NITRIC OXIDE SYNTHASE IN THE JUXTAGLOMERULAR APPARATUS

描述（申请人提供）：血管内皮依赖性松弛因子/一氧化氮（EDRF/NO）功能障碍伴盐敏感性高血压、糖尿病（DM）和心血管疾病。不对称二甲基精氨酸（ADMA）通过与NOS底物、l -精氨酸竞争通过y+系统进行膜运输，或通过竞争性抑制NOS，从而损害EDRF/NO。因此，ADMA可能是导致心血管疾病的EDRF/NO反应缺陷的基础。然而，ADMA的正常调控尚未得到广泛的研究。ADMA由二甲基精氨酸二甲氨基水解酶（DDAH-1和-2）代谢。我们发现DDAH-2在肾脏中大量表达，尤其是在黄斑致密细胞和远端肾元中。DDAH-1在血管内皮和近端小管中大量表达。我们的研究表明，血管紧张素II上调DDAH-2（从而可能降低致密斑ADMA），下调DDAH-1（从而可能增加内皮ADMA）。饮食盐限制和早期胰岛素缺乏型糖尿病都会损害EDRF/NO。这种缺陷可以通过过量的精氨酸来克服，这表明ADMA可能会抑制NO。事实上，我们发现限盐降低了血浆精氨酸，但增加了血浆ADMA，并损害了离体肠系膜抵抗血管对乙酰胆碱的EDRF/NO反应。相反，我们发现DM增加肾脏DDAH-2表达，导致肾脏ADMA水平降低。由此引起的黄斑密度NO的升高可阻断血管收缩性小管肾小球反馈（TGF）反应，从而选择性地使肾传入小动脉血管舒张，导致肾小球高滤过。我们提议测试这个假设，即精氨酸输送，肾脏和微血管抵抗血管中NOS的运输和代谢受到DDAH的差异调节，从而导致ADMA和NO在盐摄入量变化的生理适应和胰岛素缺乏型糖尿病的病理生理反应中产生位点和细胞特异性。我们在大鼠中开发了一种针对DDAH-1和-2的基因沉默策略，以测试这些亚型在肾脏抵抗血管中EDRF/NO和TGF的生理研究中的特定作用。​前两个目的是验证以下假设：由于血浆精氨酸浓度降低和内皮细胞中DDAH-1活性降低，饮食盐限制限制了血管内皮中NO的生成。这可以降低细胞内精氨酸：ADMA浓度，从而抑制EDRF/NO。我们建议研究血管紧张素和矿物皮质激素受体的具体作用。第三个目的是验证这样的假设：在早期胰岛素缺乏的糖尿病中，内皮细胞中DDAH-1的下调是血管病变前EDRF/NO缺陷的基础，而黄斑致密组织中DDAH-2的上调会增强局部NO，从而阻断小管肾小球反馈（TGF）反应，导致肾病前的高过滤过。

DESCRIPTION (provided by applicant): Dysfunction of the endothelium dependent relaxation factor/nitric oxide (EDRF/NO) in blood vessels accompanies salt sensitive hypertension, diabetes mellitus (DM) and cardiovascular disease. Asymmetric dimethylarginine (ADMA) impairs EDRF/NO either by competing with the NOS substrate, L-arginine for membrane transport via system y+, or by competitive inhibition of NOS. Therefore, ADMA could underlie defective EDRF/NO responses that contribute to cardiovascular disease. However, the normal regulation of ADMA has not been extensively studied. ADMA is metabolized by dimethylarginine dimethylaminohydrolases (DDAH-1 and -2). We have found that DDAH-2 is heavily expressed in the kidney, especially in the macula densa cells and distal nephron. DDAH-1 is heavily expressed in the vascular endothelium and proximal tubules. Our studies show that Angiotensin II upregulate DDAH-2 (and thereby may reduce macula densa ADMA) but downregulates DDAH-1 (and thereby may increase endothelial ADMA). Dietary salt restriction and early insulinopenic DM both impairs EDRF/NO. This defect can be overcome by excess arginine, suggesting that NO may be inhibited by ADMA. Indeed, we have found that salt restriction decreases plasma arginine, yet increases plasma ADMA and impairs the EDRF/NO responses to acetylcholine of isolated mesenteric resistance vessels. In contrast, we have found that DM increases renal DDAH-2 expression, leading to reduced renal ADMA levels. A consequent increase in macula densa NO could block the vasoconstrictive tubuloglomrular feedback (TGF) response and thereby vasodilate the renal afferent arteriole selectively, leading to glomerular hyperfiltration. We propose to test the hypothesis that arginine delivery, transport and metabolism by NOS in the kidneys and microvascular resistance vessels is differentially regulated by DDAH thereby leading to site- and cell-specific generation of ADMA and NO during physiologic adaptations to changes in salt intake and pathophysiologic responses to insulinopenic DM. We have developed a gene silencing strategy targeting DDAH-1 and -2 in the rat to test the specific roles of these isoforms in physiologic studies of EDRF/NO in resistance vessels and TGF in the kidney. The first two aims test the hypothesis that dietary salt restriction limits NO generation in vascular endothelium because of a reduction in plasma arginine concentration and a decrease in DDAH-1 activity in endothelial cells. This could reduce the intracellular arginine: ADMA concentration, thereby inhibiting EDRF/NO. We propose to examine the specific roles of angiotensin and mineralocorticosteroid receptors. The third aim tests the hypothesis that during early insulinopenic DM downregulation of DDAH-1 in endothelium underlies the defective EDRF/NO that precedes vasculopathy whereas upregulation of DDAH-2 in the macula densa enhances local NO that blocks the tubuloglomerular feedback (TGF) response causing hvperfiltration that precedes nephropathv.