Strategies for ACE2 Amplification to Treat Diabetic Kidney Disease

描述（由申请人提供）：ACE2是一种1型完整膜蛋白，与ACE的金属蛋白酶催化结构域具有42%的同源性。ACE促进血管紧张素II的形成，而ACE2促进血管紧张素（ANG） II降解为血管紧张素-(1-7)。通过增强血管紧张素II的降解，旨在提高ACE2活性的策略可能在肾素-血管紧张素系统中提供一个新的治疗靶点，这可能补充了目前使用ACE抑制剂抑制血管紧张素II形成或使用特异性AT1受体拮抗剂阻断其作用的方法。我们假设，由于ACE2下调，ANG II降解减少，同时ANG-(1-7)形成减少，可能在糖尿病肾病中发挥重要作用。这一假设是基于我们最近发现的糖尿病小鼠肾脏中ACE和ACE2表达的独特模式，即db/db小鼠肾小球中ACE2表达降低而ACE升高。与这一假设相一致，我们最近发现，在两种不同的糖尿病小鼠肾病模型中，使用一种特定的药理学药物MLN-4760抑制ACE2会加重蛋白尿并诱导肾小球损伤。我们建议ACE2上调将成为旨在减少蛋白尿和肾小球损伤的新疗法的靶点。我们现在有工具可以通过不同的方法直接测试这一假设：给小鼠重组ACE2，慢病毒传递ACE2和一种新的ACE2激活剂给糖尿病小鼠。假设的ACE2局部保护机制将通过足细胞特异性ACE2转基因进一步研究。本提案的具体目标是：目标1)生产小鼠重组ACE2，并建立给药方案，实现血清ACE2活性的持续增加，有效地增加ANG II降解和ANG-(1-7)的形成。目的2)创建慢病毒小鼠ACE2表达系统，并建立递送给小鼠的方案，实现血清和组织ACE2活性的持续增加，从而有效地增加全身和肾脏水平的ANG II降解和ANG-(1-7)的形成。目的3)研究小鼠重组(r)ACE2、慢病毒传递ACE2和一种新型ACE2激活剂XNT对2型糖尿病db/db模型（C57BLK和FVB背景）蛋白尿和肾小球病变的影响。通过这些不同的方法对这些参数进行ACE2扩增后，ANG II和ANG-(1-7)的相对贡献将在使用ANG-(1-7) /Mas受体的特定阻滞剂的其他实验中进行检查。目的4)验证糖尿病（db/db）肾小球ACE2过表达蛋白尿和糖尿病肾小球病变可以预防的假设，并且即使面对全身ACE2缺乏，孤立的肾小球ACE2过表达也可以实现这种保护。公共卫生相关性：肾脏疾病是1型和2型糖尿病的常见并发症。肾素-血管紧张素系统（RAS）广泛参与糖尿病肾病的发展。最近发现一种叫做ACE2的酶在血管紧张素II的处理中起作用，血管紧张素II是一种可能导致糖尿病肾病进展的激素。这一建议将更好地了解血管紧张素II，特别是糖尿病肾病中的ACE2，并有望为治疗糖尿病和肾损伤患者提供新的见解。我们推测，这一提议的结果将为开发旨在增强ACE2活性以治疗糖尿病肾病的疗法提供概念证明，就像开发ACE抑制剂以防止血管紧张素II的形成一样，现在广泛用于糖尿病和肾病患者。

DESCRIPTION (provided by applicant): ACE2 is a type 1 integral membrane protein that shares 42% homology with the metalloprotease catalytic domains of ACE. Whereas ACE promotes the formation of angiotensin II, ACE2 promotes the degradation of angiotensin (ANG) II to angiotensin-(1-7). Strategies geared at increasing ACE2 activity may provide a novel therapeutic target within the renin-angiotensin system by enhancing angiotensin II degradation that may complement the current approach of either inhibiting angiotensin II formation using ACE inhibitors or blocking its action using specific AT1 receptor antagonists. We hypothesize that decreased ANG II degradation coupled with decreased ANG-(1-7) formation owing to ACE2 downregulation may play an important role in diabetic kidney disease. This hypothesis is based on our recent findings of a distinctive pattern of ACE and ACE2 expression in kidneys from diabetic mice such that glomerular ACE2 expression is decreased and ACE is increased in the db/db mice. In keeping with this hypothesis, we have recently found that ACE2 inhibition using a specific pharmacologic agent, MLN-4760, worsens albuminuria and induces glomerular injury in two different models of nephropathy in diabetic mice. We propose that ACE2 upregulation will emerge as a target of new therapies aimed at reducing albuminuria and glomerular injury. We now have the tools to test this hypothesis directly by different approaches: the administration of murine recombinant ACE2, lentiviral ACE2 delivery and a novel ACE2 activator to diabetic mice. The postulated mechanism of local ACE2 protection will be further studied using a podocyte-specific ACE2 transgenic. The specific objectives of this proposal are: Aim 1) To produce murine recombinant ACE2 and establish protocols for delivery to mice that achieve a sustained increase in serum ACE2 activity that effectively increases ANG II degradation and ANG-(1-7) formation. Aim 2) To create a system for lentiviral murine ACE2 expression and establish protocols for delivery to mice that achieve a sustained increase in serum and tissue ACE2 activity that effectively increases ANG II degradation and ANG-(1-7) formation systemically and at the kidney level. Aim 3) To examine the effect of the administration of murine recombinant (r)ACE2, lentiviral ACE2 delivery, and a novel ACE2 activator, XNT, on albuminuria and glomerular lesions in the db/db model of type 2 diabetes (C57BLK and FVB backgrounds). The relative contribution of ANG II and ANG-(1-7) following ACE2 amplification by these various approaches on these parameters will be examined in additional experiments using a specific blocker of the ANG-(1- 7)/Mas receptor. Aim 4) To test the hypothesis that in diabetic (db/db) with glomerular ACE2 over-expression albuminuria and the glomerular lesions of diabetes can be prevented and that this protection can be accomplished with isolated glomerular ACE2 overexpression even in the face of systemic ACE2 deficiency. PUBLIC HEALTH RELEVANCE: Kidney disease is a frequent complication of both type 1 and type 2 diabetes. The renin- angiotensin system (RAS) has been widely implicated in the development of diabetic kidney disease. It has been recently found that an enzyme called ACE2 plays a role in the disposal of angiotensin II, a hormone that may be responsible for the progression of diabetic kidney disease. This proposal will provide a better understanding of angiotensin II and, in particular, ACE2 in diabetic kidney disease and has the promise to offer novel insights into ways to treat patients afflicted with diabetes and kidney damage. We surmise that results from this proposal will be proof of concept for the development of therapies aimed at enhancing the activity of ACE2 to treat diabetic kidney disease much the same way that ACE inhibitors were developed to prevent the formation of angiotensin II and are now widely used in patients with diabetes and kidney disease.