 hrs were collected for assessment of renal function with metabolic cage.These kidney tissues and blood samples were collected for further assessments after 8 weeks.Western Blot and immunohistochemical staining were used to evaluate the expression of CD36;Palmitoylation of CD36 was detected by ABE assay.HE staining and F4/80 immunohistochemistry were used to detect renal morphology and the inflammatory response.The expression of inflammatory factors and chemokines were determined by real-time PCR.Sirius red staining,immunohistochemical staining of TGF-? and ?-SMA,real-time PCR(real time-polymerase chain reaction,RT-PCR)were used to detected renal fibrosis in mice.In the second part,human renal tubular epithelial cells(HK2)were infected with the lentivirus mentioned above,the cell line were screened with puromycin.In vitro,Palmitoylation of CD36 was detected by ABE assay.The level of inflammatory factors,chemokines and fibrosis factors were detected by RT-PCR.The level of JNK phosphorylation and NF-? B were determined by Western blotting.In the third part,in vitro,DRMs method was used to isolate lipid rafts,and then western blot was used to explore the level of CD36 in DRMs.In vivo and vitro,formation of CD36/TLR4 copolymer was detected by co-immunoprecipitation(CO-IP).Results: The expression of CD36 was comparable between WTOE mice and AA-SS mice,of which either was higher levels of CD36 expression than NC mice.The renal tissues of WTOE mice showed higher palmitoylation of CD36 when compared to that of NC mice,while it was significantly downregulated of CD36 palmitoylation in AA-SS mice than WTOE mice.Urinary levels of 24 h UPRO,?2-MG,and urine volume were elevated significantly in WTOE mice.Interestingly,these damage markers already were decreased in AA-SS mice in comparison with WTOE mice.In WTOE group,the m RNA levels of the cytokines elevated significantly.Compared with WTOE group,the CD3,F4/80,MIP-1 and TNF-? m RNA decreased in AA-SS group.Sirius red staining showed more collagen deposition in WTOE group,and we observed less collagen deposition in AA-SS group.The exacerbation of fibrosis by analyzing the expression of TGF-? and ?-SMA in WTOE group,and this was particularly alleviated in our AA-SS group.Western Blot demonstrated that comparable levels of CD36 between WTOE cells and AA-SS cells,which as expected expressed more CD36 protein than this of NC cells.The results suggested the level of P-JNK and NF-? B increased in WTOE cells.The examination showed CD36 was highly enriched within the visible floating band that also contained caveolin in WTOE group,while the AA-SS protein was less apparent within the DRM fractions and was most expressed in last 6 fractions,positions that were the soluble fractions.While the AASS group and the applyment of 2BP altered the distribution.CD36-TLR4 copolymer were significantly increased in WTOE groups compared with NC groups(P<0.05).Conclusion: Our study suggests that the increased CD36 palmitoylation exacerbates the infiltration of inflammation and fibrosis degree in kidney tissues in mice.After CD36 palmitoylation was agonized,the levels of CD36 in lipid raft,CD36/TLR4,p-JNK and activation of NF-? B were decreased,which implied that the inhibition of CD36 palmitoylation can alleviate inflammation in vivo and in vitro,and protect mice from kidney injury.