TRP channels in statin reversal of diabetic nephropathy

描述（由申请人提供）：由于生活方式和饮食习惯的改变，2型糖尿病（DM）正迅速成为美国最流行的疾病之一。目前的估计表明，约有1500万人患有2型糖尿病，另有1200万人处于早期阶段（未确诊）。虽然肾功能衰竭是2型糖尿病最具破坏性的后果之一，但很少有研究检查肾脏疾病的进展和早期可能的干预措施。此外，瞬时受体电位（TRP）阳离子通道参与多种增殖性疾病，但尚未探讨其在糖尿病肾病中的作用。为了研究这一问题，我们建立了一个模拟人类早期2型糖尿病的高脂小鼠模型。在该模型中，我们发现2型糖尿病早期存在系膜增生、IV型胶原沉积、基底膜增厚、肾小球肥大和蛋白尿。然而，HMG-CoA抑制剂和降胆固醇剂辛伐他汀（SMV）通过阻止Rho的基因化而增加eNOS，逆转这些早期病变，显著减少蛋白尿。我们假设SMV通过刺激NO的产生来逆转系膜扩张，NO通过cGMP激活的激酶抑制TRPC4（形成系膜储存操作的Ca2+通道）。我们将在4个目标中检验这一假设：1。确定SMV是否可以逆转高脂肪喂养的2型糖尿病小鼠模型肾小球系膜扩张相关的肾脏病变（系膜增生、基底膜增厚和IV型胶原沉积）。在体内和体外（培养的MC系）2型糖尿病模型中，确定TRPC4 Ca2+通道在系膜增殖中的作用。确定SMV是否通过NO-cGMP刺激的TRPC4 Ca2+通道抑制逆转2型DM的系膜增殖。4. 确定TRPC4是否通过cgmp -激酶途径直接磷酸化失活。支持这一假设的初步数据包括：(1) SMV对早期糖尿病肾病（包括蛋白尿）的逆转作用，(2)TRPC4反义对高糖和高胰岛素刺激的肾小球增殖的体外抑制作用，(3)SMV处理的2型DM小鼠肾小球eNOS表达的增加，(4)硝普钠和8-Br-cGMP对肾小球SOC通道的抑制作用，(5)利用免疫染色和Western blot鉴定HMC中的PKG-1a， (6) 8-Br-cGMP对Ser239-VASP的磷酸化作用。HMC中PKG-1a的特异性底物。总结。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型糖尿病患者会患上肾脏疾病，导致他们需要透析或肾脏移植。这些研究将确定某种药物辛伐他汀是否以及如何用于治疗早期2型糖尿病患者，并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.