SGLT2抑制剂对糖尿病肾病大鼠肾脏纤维化及Ⅰ型胶原蛋白表达的影响

Effects of SGLT2 inhibitor on renal fibrosis and type Ⅰ collagen expression in diabetic nephropathy rats

目的 观察钠-葡萄糖协同转运蛋白2(SGLT2)抑制剂对糖尿病肾病大鼠肾功能、肾脏纤维化及肾组织中I型胶原蛋白表达的影响,探讨其肾脏保护作用的可能机制。方法 将28只雄性Wistar大鼠随机分为正常组8只、糖尿病肾病组10只、卡格列净组10只,糖尿病肾病组和卡格列净组采用高脂高糖饲养及腹腔注射链脲佐菌素方法造模。造模成功后,卡格列净组给予卡格列净10 mg/kg灌胃,正常组和糖尿病肾病组给予等量蒸馏水灌胃,3组持续灌胃37 d。检测各组大鼠注射链脲佐菌素第1~3天及灌胃37 d处死前1 d尿蛋白定量;灌胃37 d后取血检测血糖、尿素氮(BUN)、血肌酐(Cr)、三酰甘油(TG)、胆固醇(TC)水平,取肾脏组织进行HE染色及Masson染色,观察肾小球、肾小管及肾间质结构,电镜观察肾脏超微结构,免疫组化检测肾脏组织中I型胶原蛋白表达情况。结果 糖尿病肾病组和卡格列净组注射链脲佐菌素第1~3天及灌胃37 d处死前1 d尿蛋白定量均明显高于正常组(P均<0.05),卡格列净组灌胃37 d处死前1 d尿蛋白定量明显低于糖尿病肾病组(P<0.05)。糖尿病肾病组血糖、BUN、Cr、TG、TC水平均明显高于正常组(P均<0.05),卡格列净组上述各指标水平均明显低于糖尿病肾病组(P均<0.05)。HE染色和Masson染色显示,糖尿病肾病组肾小球部分萎缩、部分增大,毛细血管狭窄,系膜基质增生,部分肾小管上皮细胞肿胀或脱失,肾小球及间质明显纤维化;卡格列净组肾脏病理改变均较糖尿病肾病组轻。电镜下,糖尿病肾病组大鼠系膜细胞和系膜基质轻度增生,可见部分足突融合,基底膜厚度明显厚于正常组(P<0.05);卡格列净组大鼠的足突融合较糖尿病肾病组轻,基底膜厚度明显薄于糖尿病肾病组(P<0.05)。糖尿病肾病组肾组织中Ⅰ型胶原蛋白阳性表达IHC评分明显高于正常组(P<0.05),卡格列净组明显低于糖尿病肾病组(P<0.05)。结论 SGLT2抑制剂能够降低糖尿病肾病大鼠的血糖、血脂水平,减少尿蛋白排泄,改善肾脏功能,减轻肾脏纤维化,可能与其下调致纤维化因子Ⅰ型胶原蛋白的表达相关。

Objective It is to observe the effects of sodium-glucose co-transporter protein 2(SGLT2) inhibitors on renal function, renal fibrosis and type Ⅰ collagen expression in renal tissues of rats with diabetic nephropathy, and to explore the possible mechanisms of its renal protective effects. Methods Twenty-eight male Wistar rats were randomly divided into normal group with 8 rats, diabetic nephropathy group with 10 rats and canagliflozin group with 10 rats. The diabetic nephropathy group and the kaglegiline group were molded by high-fat and high-sugar feeding and intraperitoneal injection of streptozotocin. After successful modeling, the canagliflozin group was given canagliflozin 10 mg/kg by gavage, and the normal group and diabetic nephropathy group were given equal amount of distilled water by gavage, and the three groups were gavaged continuously for 37 d. The urine protein quantification of the rats in each group was measured on day 1-3 of streptozotocin injection and 1 d before sacrificing after 37 days of gavage. Their blood was collected after 37 days of gavage to detect the levels of blood glucose, urea nitrogen(BUN), blood creatinine(Cr), triacylglycerol(TG) and cholesterol(TC), and their kidney tissues were stained with HE and Masson to observe glomerular, tubular and interstitial structures, and observe renal ultrastructure by electron microscopy, and use immunohistochemistry to detect type Ⅰ collagen expression in kidney tissues. Results The urine protein quantification on day 1-3 of streptozotocin injectionand 1 d before sacrificing after 37 days of gavage were significantly higher in the diabetic nephropathy group and canagliflozin group than that in the normal group(P<0.05), and the urine protein quantification 1d before sacrificing after 37 days of gavage was significantly lower in the canagliflozin group than that in the diabetic nephropathy group(P<0.05). The levels of blood glucose, BUN, Cr, TG and TC in the diabetic nephropathy group were significantly higher than those in the normal group(all P<0.05), and the levels of all the above indexes in the canagliflozin group were significantly lower than those in the diabetic nephropathy group(all P<0.05). HE staining and Masson staining showed that the glomeruli were partially atrophied and partially enlarged, with capillary stenosis, thylakoid stroma hyperplasia, swelling or loss of some tubular epithelial cells, and obvious fibrosis in the glomeruli and interstitium;the renal pathological changes in the canagliflozin group were milder than those in the diabetic nephropathy group. Under electron microscopy, the thylakoid cells and thylakoid stroma of the rats in the diabetic nephropathy group were mildly proliferated, and some of the peduncles were fused, and the thickness of the basement membrane was significantly thicker than that of the normal group(P<0.05);the peduncle fusion of the rats in the canagliflozin group was lighter, and the thickness of the basement membrane was significantly thinner than that of the diabetic nephropathy group(P<0.05). The IHC score of positive expression of type Ⅰ collagen in kidney tissue was significantly higher in the diabetic nephropathy group than that in the normal group(P<0.05), and the canagliflozin group was significantly lower than the diabetic nephropathy group(P<0.05). Conclusion SGLT2 inhibitor can reduce blood glucose and blood lipid levels in diabetic nephropathy rats, reduce urinary protein excretion, improve renal function, and reduce renal fibrosis, which may be related to its down-regulation of the expression of fibrotic factor type Ⅰ collagen.