Strategies for ACE2 Amplification to Treat Diabetic Kidney Disease

描述（由申请人提供）：ACE 2是1型整合膜蛋白，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ACE 2促进血管紧张素II降解为血管紧张素-（1-7）。针对增加ACE 2活性的策略可以通过增强血管紧张素II降解在肾素-血管紧张素系统内提供新的治疗靶点，这可以补充当前使用ACE抑制剂抑制血管紧张素II形成或使用特异性AT 1受体拮抗剂阻断其作用的方法。我们推测由于ACE 2下调导致ANG II降解减少和ANG-（1-7）形成减少可能在糖尿病肾病中起重要作用。这一假设是基于我们最近的发现，即糖尿病小鼠肾脏中ACE和ACE 2表达的独特模式，使得db/db小鼠中肾小球ACE 2表达降低而ACE增加。与这一假设一致，我们最近发现，在糖尿病小鼠的两种不同肾病模型中，使用特定药理学试剂MLN-4760抑制ACE 2可减少蛋白尿并诱导肾小球损伤。我们认为ACE 2上调将成为减少蛋白尿和肾小球损伤的新疗法的靶点。我们现在有工具来测试这一假设直接通过不同的方法：管理鼠重组ACE 2，慢病毒ACE 2交付和一种新的ACE 2激活剂糖尿病小鼠。将使用足细胞特异性ACE 2转基因进一步研究局部ACE 2保护的假定机制。本提案的具体目标是：目的1）生产鼠重组ACE 2并建立用于递送至小鼠的方案，其实现血清ACE 2活性的持续增加，所述血清ACE 2活性有效地增加ANG II降解和ANG-（1-7）形成。目的2）建立用于慢病毒鼠ACE 2表达的系统并建立用于递送至小鼠的方案，其实现血清和组织ACE 2活性的持续增加，所述ACE 2活性有效地全身性地和在肾脏水平增加ANG II降解和ANG-（1-7）形成。目的3）在2型糖尿病db/db模型（C57 BLK和FVB背景）中检查施用鼠重组（r）ACE 2、慢病毒ACE 2递送和新型ACE 2激活剂XNT对蛋白尿和肾小球病变的影响。在使用ANG-（1 - 7）/Mas受体的特异性阻断剂的额外实验中，将检查通过这些不同方法进行ACE 2扩增后ANG II和ANG-（1-7）对这些参数的相对贡献。目的4）验证以下假设：在具有肾小球ACE 2过表达的糖尿病（db/db）中，蛋白尿和糖尿病的肾小球病变可以被预防，并且即使面对系统性ACE 2缺乏，这种保护也可以通过孤立的肾小球ACE 2过表达来实现。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）与糖尿病肾病的发生发展密切相关.最近发现，一种称为ACE 2的酶在血管紧张素II的处置中起作用，血管紧张素II是一种可能导致糖尿病肾病进展的激素。该提案将提供对血管紧张素II，特别是糖尿病肾病中ACE 2的更好理解，并有望为治疗糖尿病和肾损伤患者的方法提供新的见解。我们推测，该提案的结果将成为开发旨在增强ACE 2活性以治疗糖尿病肾病的疗法的概念验证，其方式与开发ACE抑制剂以防止血管紧张素II形成并目前广泛用于糖尿病和肾病患者的方式大致相同。

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.