炎性反应激活CXC型趋化因子配体16信号通路进而促进糖尿病肾病进展  

Activation of CXCL16 pathway by inflammation accelerates the progression of diabetic nephropathy

作　　者：胡泽波[1] 马坤岭[1] 张洋[1] 王桂花[1] 刘亮[1] 鲁荐 陈佩佩[1] 倪海峰[1] 刘必成[1] 

机构地区：[1]东南大学附属中大医院肾内科 东南大学附属中大医院肾脏病研究所,南京210009

出　　处：《中华肾脏病杂志》2016年第12期913-921,共9页Chinese Journal of Nephrology

基　　金：国家自然科学基金（81470957）;江苏省自然科学基金（BK20141343）

摘　　要：目的探讨微炎症状态下CXC型趋化因子配体16（CXC chemokine ligand 16,CXCL16）及其受体CXC型趋化因子受体6（CXC chemokine receptor6，CXCR6）途径在糖尿病肾病（diabetic nephropathy，DN）进展中的作用。方法8周龄雄性2型糖尿病小鼠（db／db小鼠）随机分为DN组（n=8）和DN微炎症组（n=8）。参照本课题组既往建立的方法，背部皮下注射10％酪蛋白制备微炎症模型。体外肾小管上皮细胞（HK-2细胞）分为高糖组和高糖＋IL-1β组，观察各组细胞中脂质沉积及细胞损伤的情况；并应用RNA于扰沉默CXCL16的表达，分为高糖＋IL-1β组、高糖＋IL-1β＋siCXCL16组、高糖＋IL-1β＋空载体组，以验证CXCL16信号通路的作用。24h尿蛋白量及尿N-乙酰-β—D-氨基葡萄糖苷酶（NAG）评估8周内小鼠。肾功能变化，电镜观察第8周肾组织的超微结构。Filipin染色及细胞内游离胆固醇定量观察肾组织及HK一2细胞中胆固醇沉积情况；免疫组化、Western印迹检测肾组织CXCL16信号通路相关分子（CXCL16、CXCR6、解整合素样金属蛋白酶10）、纤维化相关分子（纤连蛋白和α平滑肌肌动蛋白）的蛋白表达；实时定量PCR和Western印迹分别检测HK-2细胞中CXCL16信号通路相关分子、纤维化相关分子的mRNA和蛋白表达，及细胞免疫荧光检测HK-2细胞中CXCL16信号通路相关分子表达。结果微炎症组小鼠24h尿蛋白量及尿NAG高于DN组，第8周两组差异均有统计学意义（均P〈0．05）。与DN组比较，微炎症组小鼠电镜下肾小管细胞中空泡增多，同时伴有线粒体水肿、变形以及数量减少；且微炎症组小鼠肾组织中的胆固醇沉积，纤维化相关分子的蛋白表达均高于DN组（均P〈0．05）；进一步研究表明，与DN组比较，微炎症组小鼠肾组织CXCL16信号通路相关分子的蛋白表达均增加（均P〈0．05）。体外高糖＋IL-1β组细胞CXCL16信号通路相关分子、纤维Objective To investigate the potential role of CXC chemokine ligand 16 （CXCL16）/CXC chemokine receptor 6 （CXCR6） pathway in the progression of diabetic nephropathy （DN）. Methods 8- week old male db/db mice were randomly divided into DN group and DN inflamed group. 10% casein was subcutaneously injected to induce the DN mouse model with inflammation. In vitro, HK-2 cells were treated with high glucose （HG）, and IL-1β ＋ HG to investigate the effect of inflammatory stress on HK-2 cells. Further knockdown CXCL16 was mediated by RNA interference to determine the effects of CXC116, then cells were divided into HG ＋ IL-1β group, HG ＋ IL- 1β ＋ siCXCL16 group and HG ＋ IL- 1β ＋ vehicle group. Changes of renal function in mice were assessed by 24 h proteinuria and N- acetyl- β- D- glucosaminidase （NAG） during 8 weeks. The ultra- mierostructure was checked by electron microscopy at 8th week. Lipid accumulation in kidneys and HK- 2 were observed by Filipin staining and quantitative assay of intracellular free cholesterol. The protein expressions of CXC116, CXCR6, a disintegrin and metalloproteinase- 10 （ADAM10）, fibronectin and α smooth muscle actin （α- SMA） in renal tissue were detected by immunohistochemistry and Western blotting. The mRNA and protein expressions of CXC116, CXCR6, ADAM10, flbronectin and α-SMA in HK-2 cells were detected by real-time PCR and Western blotting, and protein expressions of CXC116, CXCR6 and ADAM10 in HK- 2 cells were also tested by cell immunofluorescence. Results Mice in DN inflamed group had higher 24 h proteinuria and NAG than those in DN group, and the differences between two groups shown statistical significance at 8th week （all P 〈 0.05）. Compared with DN mice, DN inflamed mice had more vacuoles within renal tubular ceils, with mitochondrial swelling, deformation and decrease. Lipid accumulation and protein expressions of fibronectin and α-SMA were increased in DN inflamed group when compared with DN group （all P 〈 0.05）. Furthe

关 键 词：糖尿病肾病 炎症 脂代谢障碍 趋化因子CXCL16 

分 类 号：R587.2[医药卫生—内分泌] R692.9[医药卫生—内科学]