摘要目的 探讨胰激肽原酶(PKK)对2型糖尿病(DM)肾病的作用及分子机制. 方法 将20只4周龄体重22～28 g的健康雄性db/db小鼠随机分为2组,PKK干预组(n=10,PKK腹腔注射60U·kg-1·d-1)和DM组(n=10,腹腔注射生理盐水1 ml/g).以同窝出生的db/m小鼠为正常对照组(n=8,腹腔注射生理盐水1 ml/g).每周检测小鼠的血糖和体重,每月监测小鼠收缩压与舒张压.干预16周后处死小鼠,留取肾脏组织标本.光镜观察各组小鼠肾小管病理学变化,电镜观察肾小管超微结构的改变.应用免疫组化方法检测肾脏组织中上皮型-钙黏附蛋白的表达水平,实时PCR方法检测小鼠肾脏组织白细胞介素1β(IL-1β)、纤维连接蛋白的mRNA表达水平.采用SPSS16.0软件进行统计学分析,多组资料用单因素方差分析. 结果 (1)干预16周后,DM组和PKK干预组小鼠血糖、体重均高于正常对照组小鼠,肾重/体重指数明显低于正常对照组,其差异有统计学意义[对照、DM、PKK组血糖分别为(5.4±0.6)、(27.4±4.6)、(26.9±3.0) mmol/L,F=86.35,P＜0.01;体重分别为(24.3±3.1)、(42.6±4.8)、(41.5±2.6)g,F=38.82,P＜0.01;肾重/体重指数分别为12.52±2.53、9.01±1.25、9.83±1.18,F=7.27,P＜0.01].DM与PKK组之间差异无统计学意义.3组之间的收缩压与舒张压差异均无统计学意义.(2)电镜提示PKK组DM小鼠肾脏近端与远端小管上皮细胞线粒体肿胀的情况、线粒体数目、近端小管上皮细胞的脂代谢障碍均较DM组改善.(3)免疫组化分析提示,与正常对照组相比,DM组E钙黏附蛋白表达明显减少,PKK干预组显著升高,分别为18.8±1.8、15.8±1.9、19.7±2.9 (F=9.70,P＜0.01).3组IL-1β的表达分别为2.30±1.22、4.14±3.40、0.92±0.59(F=11.86,P＜0.01);纤维连接蛋白的表达分别为1.67±0.53、8.45±2.02、2.04±0.79 (F=52.66,P＜0.01). 结论 胰激肽原酶对2型糖尿病小鼠的肾小管具有保护作用,且机制可能与减轻炎症、抗纤维化、减少肾小管上皮向间充质细胞转化有关.

abstractsObjective To explore the specific mechanism of pancreatic kallikrein(PKK) renal disease in type 2 diabetic mice.Methods A total number of 20(4 week-old) db/db mice were randomly divided into 2 groups depending on their body weights and blood glucose.One group was treated with PKK (PKK group,n=10,60 U· kg-1·d-1,intraperitoneal injection),while the other was given saline (DM group,n=10,1 ml/g,intraperitoneal injection).In addition,littermates db/m mice (NC group,n=8,1 ml/g,intraperitoneal injection) were given saline as non-diabetic control group.Blood glucose and body weight were measured weekly,and both systolic and diastolic blood pressure were monitored monthly.After treated for 16 weeks,mice were sacrificed and kidney tissues were collected.Light microscopy and transmission electron microscope were used to examine the tubular pathological changes and the ultrastructure,respectively.Immunohistochemistry were performed to detect the expression of epithelial-cadherin (E-cadherin).Real-time PCR was applied to determine the gene expression of interleukin-1β (IL-1β) and fibronectin in the whole kidney.Results (1) After 16 weeks,both blood glucose and body weight in DM and PKK group were significantly higher than NC group,and the kidney weight / body mass index was lower than NC group (blood glucose for NC,DM and PKK group were (5.4±0.6),(27.4±4.6),(26.9±3.0) mmol/L,expectively (F=86.35,P＜0.01); Body weights were (24.3±3.1),(42.6±4.8),(41.5 ±2.6) g,expectively (F=38.82,P＜0.01); the kidney weight/body mass index were 12.52±2.53,9.01±1.25,9.83±1.18,expectively (F=7.27,P＜0.01)).However,there wasn't any difference between DM and PKK group.Both systolic and diastolic blood pressures showed no differences between any two groups.(2)The results of transmission electron microscope showed that PKK treatment could significantly improve cellular mitochondrial swelling which was present in both the proximal and distal tubular epithelial cells in DM group.Meanwhile,PKK could also increase the number of mitochondria and ameliorate lipid metabolism disorders in proximal tubular cells as compared to DM group.(3) Compared with NC group,E-cadherin expression was significantly decreased in DM group,while the PKK treatment significantly increased the expression of E-cadherin.Quantitative PCR showed that PKK could significantly decrease the gene expression of IL-1β and fibronectin as compared with DM group.Conclusions PKK has protective effects on tubules of db/db mice,which was independent of improvements in blood pressure and blood glucose.The renoprotective effect of PKK might be mediated by reducing inflammation,anti-fibrosis and decreasing the epithelial to mesenchymal transition.