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

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）正在迅速成为美国最流行的疾病之一。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干预的可能性。此外，瞬时受体电位（TRP）阳离子通道，这是参与各种增殖性疾病，在糖尿病肾病的作用还没有探讨。为了研究这个问题，我们开发了一种模拟人类早期2型糖尿病状况的高脂肪小鼠模型。利用此模型，我们发现系膜增生，IV型胶原沉积，基底膜增厚，肾小球肥大，蛋白尿在2型糖尿病的早期阶段。然而，HMG-CoA抑制剂和降胆固醇剂辛伐他汀（SMV）通过阻止Rho的gerenylation增加eNOS，逆转了这些早期病变并显著减少了白蛋白尿。我们假设SMV通过刺激NO的产生逆转系膜扩张，NO通过cGMP激活的激酶抑制TRPC 4（形成系膜钙库操纵的钙通道）。我们将在4个目标中测试这个假设：1。确定SMV是否可以逆转与高脂喂养的2型DM小鼠模型的系膜扩张（系膜增殖、基底膜增厚和IV型胶原沉积）相关的肾脏病变。2.在2型DM的体内和体外（培养的MC系）模型中确定TRPC 4 Ca 2+通道在系膜增殖中的作用。3.确定SMV是否通过NO-cGMP刺激TRPC 4 Ca 2+通道抑制来逆转2型DM的系膜增殖。4.确定TRPC 4是否通过cGMP-激酶途径直接磷酸化而失活。支持这一假设的初步数据包括：（1）通过SMV逆转早期糖尿病肾病，包括蛋白尿，（2）通过TRPC 4反义体外减弱高糖和高胰岛素刺激的系膜增殖，（3）在SMV处理的2型DM小鼠的肾小球中eNOS表达增加，（4）通过硝普钠和8-Br-cGMP抑制系膜SOC通道，（5）免疫组化和Western blot鉴定HMC中PKG-1a的存在;（6）PKG-1a的特异性底物Ser 239-VASP被8-Br-cGMP磷酸化。Lay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型糖尿病患者发展为肾病，导致他们需要透析或肾移植。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2型糖尿病早期患者，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.