Effect Of MsrA On Kidney Fibrosis In Western Diet-fed SR-BI Deficient Mice

Objective:As an important excretory and endocrine organ of human body,the kidney plays an important role in maintaining the internal environment.Lipid metabolism disorder is a major risk factor for metabolic diseases,which related closely to the occurrence and development of atherosclerosis,fatty liver,and diabetes.Recent studies have shown that disorders of lipid metabolism put the body into a state of chronic inflammation and oxidative stress,leading to impaired kidney function.High-density lipoprotein(HDL)has attracted much attention as a prophylactic factor for atherosclerosis(As).In recent years,it was found that HDL dysfunction can exacerbate inflammation and oxidative stress in vivo.However,group B type I scavenger receptors(SR-BI)are functional receptors of HDL and participate in the regulation of lipid metabolism,the deletion of which could cause dyslipidemia,HDL dysfunction and renal tissue damage in mice.Methionyl sulfoxide reductase A(MsrA)is an intracellular-specific reductase,and its antioxidative function is essential to maintain the homeostasis of the redox state in the body.Upon the previous experiments of the laboratory,the level of MsrA was found to have significantly increased in liver,heart,kidney and other tissues of the mice injected with Lv-MsrA-GFP,and can reduce the blood lipid levels,improve the inflammation and oxidation status and delay the progression of AS in ApoE-/-mice.In this study,we used SR-BI-/-mice fed with a high-fat diet as the HDL dysfunctional animal model.Regarding HDL as a regulatory target,we observed its effects in kidney fibrosis and summarized the related mechanisms by over expression of hMsrA interfered with the overall oxygen status of the body to improve the function of HDL.METHODS:Chosen from WT and SR-BI-/-female mice,which were fed to 10-11 months of age,the basal states were compared and the total cholesterol(TC),free cholesterol(FC)and triglyceride(TG)in the plasma were measured by the biochemical enzyme kit;mouse plasma paraoxonase 1(PON1)activity was measured by continuous dynamic assay;kidney morphology changes were observed to analyze renal fibrosis by Masson staining;the expression of oxidative inflammation and fibrosis-related genes in two mouse kidneys,such as tumor necrosis factor alpha(TNF-alpha),type Ⅳcollagen(COL IV),matrix metalloproteinase 2(MMP 2),matrix metalloproteinase 9(MMP 9)and so on,were detected by qPCR.Lv-GFP and Lv-GFP-MsrA,the lentiviral particles,were obtained by lentivirus packaging using the recombinant plasmid pWPI-GFP and pWPI-GFP-MsrA constructed by the laboratory.The SR-BI-/-mice were randomly divided into two groups with each group of seven,and the lentivirus particles were injected to the mice by means of retrobulbar injection.After two weeks of general diet,replacement of a high-fat diet could accelerate the dyslipidemia in the mice.After eight months of high-fat diet,the mice were anesthetized by isoflurane for blood collection and then were dissected to obtain the tissues.The high expression of MsrA in the kidney was verified by qPCR and western blotting;plasma total cholesterol(TC),free cholesterol(FC)and triglyceride(TG)were detected by biochemical enzymatic kit;the continuous dynamic method was used to determine paraoxonase 1(PON1)activity in the plasma;kidney tissue paraffin sections were visualized by Masson staining to observe renal morphological changes and to observe the renal fibrosis in·mice;qPCR and western blotting were used to detect fibrosis-related molecules,such as fibronectin(Fn),type I collagen(COL I),type IV collagen(COL IV),transforming growth factor-(β(TGF-β),matrix metalloproteinase 2(MMP2),matrix metalloproteinase 9(MMP9),and the mRNA and protein expression of inflammatory oxidative-related molecules,such as PON1,IL-1β,IL-6 and so on.Results:Under basal conditions,the content of TC and FC in the plasma of SR-BI-/-mice was significantly higher than that in wild-type mice,while TG had no significant difference,and antioxidant protein PON1 content was significantly lower than that in WT mice.Masson staining showed that the renal fibrosis in SR-BI-/-mice was more severe than that in WT mice(P<0.05).Compared with WT mice,the expression of MsrA was decreased and TNF-a was increased(P<0.01)in SR-BI-/-mice.The expression of Collagen IV(a target of renal fibrosis)was higher in kidneys of SR-BI-/-mice than in WT mice(P<0.05),while the expression levels of proteasome MMP 2 and MMP 9 in extracellular matrix were significantly lower in SR-BI-/-mice than those in WT mice(P<0.05).The SR-BI-/-mice were infected with lentivirus particles Lv-GFP and Lv-GFP-MsrA and euthanized after eight-month high-fat diet.It was confirmed by qPCR and western blotting that renal MsrA was highly expressed and a high expression model was successfully established.Compared with LV-GFP group,TC,FC and TG levels were significantly decreased(P<0.01)and PON]activity was significantly higher(P<0.01)in the plasma of LV-MsrA group.Masson staining showed that the degree of fibrosis was significantly reduced in the kidney of Lv-MsrA group.The results of qPCR showed that the inflammatory cytokines IL-1β,IL-6,and TNF-a mRNA were significantly decreased(P<0.05)in the kidney.The expression of PON1,MMP2 and MMP9 was significantly increased(P<0.05)in Lv-MsrA group,while the relative mRNA expression of Fn,COL 1,COL IV,TGF-β and other fibrosis-related genes were significantly decreased(P<0.05).Conclusion:We have successfully constructed the Lv-GFP-MsrA lentiviral vector and infected SR-BI-/-mice to highly express MsrA in mice.Due to its anti-oxidation,it can regulate the dyslipidemia,reduce the body’s inflammation level,improve the oxidative stress and HDL’s function,extenuate renal fibrotic lesions,and delay the progression of kidney damage.