肾素抑制剂Aliskiren与血管紧张素1-7单独或联合应用对糖尿病肾病的保护作用及机制

Diabetes mellitus is becoming the leading cause of cardiovascular and renal diseases in China and most of the world. The pathogenesis of diabetic complications are multifactorial, recent evidences showed that endoplasmic reticulum stress (ER stress) and innate immune response (e.g.TLR, NLR families) played important roles in development of diabetic nephropathy. The benefits of blockade of the renin-angiotensin-aldosterone system (RAAS) in diabetes (mainly by inhibiting effects of angiotensin II in the kidney), namely angiotensin-converting enzyme inhibitors (ACEi) and angiotensin II type 1 receptor blockers (ARB) are well established. However, ACEi or ARB treatment does not prevent development of diabetic nephropathy which is at least, in part, attributed to feedback effects arising from the blockade of the RAAS. Recently a renin direct inhibitor aliskiren was developed which may cause more complete inhibition of the RAAS. It reduces angiotensin II levels and plasma renin activity (PRA) without stimulating compensatory increases in PRA or angiotensin II that is seen in ACEi and ARBs treatments. Use of aliskiren in combination with other agents that antagonize the RAAS is considered as a treatment stratagy. Angiotensin 1-7 (Ang 1-7) has been shown to increase insulin sensitivity，mice with genetic ablation of the Ang 1-7 Mas receptor develop features of metabolic syndrome. Therefore aiming at amplifying effects of Ang 1-7-Mas axis should be investigated in the prevention and treatment of diabetes and its complications. Our hypothesis is that aliskiren treatment with Ang 1-7 could have synergistic effects in preventing diabetic nephropathy and its complications, by inhibiting ER stress and innate immunity response in the diabetic kidneys. Expression and regulation of markers of ER stress and innate immunity response as well as their signaling pathways in cultured human podocytes, in cultured proximal tubular cells (HK2), and in the kidneys of diabetic mice, will be investigated. Specific Aim 1: To determine protection and molecular mechanism of aliskiren and /or Ang 1-7 on cultured human podocytes and tubular cells in response to palmitic acid-induced ER stress or high glucose-induced inflammation (TLR and NLR). Specific Aim 2: To determine and compare effects and molecular mechanisms of renal protection of aliskiren and/or Ang 1-7, in streptozotocin(STZ)-induced type I diabetic DBA/2J mice, in STZ-induced type I diabetic AT1R knockout mice, and in type II KKAy/KKaa mice. The proposed studies will therefore establish physiological, cellular, and molecular effects of renin inhibitor and ANG1-7 in the prevention of diabetic nephropathy and will add novel therapies for treatment of diabetes complications.

糖尿病肾病是糖尿病重要的微血管病变并发症，是引起糖尿病患者终末肾疾病甚至死亡的主要原因。糖尿病时肾脏细胞内质网应激反应和天然免疫系统相关的炎症反应近来被认为参与糖尿病肾病的病理生理过程，但肾素-血管紧张素系统（RAS）过度激活或抑制在此过程中的作用仍不清楚。与ACEi或ARB相比，肾素直接抑制剂能够更完整的抑制RAS系统，减少由于应用ACEi或ARB所带来的负反馈作用，延缓糖尿病肾病的发展。本课题拟在初步发现肾素抑制剂或血管紧张素1-7（Ang1-7）缓解肾细胞内质网应激反应和抑制天然免疫相关的炎症反应的基础上，采用生理学，免疫学和分子生物学技术，在细胞，器官和动物整体水平上，深入研究肾素抑制剂和/或Ang1-7在糖尿病肾病时对肾脏功能和肾脏细胞的保护作用，特别着重于对内质网应激反应和天然免疫诱导的炎症反应的抑制作用的研究，并阐明其分子细胞机制，为治疗糖尿病肾病提供新的思路和理论依据。

糖尿病肾病是糖尿病全身性微血管病变并发症之一，晚期出现严重肾功能衰竭，是糖尿病患者的主要死亡原因之一。了解糖尿病肾病发病机制，研究影响病程的相关因素以及相应合理的干预治疗具有重要的社会意义。肾脏局部肾素-血管紧张素II（renin-angiotensin system, RAS）的过度激活特别是血管紧张素II参与糖尿病肾脏损害。直接肾素抑制剂aliskiren能够抑制血浆肾素活性，理论上能够更完整的抑制血管紧张素II的产生，是比较理想的RAS系统抑制药物。在本项目资助下，我们发现：1）在糖尿病肾病小鼠，aliskiren能够减轻肾小球硬化，降低蛋白尿，抑制肾脏炎症、纤维化及内质网应激反应，延缓糖尿病肾脏损害。2）在体外培养的肾脏上皮细胞中，aliskiren或血管紧张素II受体阻断剂valsartan抑制饱和脂肪酸棕榈酸诱导的细胞内质网应激反应及细胞凋亡；RAS系统拮抗防止高脂饮食诱导的小鼠肾脏脂质损害，减弱内质网应激反应，改善肾脏功能。3）Aliskiren与丝氨酸蛋白酶抑制剂chymostatin联合应用能更好的减弱棕榈酸诱导的肾脏细胞损害。4）血管紧张素1-7的受体Mas激动剂加重棕榈酸诱导的肾脏上皮细胞损害，诱发细胞内质网应激与自噬反应，Mas受体阻断剂或Mas受体敲除减轻棕榈酸诱导的细胞损害，促进细胞增殖与存活，减弱内质网应激与自噬，这些保护性反应可能与细胞内钙离子水平的变化有关。.肾源性尿崩症是指肾脏对抗利尿激素不敏感，尿浓缩功能障碍，排出大量低渗尿，分子基础是肾脏集合管水通道蛋白-2（AQP2）表达下降，促进AQP2蛋白表达是治疗尿崩症的关键措施。我们发现，直接肾素抑制剂aliskiren可以通过cAMP-PKA细胞信号系统，刺激肾脏集合管主细胞AQP2蛋白顶膜穿梭和表达，增加锂诱导的肾源性尿崩症小鼠尿渗透压，减少尿量，改善肾脏尿浓缩功能，可能是潜在的治疗尿崩症的药物之一。.在本项目的资助下，我们还发现：在尿路梗阻动物模型中，直接肾素抑制剂aliskiren可以抑制NLRP3炎症小体的激活，改善炎症诱导的肾脏水钠代谢紊乱；锂诱导的肾源性尿崩症模型中，4-苯基丁酸通过缓解内质网应激增加AQP2蛋白表达，改善肾脏尿浓缩功能。