基于转基因模型的激肽释放酶结合蛋白在糖尿病肾病肾小管间质病变中的作用和分子机制研究

Current studies on biological markers and therapeutic targets of diabetic nephropathy (DN) focus on the renal tubulointerstitial lesions. Our previous study has shown that kallikrein binding protein (KBP) was significantly higher in diabetic patients with microalbuminuria and was associated with urinary albumin excretion and renal dysfunction, suggesting that KBP may be a potential early diagnostic and progression predictors of DN. The activation of Wnt signaling pathway plays an important role in tubulointerstitial fibrosis of DN.We confirmed that KBP is an inhibitor of Wnt signaling pathway through original KBP transgenic mice, suggesting that KBP may protect DN through Wnt signaling pathway.However, the traditional DN animal model cannot mimic the advanced process of DN which limits the research progress. This study aims to evaluate the significance of KBP as a predictor of early diagnosis and progression of DN by observing the expression level of KBP in DN patients and animal models. We will further investigate the role of KBP in DN through a stable KBP over-expression transgenic mouse model with progressive DN and clarify the molecular mechanism by which the KBP participate in tubulointerstitial fibrosis through the classical Wnt signal pathway, thus providing a solid evidence for the early diagnosis of DN and the development of new therapeutic target.

围绕肾小管间质病变寻找糖尿病肾病(DN)干预靶点是目前DN的研究热点。课题组前期发现激肽释放酶结合蛋白(KBP)在糖尿病合并微量白蛋白尿患者血清中显著升高，并与尿白蛋白排泄水平及肾功能不良呈正相关，提示KBP可能作为DN早期诊断生物学标志和进展预测因子并参与DN的发生。Wnt信号通路异常活化是肾小管间质纤维化的重要机制之一，我们通过原创的转基因小鼠模型证实KBP是Wnt信号通路抑制剂，由此推测KBP可能通过Wnt信号通路对DN发挥保护作用。然而，由于传统的糖尿病动物模型难以模拟DN进展期病变，限制了其机制研究。本研究拟在前期成功构建的高度模拟人DN进展过程并长期稳定过表达KBP的转基因OVE小鼠模型基础上，结合原代近端肾小管上皮细胞培养，从组织、细胞、分子水平探索KBP参与肾小管间质纤维化的具体机制，并评价其作为DN早期诊断生物学标志物的重要意义，为开发DN新的治疗靶点提供坚实理论依据。

激肽释放酶结合蛋白Kallistatin是组织激肽释放酶抑制因子，同时也是Wnt信号通路的抑制剂。它在糖尿病肾病中的作用尚未阐明。我们发现与健康人和没有肾脏损伤的糖尿病患者相比，糖尿病肾病患者血清中Kallistatin的水平显著升高，并且与尿蛋白水平呈正相关。同时我们发现，在1型糖尿病动物模型（Akita和OVE26小鼠）和2型糖尿病动物模型(db/db小鼠)中，肾脏Kallistatin水平是明显升高的。为了进一步明确其在糖尿病肾病发病中的作用机制，我们构建了过表达Kallistatin的转基因OVE26小鼠模型，发现过表达Kallistatin可以加重蛋白尿、肾脏纤维化、炎症和氧化应激。与野生型OVE26糖尿病小鼠相比，Kallistatin过表达转基因糖尿病小鼠的激肽释放酶的活性被抑制，但是RAS系统活性增高。通过免疫组化染色我们发现肾脏组织内血管密度下降，同时HIF-1水平明显升高。综上，Kallistatin水平增高可能通过诱导RAS过度活化并导致组织缺氧，从而加重了糖尿病肾损伤。本研究显示Kallistatin水平与糖尿病肾病呈正向相关，其可能作为一个潜在的提示糖尿病肾病进展的生物学标记。