腹腔注射帕立骨化醇的糖尿病肾病大鼠肾脏病理变化及自噬相关蛋白表达观察

Renal pathological changes and autophagy-related protein expression in diabetic nephropathy rats after intraperitoneal injection of paricalcitol

目的 观察腹腔注射帕立骨化醇的糖尿病肾病(DN)大鼠肾脏病理变化及自噬相关蛋白[Klotho蛋白、微管相关蛋白1轻链3(LC3)-Ⅱ/LC3-Ⅰ)]表达情况。方法 18只雄性SD大鼠随机分为对照组(A组)、DN组(B组)、DN+帕立骨化醇组(C组),B、C组大鼠采用一次性腹腔注射链脲佐菌素(STZ)60mg/kg的方法制作DN模型。C组大鼠每周3次腹腔注射低剂量帕立骨化醇0.4μg/kg,A、B组同期腹腔注射等量生理盐水。各组大鼠腹腔注射12周后,取内眦静脉血后,放入代谢笼中禁食水并收集24h尿液,然后处死大鼠,取大鼠肾脏组织分离肾皮质备检。第12周末称量各组大鼠体质量;取内眦静脉血,采用苦味酸法检测大鼠血肌酐(Scr),采用快速血糖仪检测空腹血糖(FPG);取收集的24h尿液,采用比浊法测定尿蛋白。取大鼠肾脏组织分离肾皮质,制备切片,PASM染色后,显微镜下观察各组大鼠肾组织病理形态学变化;采用免疫组织化学法检测各组大鼠肾小管间质E-cadherin;采用免疫荧光法检测各组大鼠肾小管上皮细胞Klotho蛋白;采用Westernblotting法检测各组大鼠肾皮质LC3-Ⅱ/LC3-Ⅰ。结果 与A组相比,B、C组大鼠体质量降低,Scr、FPG及尿蛋白水平均显著升高(P均<0.05);与B组相比,C组大鼠Scr及尿蛋白水平均显著降低(P均<0.05)。A组肾小球及肾小管间质无明显病理改变;B组肾小球体积增大,系膜细胞基质增多,肾小管细胞肥大,出现泡沫样变、小灶性萎缩、轻度炎性细胞浸润;C组肾小管及肾小管间质损害较B组明显减轻,肾小管萎缩及间质纤维化不明显。A、B、C组大鼠肾小管间质E-cadherin相对表达量分别为222±37、139±19、192±38,肾小管上皮细胞Klotho蛋白荧光强度分别为3.5±0.3、1.5±0.3、2.8±0.6,肾皮质LC3-Ⅱ/LC3-Ⅰ分别为1.5±0.2、1.0±0.2、2.8±0.6,组间相比,P均<0.05。结论 帕立骨化醇能改善DN大鼠肾损伤,其机制与升高肾小管间质E-cadherin缓解EMT、上调自噬相关蛋白(肾小管上皮细胞Klotho蛋白、肾皮质LC3-Ⅱ/LC3-Ⅰ)表达增强自噬作用有关。

Objective To observe the renal pathological changes and autophagy-related proteins [Klotho and microtubule-associated protein 1 light chain 3 (LC3)-Ⅱ/LC3-Ⅰ] expression in diabetic nephropathy (DN) rats after intraperitoneal injection of paricalcitol. Methods Eighteen male Sprague-Dawley rats were randomly divided into three groups: control group (group A), DN group (group B), and DN+paricalcitol group (group C). DN models were established by a single intraperitoneal injection of streptozotocin (STZ) 60 mg/kg in the groups B and C. Rats in the group C were injected intraperitoneally with paricalcitol at a low-dose of 0.4 μg/kg (3 times a week). Rats in the group A and B were treated with the same amount of normal saline. After 12 weeks of treatment, inner canthus blood was used for measuring the level of serum creatinine (Scr) by picric acid method, and fasting blood glucose (FPG) by rapid glucometer. Metabolic cages were used for collecting 24 h urine, and 24 h urine protein was determined by turbidimetric method. Then all rats were sacrificed for measurement of body weight and renal cortex. PASM staining was used to observe the pathological changes of the kidneys. E-cadherin expression in the renal tubular interstitial was examined by immunohistochemistry. Klotho in renal tubular was detected by immunofluorescence. The renal cortex LC3 -Ⅱ/LC3 -Ⅰ was detected by Western blotting. Results Compared with group A, the body weight of rats decreased, and Scr, FPG and urinary protein levels significantly increased in the groups B and C (all P <0.05). Compared with group B, Scr and urinary protein levels decreased in the group C (both P <0.05). There were no significant pathological changes in glomerulus and tubulointerstitium in the group A. In the group B, glomerular volume increased, mesangial cell matrix increased, and renal tubular cell hypertrophy, foam-like degeneration, focal atrophy, and mild inflammatory cell infiltration were found. Renal tubular atrophy and interstitial fibrosis were not obvious in the group C as compared with those of group B. The relative expression levels of E-cadherin in the groups A, B and C were 222±37, 139±19, and 192±38, respectively. The fluorescence intensities of Klotho were 3.5±0.3, 1.5±0.3, and 2.8±0.6, respectively. The LC3 -Ⅱ/LC3 -Ⅰ ratios were 1.5±0.2, 1.0±0.2, and 2.8±0.6, respectively;significant difference was found between groups (all P <0.05). Conclusion Paricalcitol can improve renal injury in DN rats, which may be related to the elevation of E-cadherin in renal tubulointerstitial, thereby alleviating EMT, and up-regulation of autophagy-related proteins expression (renal tubular epithelial cells Klotho, renal cortex LC3-Ⅱ/LC3-Ⅰ) and enhancement of autophagy.