The Role And Mechanism Of Bruton Tyrosine Kinase By Regulating NLRP3 In Inflammation Of Diabetic Nephropathy

Background and purpose Diabetic nephropathy(DN)is the one of the main causes.of end-stage renal failure.In recent years,numerous evidences have shown that the infiltration and activation of macrophages in the kidney trigger the release of inflammatory factors and accelerate the development of DN.Bruton tyrosine kinase,a non-receptor tyrosine kinase,is a member of the Tec family.It is expressed mainly in the myeloid cell surface,such as B lymphocytes,basophils,monocytes.Previous studies have confirmed that Btk is involved in the regulation of cell division,proliferation and apoptosis,and plays an important role in the occurrence of tumors,ischemia-reperfusion injury,rheumatoid arthritis and other diseases.Our previous experiments showed that the expression of p Btk was increased in macrophages cultured with high glucose,and Btk inhibitors could inhibit the activation of macrophages under high glucose environment.NLRP3 inflammasome belongs to the NLRs family and is located in the cytoplasm,including NLRP3,ASC and caspase-1.NLRP3 recruits ASC through PYD domain,and then activates the caspase-1 precursor(45).It cleaved itself into the activated form of caspase-1(20),which can hydrolyze and cut the IL-1?precursor into the mature form of IL-1?.This study intends to adopt the STZ-induced diabetes model in vivo and use Btk gene knockout mice and other means to determine whether there is the expression and activation of NLRP3 inflammsome in diabetic nephropathy patients,to explore the regulatory effect of Bruton tyrosine kinase on the kidney NLRP3 of diabetic mice,and to seek new means to improve the inflammation of diabetic kidney.Methods SPF-class C57BL/6J mice and healthy male C57BL/6J myeloid cell btk gene knockout micewere selected and injected intraperitoneally with 50mg/kg streptozotocin.The control group was injected with the corresponding volume of sodium citrate solution for5 consecutive days.Blood glucose was detected one week later.The mice were randomly divided into normal control group,diabetes model group,Btk-/-control group and Btk-/-diabetes model group,there were seven mice in each group.Blood,urine and kidney tissue samples of mice were collected at the end of 12 weeks for the measurement of blood glucose,kidney weight,body weirht and 24-hour urinary albumin excretion rate.Pathological damage of renal tissue were observed by light microscope.Immunohistochemical method was used to detect activated macrophages(i NOSpositive cells),the protein of NLRP3,TNF-?,MCP-1 and IL-1?,method was used to detect CD68,i NOS,Btk,NLRP3 co-expression,Western blot was used to detect the protein expression of Btk,p-Btk,i NOS,NLRP3,caspase-1,ASC,IL-1?,TNF-?,MCP-1 in renal tissue,and real-time quantitative PCR was used to detect the m RNA expression of TNF-?,MCP-1,IL-1?.The co-immunoprecipitation was used to detect the interaction between ASC and NLRP3.Results1.Twelve-week-old diabetes model group the blood glucose,renal weight/body weight and 24-hour urine protein excretion rate of the mice were significantly higher than those in the normal control group(p<0.05),and the renal weight/body rate and 24-hour urine protein excretion rate of the mice in the Btk-/-diabetes model group were significantly lower than those in the diabetes model group(p<0.05).2.Compared with the normal control group at the end of 12 weeks,the mesangial expansion index and renal tubular interstitial injury index score of the diabetic model group were significantly increased(p<0.05),and the pathological changes of the Btk-/-diabetic model group were significantly improved(p<0.05).3.Immunohistochemistry showed that the expression of NLRP3 in renal tissue of the model group of diabetes mellitus was significantly higher than that of the normal control group at the end of twelve weeks,while the expression of NLRP3 in renal tissue of the model group of Btk-/-diabetes mellitus was significantly lower than that of the model group of diabetes mellitus(p<0.05).4.Laser confocal imaging showed that the expression of CD68,Btk and NLRP3 in renal tissues of the diabetic model group was significantly higher than that of the normal control group,while the expression of CD68,Btk and NLRP3 in renal tissues of the Btk-/-diabetic model group was significantly lower than that of the diabetic model group.5.Westernblot showed that the relative protein expression levels of p-Btk,i NOS,NLRP3,caspase-1,ASC,IL-1?,TNF-?,and MCP-1 in the 12-week diabetes model group were significantly higher than those in the normal control group(p<0.05),while that in the Btk-/-diabetes model group was significantly lower than that in the diabetes model group(p<0.05).6.Real-time quantitative PCR results showed that the expression of TNF-?,MCP-1 and IL-1?m RNA in renal tissues of the diabetic model group was significantly higher than that of the normal control group(p<0.05),while that of the Btk-/-diabetes model group was significantly lower than that of the diabetic model group(p<0.05).7.Co-immunoprecipitation showed that the combination of ASC and NLRP3 in the diabetic model group was significantly higher than that in the normal control group(p<0.05),while the Btk-/-diabetes model group was significantly lower than the diabetic model group(p<0.05).Conclusions The protein expression of p-btk,i NOS,NLRP3,caspase-1,ASC,IL-1?,TNF-?,MCP-1was up-regulated and the relative expression of TNF-?,MCP-1,IL-1?was increased in the renal tissue of diabetic mice.The knockout of Btk gene could down-regulate the expression of NLRP3 signaling pathway and inflammatory factors,suggesting that Btk may reduce the damage of diabetic kidney by down-regulating the inflammatory response of NLRP3.And the possible mechanism is to influence the interaction between ASC and NLRP3.