受体介导的前肾素在糖尿病肾病中的致纤维化作用及机制研究

Diabetic nephropathy (DN) is one of leading causes of end-stage renal disease in China. Understanding the pathogenesis of the systemic complications of diabetes is essential to devise new effective therapeutic strategies. Prorenin is the biosynthetic precursor of renin. A number of studies have shown that plasma prorenin levels are commonly elevated in diabetic patients and appear to predict the development of DN. However, the potential pathogenic role of prorenin in DN is unclear. .Recently, a novel prorenin receptor ((p)RR) has been discovered. (p)RR is expressed in multiple organs, including the kidney, and binds prorenin and renin with equivalent affinities. When bound to (p)RR, prorenin cleaves angiotensinogen without being proteolytically converted to renin. We recently generated a conditional cyp1a1 rat prorenin transgenic rat model. Preliminary data showed that high levels of circulating prorenin of hepatic origin caused hypertension, albuminuria and renal fibrosis via induction of inflammation, oxidative stress and the Wnt/ß-catenin and PI3K/Akt-mediated signaling pathways. Of note, treatment with enalapril (an ACEI) prevented prorenin-induced hypertension and ameliorated, but did not prevent, prorenin-induced podocyte injury, albuminuria and renal fibrosis, suggesting the involvement of angiotensin (Ang) II-dependent and Ang II-independent pathways. To exclude the possibility that the partially protective effect of enalapril can be due to incomplete blockage of renin-angiotensin system (RAS), a novel mutant prorenin transgenic rat model, which lacks the enzymatic activity, has been generated in our laboratory. Importantly, the increased mutant prorenin expression caused significant albuminuria in the absence of RAS activation and hypertension, suggesting Ang II-independent effects. Based on our state-of-the-art models and exciting data, we hypothesize that prorenin, via pRR, activates Ang II-dependent and -independent signal mechanisms to promote renal fibrosis in diabetes..We propose to 1) investigate whether the high levels of prorenin accelerate the development of diabetic nephropathy using the inducible prorenin transgenic and diabetic rat model; 2) investigate the potential signaling mechanisms of action of prorenin in diabetes by investigating whether the pathological effect of prorenin is mediated by prorenin receptor, whether it depends on renin's enzymatic activity to augment Ang II production or whether both pathways are involved; 3) test the prorenin receptor blocker therapeutically in a model of diabetic nephropathy, compared with and in combination with Ang II blockade. The proposed studies will further our understanding of the role and signal mechanisms of prorenin and its cellular receptor in the development of DN. Positive results could lead to the development of new therapeutic strategies by adding the prorenin receptor antagonists to RAS blockers to prevent the development of renal or cardiovascular complications associated with diabetes.

最近，人们成功地克隆出一种特异性的肾素／前肾素的受体（PRR)，体内多脏器包括肾脏细胞表达该受体。我们在前期的研究中发现在体外前肾素与受体结合后通过分子变构而非去前体肽获得将血管紧张素原转化为血管紧张素I的活性，并激活细胞内致纤维化信号通路。在体内诱导型转基因大鼠其肝脏高表达前肾素而非肾素，导致高血压、蛋白尿及肾纤维化，而这种作用并不能被依那普利完全阻断；诱导型转基因大鼠其肝脏高表达无酶活性变异型前肾素，未有高血压但有明显蛋白尿。基于此研究结果，拟通过建立前肾素及变异型前肾素转基因的糖尿病大鼠模型,探讨前肾素在糖尿病肾病 (DN)中的病理作用及机制。此外，通过将纳米技术和siRNA技术整合，我们已构建出基于肾小球内细胞PRR表达靶向特异性纳米颗粒，本研究还将探讨该纳米颗粒抑制PRR表达及其介导的前肾素的病理效应对DN的治疗作用，为临床DN的治疗开劈新的道路。

糖尿病肾病是我国慢性肾衰的常见病因，因此研究糖尿病肾病的发生机制及开发治疗手段是学术界的重点课题。其中前肾素一直被学者们认为是预测和判断糖尿病血管并发症特别是糖尿病肾病病变的生物指标，但其是否致病以及如何致病尚未明确。最近，人们成功地克隆出特异性的前肾素的受体（PRR)，体内多脏器包括肾脏细胞表达该受体。我们在前期的研究中发现在体外前肾素与受体结合后通过分子变构而非去伪肽获得将血管紧张素原(AGT)转化为血管紧张素(Ang)I的活性，并激活受体后细胞内致纤维化信号通路。基于此研究结果，我们提出如下假说：在糖尿病进程中，升高的前肾素与肾脏局部前肾素受体特异性结合，一方面获得了转化AGT的能力，使Ang II产生增多，另一方面激活受体后通路，产生非Ang II依赖的致病效应，从而参与糖尿病肾病的发生和发展。我们的研究内容包括1）利用我们自主建立的肝源性、诱导型前肾素转基因大鼠建立1型糖尿病模型探讨升高血浆前肾素在糖尿病肾病发病中的作用；2）探讨前肾素与其受体通路在糖尿病肾病发病中的作用机制；3）探讨阻断PRR对糖尿病肾病的治疗作用。通过4年的研究，我们成功完成了诱导型前肾素转基因的型糖尿病模型制备，并完成了升高血浆前肾素水平对糖尿病肾脏病变影响的研究以及阿莫地平和依那普利对诱导型前肾素转基因的I 型糖尿病肾病的干预及前肾素的AngII 依赖作用机制的论证。我们进一步完成了诱导型变异前肾素转基因的I型糖尿病的模型制备，并完成升高的无酶活性的变异型前肾素对糖尿病肾脏病变影响的观察以及前肾素的AngII非依赖性作用机制的论证。最后，我们对PRR的阻断治疗进行了初步探讨性研究。研究结果表明，糖尿病状态下，升高的前肾素通过导致高血压、刺激肾内炎症和氧化应急通路参与糖尿病肾病的发病和进展，其致病作用是通过结合前肾素受体由AngII介导的以及非AngII介导的双重机制参与糖尿病肾病病变。特异性PRR的siRNA或中和抗体以及特异性RNA-结合蛋白HuR的抑制剂有望进一步阻断前肾素在糖尿病肾病中的致病作用。