巨噬细胞Bruton酪氨酸激酶基因特异性敲除减轻糖尿病小鼠肾脏损害的作用及机制

Bruton's tyrosine kinase knockout in macrophages attenuates diabetic kidney disease in the streptozotocin-induced mice

目的探讨巨噬细胞Bruton酪氨酸激酶(Btk)基因特异性敲除对糖尿病小鼠肾脏损害的作用及机制。方法选用巨噬细胞Btk基因特异性敲除(Btk-/-)小鼠和C57BL/6N小鼠链脲菌素(STZ,50 mg/kg)造模成功后随机分为正常组、糖尿病组、Btk-/-组和Btk-/-糖尿病组。12周后测定小鼠一般指标,观察肾组织病理学改变,免疫荧光检测肾组织巨噬细胞标志物CD68表达,免疫组化检测足细胞标志物WT1和Nephrin的表达,Western印迹检测细胞外基质纤维连接蛋白(FN)、IV型胶原(IV-Col)及转化生长因子β1(TGF-β1),巨噬细胞激活标志物诱导型一氧化氮合酶(iNOS)、磷酸化(p)-Btk,炎性因子白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)和丝裂原活化蛋白激酶(MAPK)信号通路p-p38MAPK、p-JNK、p-ERK以及核因κB(NF-κB)通路p-p65、p-IκB蛋白水平变化;实时荧光定量PCR检测炎性因子IL-1β、TNF-α、单核细胞趋化蛋白1(MCP-1)mRNA表达。结果与糖尿病组相比,Btk-/-糖尿病组尿白蛋白明显减少,肾组织损伤明显减轻,肾脏巨噬细胞CD68表达明显减少,足细胞标志物WT1及Nephrin表达明显增加,细胞外基质FN、IV-Col及TGF-β1表达明显减少,炎性因子IL-1β、TNF-α及MCP-1表达明显降低,p-JNK、p-ERK、p-p38MAPK及p-p65、p-IκB表达明显下调(均P<0.05)。结论巨噬细胞Btk特异性敲除可能通过MAPK、NF-κB通路降低炎性因子的表达从而对糖尿病小鼠肾脏起到保护作用。

Objective To investigate whether Bruton's tyrosine kinase knockout(Btk-/-)in macrophages attenuates diabetic kidney disease in the streptozotocin(STZ)-induced mice.Methods Macrophages-specific Btk-/-mice and control mice(C57BL/6N)were randomly divided into WT group,diabetic group,Btk-/-group and Btk-/-diabetic group.The diabetic models were induced by STZ(50 mg/kg).After 12 weeks,relevant biochemical parameters and the histological changes of kidneys were detected.The expression of macrophages marker CD68 were detected by immunofluorescence,and the immunohistochemistry was employed to detect the expression of WT1 and Nephrin on renal podocytes.In addition,the expression of fibronectin(FN),collagen type IV(IV-Col),transforming growth factor-β1(TGF-β1),iNOS,phospho(p)-Btk,interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),MAPK and NF-κB signaling pathway were detected by Western blotting.RT-PCR was used to detect the mRNA of IL-1β,TNF-αand monocyte chemotactic protein-1(MCP-1).Results Compared with diabetic group,the mice in Btk-/-diabetic group had reduced albuminuria and attenuated kidney histopathology significantly,significantly increased WT1 and Nephrin,significantly decreased expression of CD68,FN,IV-Col and TGF-β1,and these changes were correlated with decreased of renal inflammatory cytokines such as IL-1β,TNF-α,MCP-1 and down-regulating MAPK and NF-κB signaling pathway(all P<0.05).Conclusion Macrophages-specific Btk-/-may protect the kidney of diabetic mice by reducing the expression of renal inflammatory cytokines in MAPK and NF-κB signaling pathway.