Renal Cortical Oxidative & Nitrosative Stress in IDDM

在大鼠1型糖尿病(DM)的早期阶段，超氧阴离子(O2-)的产生加速了一氧化氮(NO)通过过氧亚硝酸盐(ONOO)的降解。形成，导致蛋白质酪氨酸硝化和血管反应性改变。糖尿病肾脏氧化应激的发生机制尚不清楚。我们发现糖尿病大鼠的循环血管紧张素II(Ang11)水平和肾皮质Ang111型(AT1)受体蛋白水平均显著升高。AT1受体的激活可以通过激活NAD(P)H氧化酶来刺激O2.-的产生。我们还发现了糖尿病大鼠肾皮质热休克蛋白90水平降低的证据。新的证据表明，Hsp90代表着NO产生的一个重要步骤，其功能至少部分地限制了O2。-由NO合成酶(NOS)产生。在这些初步观察的基础上，我们推测NAD(P)H氧化酶的激活和NOS的解偶联参与了糖尿病肾皮质的氧化和亚硝化应激，从而影响了NO的生物利用度和最终减轻其对肾功能的影响。STZ诱导的大鼠DM模型将被用来评估：1)依赖Ang11的NAD(P)H氧化酶激活在肾皮质O2中的作用。 糖尿病时产生；2)Hsp90或BH4依赖的一氧化氮合酶解偶联蛋白对肾脏的贡献 糖尿病时皮质氧的产生；以及3)鸟氨酰环化酶酪氨酸硝化在糖尿病时肾微血管功能改变中的潜在作用。为了符合RFA-DK-O2-O23，本申请请求支持两个独立、成熟的研究人员之间的合作伙伴关系--帕梅拉·K·卡明斯(在糖尿病肾脏微血管并发症方面有专长的肾脏生理学家)和詹妮弗·S·波洛克(在一氧化氮合酶方面有特殊专长的蛋白质化学家)。卡明斯博士和波洛克博士最近在一份联合出版物中记录了他们作为合作者取得的成功，为该项目提供了许多初步数据。拟议工作的完成将使波洛克博士成为一名活跃的糖尿病研究人员。这一研究伙伴关系的资助还将通过促进获得专门知识和技术来加强卡明斯博士的实验室正在进行的努力，这些专门知识和技术是对她的实验室目前可用于研究糖尿病的肾脏微血管功能和氧化应激的技术的补充。因此，拟议的合作使生化、细胞和功能方法能够揭示治疗干预的新靶点，从而限制糖尿病早期阶段氧化和亚硝化应激的发展或后果。 延缓或预防糖尿病肾病的发展。

During the early stage of type 1 diabetes mellitus (DM) in the rat, accelerated superoxide anion (O2 -) production fuels degradation of nitric oxide (NO) via peroxynitrite (ONOO.) formation, resulting in protein tyrosine nitration and alterations in vascular reactivity. The mechanisms engendering renal oxidative stress in DM have not been established. We have found that rats with DM exhibit significant increases in both circulating angiotensin II (Angll) levels and renal cortical levels of the Angll type 1 (AT 1) receptor protein. AT 1 receptor activation can provoke O2.- production via NAD(P)H oxidase activation. We have also found evidence of reduced Hsp90 levels in the renal cortex of rats with DM. Emerging evidence indicates that Hsp90 represents an essential step in NO production, functioning at least in part to limit O2.- production by NO synthase (NOS). On the basis of these preliminary observations, we hypothesize that NAD(P)H oxidase activation and NOS-uncoupling contribute to renal cortical oxidative and nitrosative stress in DM, with consequent effects on NO bioavailability and action that ultimately diminish its impact on renal function. The STZ-induced model of DM in the rat will be utilized to evaluate: 1) the role of Angll-dependent NAD(P)H oxidase activation in the renal cortical O2.- production during DM; 2) the contribution of Hsp90- or BH4-dependent NOS-uncouplin9 to renal cortical O2.- production during DM; and 3) the potential role of tyrosine nitration of guanylyl cyclase in altering in renal microvascular function during DM. Tin accord with RFA-DK-O2-O23, this application requests support for a collaborative partnership between two independent, established investigators - Pamela K. Carmines (a renal physiologist with expertise in the renal microvascular complications of DM)) and Jennifer S. Pollock (a protein chemist with particular expertise in NOS). Drs. Carmines and Pollock recently documented their success as collaborators in a joint publication providing much of the preliminary data for this project. Completion of the proposed work should position Dr. Pollock as an active diabetes researcher. Funding of this research partnership would also strengthen the ongoing efforts of Dr. Carmines' laboratory by facilitating access to expertise and technologies that are complementary to those currently available to her laboratory for studies of renal microvascular function and oxidative stress in DM. The proposed collaboration thus allows a biochemical, cellular, and functional approach that could unveil novel targets for therapeutic interventions that limit the development or consequences of oxidative and nitrosative stress during the early stage of DM, thereby delaying or preventing development of diabetic nephropathy.