高糖对肾脏固有细胞蛋白泛素化修饰及内质网应激的影响

Ubiquitination and endoplasmic reticulum stress of renal intrinsic cells in hyperglycemia

目的观察糖尿病小鼠中肾脏固有细胞（足细胞、近端肾小管上皮细胞）的泛素化修饰和内质网应激的情况。方法建立链脲菌素小鼠糖尿病模型，成模16周后取肾组织，免疫荧光法检测泛素化蛋白和内质网应激蛋白葡萄糖调节蛋白94（GRP94）在肾脏不同区段的表达和分布。流式细胞术分离培养原代小鼠足细胞和近曲小管上皮细胞，高糖（30mmol／L）$1J激不同时间（1d、3d、7d），Western印迹检测蛋白泛素化蛋白和GRP94蛋白的表达。结果糖尿病小鼠出现微量蛋白尿和轻度系膜区增宽，泛素化蛋白在肾小球主要分布于足细胞，并散在分布于肾小管上皮（近端、远端）的胞质和胞核，糖尿病组肾小球和近端肾小管的泛素化蛋白表达均高于对照组（均P〈0．05）。高糖刺激后，原代足细胞和近曲小管上皮细胞泛素化蛋白均呈时间依赖方式升高，刺激3d、7d高糖组均高于正常糖组（均P〈0．05）。GRP94散布在肾小球和肾小管，糖尿病组肾小球GRP94表达均高于对照组（P〈0．05），但近端肾小管GRP94表达与对照组差异无统计学意义。足细胞GRP94表达在高糖刺激后随时间延长而升高，3d、7d时高糖组与正常糖组差异均有统计学意义（均P〈0．05）；近曲小管上皮细胞在高糖刺激后GRP94未发生明显变化，与正常糖组比较差异无统计学意义。结论高糖可诱导肾组织蛋白泛素化水平增加。而足细胞内的蛋白质代谢失衡和内质网应激可能参与糖尿病肾病早期足细胞损伤。

Objective To investigate the effects of hyperglycemia on ubiquitination and endoplasmic reticulum stress in renal intrinsic cells （podocytes and proximal tubular epithelial cells） and its role in patbogenesis of diabetic nephropathy. Methods Diabetic mice were induced by streptozotocin injection. After 16 weeks of hyperglycemia, immunofluorescence was used to detect the expressions of ubiquitination and glucoseregulating protein 94 （GRP94） in renal cortex and medulla area of kidney sections. Primary mouse podocyte and proximal tubular epithelial cells were isolated by flow cytometry, and exposed to 30 mmol/L glucose for indicated time （1 d, 3 d and 7 d）. Their ubiquitination and GRP94 expressions were evaluated by Western blotting. Results Diabetic mice presented microalbuminuria and slightly widened mesangium was found in glomerular area. Ubiquitinated proteins, mainly localized in podocytes and tubular epithelial cells, exhibited an apparently higher expression in diabetic mice than control mice （all P 〈 0.05）. Hyperglycemia promoted the ubiquitination in a time- dependent manner. Compared with their normal cells, primary mouse podocyte and primary tubular epithilial cells treated with high glucose for 3 d and 7 d showed increased ubiquitinated protein （all P 〈 0.05）. GRP94 was interspersed in podocytes and proximal tubular epithelial cells. Expression of GRP94 was significantly increased in glomerular area of diabetic mice and podocyte with 3 and 7 day-high glucose as compared with those in their control groups （all P 〈 0.05）. GRP94 expression had no significant change in tubular area and tubular epithilial cells treated with high glucose. Conclusions Hyperglycemia may lead to accumulation of ubiquitinated proteins in intrinsic kidney cells. The imbalance of protein homeostasis in podocyte may contribute to podocyte injury during the onset of diabetic nephropathy.