人组织激肽释放酶基因治疗对2型糖尿病大鼠胰岛素抵抗和肾脏并发症的影响

Effect of human tissue kallikrein gene therapy on insulin resistance and nephropathy in type 2 diabetic rats

目的探讨人组织激肽释放酶（HK）基因对2型糖尿病大鼠胰岛素抵抗和慢性肾脏并发症的治疗作用及其机制。方法在雄性Wistar大鼠以高脂高糖饲料加小剂量链脲佐菌素建立2型糖尿病动物模型。以重组腺相关病毒为载体介导HK基因（HK组）或对照基因LacZ（LacZ组）在糖尿病大鼠体内表达，观察实验动物血压、血糖、血胰岛素、尿微量白蛋白、尿渗透压的变化。计算稳态模型评估的胰岛素抵抗指数（HOMA-IR）、内生肌酐清除率（Ccr）和尿白蛋白排泄率（UAER）。Western印迹法检测肝脏、骨骼肌磷脂酰肌醇3激酶（PI3K）的110亚基（p110）和蛋白激酶B（Akt／PKB）第308位苏氨酸（pThr308）的磷酸化水平。肾脏切片作形态学分析。结果HK组第2周开始出现血压下降，并一直持续到实验结束（12周时），而LacZ组血压无明显下降。HK组和LacZ组大鼠空腹血糖无明显变化[（13．09±3．01vs13．58±2．88）mmol／L]，但两组空腹血胰岛素水平[（8．19±2．45vs13．85±3．76）mIU／L，P〈0．01]和HOMA—IR（4．76±0．33vs8．36±0．48，P〈0．01）差异有统计学意义。HK组大鼠肌肉和肝脏p110和pThr308的表达明显增加。HK组UAER[（7．90±2．76vs19．07±9．17）mg／24h，P〈0．01]和Ccr（0．16±0．12vs0．43±0．25，P〈0．01）明显低于LacZ组，而尿渗透压明显高于LacZ组[（1150．3±301．9vs737．8±184．5）mmol／L，P〈0．05]。肾脏病理学结果显示LacZ组肾小球和肾小管损害明显，而HK组明显减轻。结论重组腺相关病毒介导HK基因表达可能通过增加PI3K／Akt磷酸化明显改善2型糖尿病大鼠胰岛素抵抗，同时明显减轻糖尿病肾脏损害。

Objective To evaluate the therapeutic effect of recombinant adeno-associated viral vector （rAAV） expressing human tissue kallikrein gene（rAAV-HK） on insulin resistance and renal complications in type 2 diabetic rats. Methods Male Wistar rats were injected low dose streptozotocin and fed with high fat and sucrose diets to form type 2 diabetic model, rAAV mediated HK gene （ HK group） or LacZ gene （ LacZ group） were introduced to the diabetic rats, and their systolic blood pressure, fasting blood glucose and insulin, serum creatinine, urine creatinine, urine osmolarity and urine microalbumin were measured. The homeostasis model assessment of insulin resistance （HOMA-IR） , urinary albumin excretion rate （UAER） and creatinine clearance rate （Ccr） were calculated. The expression of PI3-kinase p110 catalytic subunit （p110） and Akt phosphorylation on Thr-308 were detected by Western blot. The morphology of kidney was observed. Results Delivery of rAAV-HK resulted in a reduction in blood pressure at 2 weeks and the hypotensive effect lasted for the duration of the study. The HOMA-IR was significantly lower in HK group than LacZ group（4.76 ±0.33 vs 8.36±0.48, P 〈 0.01） atthe end of the study, fasting insulin level was reduced [ （8.19 ±2.45 vs 13.85±3.76）mIU/L,P〈 0.01 ], but there was no significant change in fasting blood glucose[ （ 13.09 ± 3.01 vs 13.58 ± 2.88 ） mmol/L]. The phosphorylation of p110 and Akt Thr-308 were significantly decreased in skeletal muscle and liver in LacZ group and were almost corrected by HK gene therapy. The UAER and Ccr were significantly lower and urinary osmolarity were higher in HK-treated rats compared with LacZ rats. Histological assessment indicated that the renal complication was relieved by HK gene delivery. Conclusion The rAAV-mediated HK gene delivery efficiently attenuated insulin resistance partly through PI3K/Akt pathway and diabetic nephropathy in type 2 diabetic rats.