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

描述（由申请人提供）：氧化亚硝胺应激在糖尿病周围神经病变（PDN）中的重要性的证据正在出现，但其机制尚未得到很好的理解。Na？交换器-1 (NHE-1)，先前在糖尿病并发症的几个组织部位被证实，引起细胞质ph值的增加和糖酵解上部的相关激活。该建议的总体假设是NHE-1在PDN中起重要作用，因为在糖尿病诱导的抑制（内皮细胞）或甘油醛- 3-磷酸脱氢激活不足（雪旺细胞，SC）的情况下，NHE-1驱动糖酵解上部的激活，导致过量的糖酵解流量转向形成a-甘油磷酸和甲基乙二醛。从而激活两种主要的自由基生成机制，即非酶糖基化和NAD(P)H氧化酶。为了支持这一假设，我们的初步研究表明：1)NHE-1在大鼠和小鼠周围神经、大鼠DRG神经元和人SC中大量表达；2)高糖时HSC基础ph和NHE-1表达及活性升高；3)糖尿病周围神经糖酵解上半部分被激活；4)特异性NHE-1抑制剂cariporide至少部分预防stz -糖尿病大鼠神经传导缺损、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小鼠发生PDN的严重程度低于stz -糖尿病野生型小鼠。本提案的目的是评估NHE-1在1型和2型糖尿病动物模型中PDN中的作用。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)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；3)评价NHE-1对糖尿病大鼠周围神经、血管神经、脊髓和DRG神经元以及高糖暴露培养的HSC和共培养大鼠SC和DRG神经元氧化亚硝化应激的贡献。该项目结合了动物模型的生理、行为、生化、免疫组织化学和结构研究，以及培养的HSC和共培养的大鼠SC和DRG神经元的分子研究。这些发现将提供关于NHE-1在两种糖尿病PDN中的作用的新信息，并可能为开发NHE-1抑制剂和含NHE-1抑制剂的药物组合提供理论依据。公共卫生相关性：糖尿病周围神经病变（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中的重要性在动物和人类研究中都有证据，但其机制尚不清楚。Na?的上调交换器-1 (NHE-1)，先前在糖尿病动物的几个组织中被证实，引起细胞质ph值的增加和糖酵解上部的相关激活。该建议的总体假设是NHE-1在PDN中起重要作用，因为在糖尿病诱导的抑制（内皮细胞）或甘油醛-3-磷酸脱氢酶激活不足（雪旺细胞，SC）的情况下，NHE-1驱动糖酵解上部的激活，导致过量的糖酵解流量转向两种主要的自由基生成途径，即非酶糖化和NAD (P) H氧化酶。我们的初步数据支持了这一假设：1)NHE-1在大鼠和小鼠周围神经、大鼠DRG神经元和人SC中大量表达；2)高糖升高HSC基础ph、NHE-1表达及活性；3)糖酵解上半部分在糖尿病神经活化；4)特异性NHE- 1抑制剂cariporide至少对stz -糖尿病大鼠PDN有部分预防作用；5)与NHE-1含量正常的糖尿病小鼠相比，糖尿病小鼠发生PDN的严重程度较轻。本提案的目的是评估NHE-1在1型和2型糖尿病动物模型中PDN中的作用。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)探讨糖尿病诱导的NHE-1过表达机制；3)评估NHE-1对糖尿病大鼠周围神经系统氧化亚硝化应激的贡献，以及高糖暴露培养的HSC和共培养的大鼠SC和DRG神经元。该项目结合了动物模型的生理、行为、生化、免疫组织化学和结构研究以及细胞培养的分子研究。这些发现将提供关于NHE-1在两种糖尿病中PDN中的作用的新信息，并可能为开发用于预防和治疗PDN的NHE-1抑制剂和含NHE-1抑制剂的药物组合提供理论依据。

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.