Na+/H+-exchanger-1 and Diabetic Neuropathy

描述(由申请人提供)：氧化亚硝胺应激在周围型糖尿病神经病变(PDN)中的重要性的证据正在出现，但其机制尚不清楚。上调钠？交换器-1(NHE-1)以前在糖尿病并发症的几个组织部位被证实，导致胞浆ph值增加和糖酵解上部的相关激活。这一建议的总体假设是，NHE-1在PDN中扮演着重要的角色，因为NHE-1驱动的糖酵解上部的激活，在糖尿病引起的3-磷酸甘油醛脱氢酸抑制(内皮细胞)或激活不足的条件下(雪旺细胞，SC)，是将多余的糖酵解通量转向形成α-甘油磷酸和甲乙醛的基础，从而激活了两个主要的自由基产生机制，即非酶糖基化和NAD(P)H氧化酶。为支持这一假说，我们的初步研究表明：1)NHE-1在大鼠和小鼠周围神经、大鼠背根神经节神经元和人SC中大量表达；2)HSC基础pH和NHE-1的表达和活性在高糖刺激下增加；3)糖酵解的上部在糖尿病周围神经中被激活；4)NHE-1的特异性抑制剂cariporide至少部分地阻止了STZ糖尿病大鼠的神经传导障碍、感觉神经病变、神经血管功能障碍和代谢失衡；5)STZ糖尿病NHE-1小鼠与STZ糖尿病野生型小鼠相比，发展成严重的PDN。该建议的目的是评估NHE-1在1型和2型糖尿病动物模型中的PDN中的作用。其具体目的是1)阐明抑制NHE-1是否能逆转STZ糖尿病和ZDF大鼠的PDN；2)确定HIF-1a和醛固酮在糖尿病和高糖诱导的NHE-1过表达中的作用；以及3)评估NHE-1在糖尿病大鼠以及高糖暴露的培养的HSC和共培养的大鼠SC和DRG神经元的氧化-亚硝化应激中的作用。该项目结合了动物模型中的生理、行为、生化、免疫组织化学和结构研究，以及培养的HSC和共同培养的大鼠SC和DRG神经元的分子研究。这些发现将产生关于NHE-1在两种类型糖尿病的PDN中的作用的新信息，并可能为开发NHE-1抑制剂和含有NHE-1抑制剂的药物组合提供理论基础。公共卫生相关性：外周糖尿病神经病变(PDN)是糖尿病最具破坏性的并发症，也是导致足部截肢的主要原因。自由基和氧化剂在PDN中的重要性的证据正在从动物和人类研究中涌现，但其机制尚不清楚。钠的上调？交换器-1(NHE-1)以前在糖尿病动物的几个组织中被证实，导致细胞内ph值的增加和糖酵解上部的相关激活。这一建议的总体假设是NHE-1在PDN中起重要作用，因为在糖尿病引起的3-磷酸甘油醛脱氢酶抑制(内皮细胞)或不足激活(Schwann细胞，SC)的情况下，NHE-1驱动的糖酵解上部的激活是将过量的糖酵解通量转向两条主要的自由基产生途径，即非酶糖基化和NAD(P)H氧化酶。我们的初步数据支持这一假说：1)NHE-1在大鼠和小鼠周围神经、大鼠背根神经节神经元和人SC中大量表达；2)高糖刺激HSC基础PH值及NHE-1的表达和活性；3)糖尿病神经中激活了糖酵解的上部；4)NHE-1的特异性抑制剂cariporide至少部分阻止了STZ-糖尿病大鼠的PDN；5)与NHE-1含量正常的糖尿病小鼠相比，糖尿病NHE-1小鼠发展为不那么严重的PDN。该建议的目的是评估NHE-1在1型和2型糖尿病动物模型中的PDN中的作用。本研究的具体目的是：1)阐明NHE-1抑制是否能逆转STZ糖尿病和ZDF大鼠的PDN；2)评估糖尿病诱导的NHE-1过度表达的机制；3)评估NHE-1在糖尿病大鼠和高糖暴露的培养的HSC和共同培养的大鼠SC和DRG神经元的周围神经系统氧化-亚硝化应激中的作用。该项目将动物模型中的生理、行为、生化、免疫组织化学和结构研究与细胞培养中的分子研究相结合。这些发现将产生关于NHE-1在PDN中在两种类型糖尿病中的作用的新信息，并可能为开发NHE-1抑制剂和含有NHE-1抑制剂的药物组合预防和治疗PDN提供理论基础。

DESCRIPTION (provided by applicant): Evidence for importance of oxidative-nitrosamine stress in peripheral diabetic neuropathy (PDN) is emerging, but the mechanisms are not well understood. Up regulation of Na? exchanger-1 (NHE-1), previously demonstrated in several tissue-sites for diabetes complications, causes an increase in cytosolic ph and related activation of the upper part of glycolysis. The overall hypothesis of this proposal is that NHE-1 plays an important role in PDN, because NHE-1-driven activation of the upper part of glycolysis, under conditions of diabetes-induced inhibition (endothelial cells) or insufficient activation (Schwann cells, SC) of glyceraldehyde- 3-phosphate dehydrogenate, underlies diversion of the excessive glycolytic flux towards formation of a-glycerophosphate and methylglyoxal, with resulting activation of two major free radical-generating mechanisms i.e., non-enzymatic glycation and NAD(P)H oxidase. In support of this hypothesis, our preliminary studies have shown that 1) NHE-1 is abundantly expressed in rat and mouse peripheral nerve, rat DRG neurons, and human SC; 2) HSC basal ph and NHE-1 expression and activity increase in response to high glucose; 3) the upper part of glycolysis is activated in the diabetic peripheral nerve; 4) the specific NHE-1 inhibitor cariporide, at least, partially prevents nerve conduction deficits, sensory neuropathy, neurovascular dysfunction, and metabolic imbalances in STZ-diabetic rats; 5) STZ-diabetic NHE-1 mice develop less severe PDN than STZ-diabetic wild-type mice. The OBJECTIVE of this proposal is to evaluate the role of NHE-1 in PDN in animal models of Type 1 and Type 2 diabetes. The specific aims are 1) elucidate if NHE-1 inhibition reverses PDN in STZ- diabetic and ZDF rats; 2) determine the roles of HIF-1a and aldosterone in diabetes- and high glucose-induced NHE-1 overexpression; and 3) evaluate the contribution of NHE-1 to oxidative-nitrosative stress in peripheral nerve, vasa nervorum, spinal cord, and DRG neurons of diabetic rats and high glucose-exposed cultured HSC and co-cultured rat SC and DRG neurons. The project combines physiological, behavioral, biochemical, immunohistochemical, and structural studies in animal models with molecular studies in cultured HSC and co- cultured rat SC and DRG neurons. The findings will generate new information on the role for NHE-1 in PDN in two types of diabetes and may provide rationale for development of NHE-1 inhibitors and NHE-1 inhibitor- containing drug combinations. PUBLIC HEALTH RELEVANCE: Peripheral diabetic neuropathy (PDN) is the most devastating complication of diabetes mellitus, and a leading cause of foot amputation. Evidence for importance of free radicals and oxidants in PDN is emerging from both animal and human studies, but the mechanisms are not well understood. Upregulation of Na? exchanger-1 (NHE-1), previously demonstrated in several tissues of diabetic animals, causes an increase in cytosolic ph and related activation of the upper part of glycolysis. The overall hypothesis of this proposal is that NHE-1 plays an important role in PDN, because NHE-1-driven activation of the upper part of glycolysis, under conditions of diabetes-induced inhibition (endothelial cells) or insufficient activation (Schwann cells, SC) of glyceraldehyde-3-phosphate dehydrogenase, underlies diversion of the excessive glycolytic flux towards two major free radical-generating pathways i.e., non-enzymatic glycation and NAD (P) H oxidase. The hypothesis is supported by our preliminary data demonstrating that 1) NHE-1 is abundantly expressed in rat and mouse peripheral nerve, rat DRG neurons, and human SC; 2) HSC basal ph and NHE-1 expression and activity are increased by high glucose; 3) the upper part of glycolysis is activated in the diabetic nerve; 4) the specific NHE- 1 inhibitor cariporide, at least, partially prevents PDN in STZ-diabetic rats; 5) diabetic NHE-1 mice develop less severe PDN than diabetic mice with normal NHE-1 content. The OBJECTIVE of this proposal is to evaluate the role of NHE-1 in PDN in animal models of Type 1 and Type 2 diabetes. The SPECIFIC AIMS are 1) elucidate if NHE-1 inhibition reverses PDN in STZ-diabetic and ZDF rats; 2) evaluate the mechanisms of diabetes-induced NHE-1 over expression; and 3) assess the contribution of NHE-1 to oxidative-nitrosative stress in peripheral nervous system of diabetic rats and high glucose-exposed cultured HSC and co-cultured rat SC and DRG neurons. The project combines physiological, behavioral, biochemical, immunohistochemical, and structural studies in animal's models with molecular studies in cell cultures. The findings will generate new information on the role for NHE-1 in PDN in two types of diabetes and may provide rationale for development of NHE-1 inhibitors and NHE-1 inhibitor-containing drug combinations for prevention and treatment of PDN.