Mechanisms of tubular atrophy

描述（由申请人提供）：美国有超过2600万人患有慢性肾脏疾病，最常见的是糖尿病肾小球损伤。然而，进展到终末期与近端小管消失（小管萎缩）紧密相关，这是由细胞凋亡引起的。在之前的资助期内，我们确定了非酯化脂肪酸（NEFA）代谢物长链酰基辅酶a （LC-CoA）的细胞内积累通过抑制NHE1 Na+/H+交换剂依赖性细胞存活的机制刺激管状萎缩。过量的LC-CoAs被储存为细胞质脂滴，以保护细胞免受脂肪毒性，尽管这种NEFA缓冲能力在近端小管中受到限制。多种因素共同导致糖尿病肾病中NEFA的积累，但最显著的是与NEFA结合的过滤白蛋白的重吸收。我们假设脂肪酸转运蛋白-2 （FATP2）摄取NEFA导致近端小管脂肪凋亡，从而导致小管萎缩和糖尿病肾病的进展。该假设将有以下具体目的：(1)为了确定FATP2是否是近端管内NEFA摄取的主要转运体，将在FATP2缺陷小鼠的离体灌注近端小管中测量荧光标记的NEFA摄取，并在用针对FATP2的shRNAs处理的培养近端小管细胞中检测细胞凋亡；(2)为了确定FATP2介导的NEFA内化与糖尿病肾病的病理生理相关性，我们将在易患糖尿病肾病的eNOS-/-db/db小鼠体内检测13c标记的NEFA摄取，并测量胰岛素对近端小管细胞中FATP2依赖性NEFA摄取的影响；(3)遗传学和药理学工具将用于测试近端小管NEFA摄取是否调节eNOS-/-db/db小鼠糖尿病肾病的进展。在完成这些实验后，我们希望FATP2将成为治疗糖尿病肾病的可药物靶点。

DESCRIPTION (provided by applicant): Over 26 million people in the U.S. have chronic kidney diseases, most commonly from diabetic glomerular injury. However, progression to end stage is more tightly coupled to proximal tubule disappearance (tubular atrophy), which is caused by apoptosis. In the previous funding period we determined that intracellular accumulation of a non-esterified fatty acid (NEFA) metabolite, long-chain acyl-coenzyme A (LC-CoA), stimulates tubular atrophy by a mechanism that involves inhibition of NHE1 Na+/H+ exchanger-dependent cell survival. Excess LC-CoAs are stored as cytoplasmic lipid droplets to shield cells from lipotoxicity, though this NEFA buffering capacity is limited in the proximal tubule. Multiple factors converge to cause NEFA accumulation in diabetic nephropathy, but most notably reabsorption of filtered albumin bound to NEFA. We hypothesize that luminal NEFA uptake by fatty acid transporter-2 (FATP2) causes proximal tubule lipoapoptosis, which contributes to tubular atrophy and progression of diabetic nephropathy. The hypothesis will be pursued with the following specific aims: (1) To determine whether FATP2 is the major transporter for luminal proximal tubule NEFA uptake, fluorescently labeled NEFA uptake will be measured in isolated perfused proximal tubules from FATP2-deficient mice, and apoptosis will be assayed in cultured proximal tubule cells treated with shRNAs directed against FATP2; (2) To determine the pathophysiological relevance of FATP2- mediated NEFA internalization in diabetic nephropathy 13C-labeled NEFA uptake will be assayed in vivo in eNOS-/-db/db mice prone to diabetic nephropathy, and the effects of insulin on FATP2-dependent NEFA uptake in proximal tubule cells will be measured; (3) Genetic and pharmacologic tools will be employed to test whether proximal tubule NEFA uptake regulates diabetic nephropathy progression in eNOS-/-db/db mice. Upon completion of these experiments we are hopeful that FATP2 will emerge as a druggable target for the treatment of diabetic nephropathy.