Role of TRP Channels in Statin Reversal of Nephropathy in Early Stage Type 2 DM

描述(申请人提供)：由于生活方式和饮食习惯的改变，2型糖尿病(DM)正迅速成为美国最常见的疾病之一。目前的估计表明，大约有1500万人患有2型糖尿病，另有1200万人(未确诊)处于早期阶段。虽然肾功能衰竭是2型糖尿病最具破坏性的后果之一，但很少有研究研究肾脏疾病的进展以及早期可能的干预措施。此外，参与多种增殖性疾病的瞬时受体电位(Trp)阳离子通道在糖尿病肾病中的作用尚未被探讨。为了研究这个问题，我们建立了一个模拟人类早期2型糖尿病的高脂肪小鼠模型。使用该模型，我们发现在2型糖尿病早期，肾小球系膜增生、IV型胶原沉积、基底膜增厚、肾小球肥大和蛋白尿。然而，HMG-CoA抑制剂和降胆固醇药辛伐他汀(SMV)通过阻止Rho的杰烯基化而增加eNOS，逆转了这些早期病变，并显著减少了蛋白尿。我们假设SMV通过刺激NO的产生来逆转系膜扩张，而NO通过cGMP激活的激酶抑制TRPC4(形成系膜储备库操作的钙通道)。我们将从四个方面验证这一假说：1.确定SMV是否能逆转高脂喂养的2型糖尿病小鼠肾小球系膜扩张相关的损害(系膜增生、基底膜增厚和IV型胶原沉积)。2.探讨TRPC4钙通道在2型糖尿病肾小球系膜细胞增殖中的作用。3.确定SMV是否通过NO-cGMP抑制TRPC4钙通道而逆转2型糖尿病大鼠系膜细胞的增殖。4.确定TRPC4是否通过cGMP-激酶途径直接磷酸化而失活。支持这一假说的初步数据包括：(1)SMV逆转包括蛋白尿在内的早期糖尿病肾病，(2)TRPC4反义寡核苷酸体外抑制高糖和高胰岛素刺激的系膜细胞增殖，(3)SMV治疗的2型DM小鼠肾小球eNOS表达增加，(4)硝普钠和8-BR-cGMP抑制系膜SOC通道，(5)免疫染色和Western印迹法检测HMC中PKG-1a的表达，以及(6)HMC中PKG-1a的特异性底物Ser239-Vasp的8-BR-cGMP磷酸化。Laye摘要。由于饮食习惯和缺乏锻炼，2型糖尿病正在成为美国最大的健康问题。大约30%的2型糖尿病患者患有肾脏疾病，导致他们需要透析或肾脏移植。这些研究将确定是否以及如何使用某种药物辛伐他汀来治疗2型糖尿病早期患者，预防或减缓肾脏疾病的进展。

DESCRIPTION (provided by applicant): Because of changing lifestyle and dietary habits, type 2 diabetes mellitus (DM) is rapidly becoming 1 of the most prevalent diseases in the United States. Current estimates indicate that approximately 15 million have type 2 DM and another 12 million are (undiagnosed) in the early stages. Although renal failure is 1 of the most devastating consequences of type 2 DM, few studies have examined the progression of renal disease and a possible intervention in the early stages. Moreover, the role of transient receptor potential (TRP) cation channels, which are involved in a variety of proliferative diseases, has not been explored in diabetic nephropathy. To study this problem, we developed a high fat mouse model that mimics the condition of human early stage type 2 DM. Using this model, we found mesangial proliferation, collagen IV deposition, basement membrane thickening, glomerular hypertrophy, and albuminuria in the early stages of type 2 DM. However, the HMG-CoA inhibitor and cholesterol-lowering agent, simvastatin (SMV), which increases eNOS by preventing the gerenylation of Rho, reversed these early lesions and significantly reduced the albuminuria. We hypothesize that SMV reverses mesangial expansion by stimulating production of NO, which inhibits TRPC4 (which forms mesangial store-operated Ca2+ channels) via cGMP activated kinase. We will test this hypothesis in 4 Aims: 1. Determine if SMV can reverse the renal lesions associated with mesangial expansion (mesangial proliferation, basement membrane thickening and collagen IV deposition) of a high fat fed mouse model of type 2 DM. 2. Determine^ the role of TRPC4 Ca2+ channels in mesangial proliferation in an in vivo and an in vitro (cultured MC line) model of type 2 DM. 3. Determine whether SMV reverses the mesangial proliferation of type 2 DM via NO-cGMP stimulated inhibition of TRPC4 Ca2+ channels. 4. Determine whether TRPC4 is inactivated via direct phosphorylation by the cGMP-kinase pathway. The preliminary data supporting this hypothesis include: (1) reversal of early stage diabetic nephropathy, including albuminuria, by SMV, (2) in vitro attenuation by TRPC4 antisense of high glucose and high insulin-stimulated mesangial proliferation, (3) an increase in eNOS expression in glomeruli of SMV- treated type 2 DM mice, (4) inhibition of mesangial SOC channels by sodium nitroprusside and 8-Br-cGMP, (5) the identification of PKG-1a in HMC using immunostaining and Western blot and (6) the phosphorylation by 8-Br-cGMP of Ser239-VASP, a specific substrate for PKG-1a in HMC. Lay Summary. Because of dietary habits and lack of exercise, type 2 diabetes mellitus is becoming the biggest health issue in the US. Approximately 30% of type 2 diabetics develop kidney disease causing them to require either dialysis or a kidney transplant. These studies will determine if and how a certain drug, simvastatin, can be used to treat patients in the early stage of type 2 diabetes mellitus and prevent or slow the progression of kidney disease.