Mechanisms of tubular atrophy

描述(申请人提供)：在美国，超过2600万人患有慢性肾脏疾病，最常见的原因是糖尿病肾小球损伤。然而，进展到终末期与近端小管消失(肾小管萎缩)更紧密地联系在一起，这是由细胞凋亡引起的。在之前的资助期间，我们确定了非酯化脂肪酸(NEFA)代谢产物长链酰基辅酶A(LC-CoA)在细胞内的积累通过抑制NHE1 Na+/H+交换器依赖的细胞存活的机制来刺激肾小管萎缩。过量的LC-COA以细胞质脂滴的形式储存，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有限。多种因素汇聚在一起，导致糖尿病肾病的NEFA积聚，但最显著的是过滤后的白蛋白与NEFA结合后的重吸收。我们假设脂肪酸转运蛋白-2(FATP2)的腔内NEFA摄取导致近端小管脂细胞凋亡，从而导致肾小管萎缩和糖尿病肾病的进展。(1)为了确定FATP2是否是管腔近端小管摄取NEFA的主要转运体，将检测FATP2缺陷小鼠的离体灌流近端小管对NEFA的摄取，并用针对FATP2的shRNA处理培养的近端小管细胞，检测细胞凋亡；(2)为了确定FATP2介导的NEFA内化在糖尿病肾病中的病理生理学相关性，将在体内检测易患糖尿病肾病的eNOS/db/db小鼠对13C标记的NEFA摄取，以及胰岛素对近端小管细胞摄取依赖FATP2的NEFA的影响；(3)遗传学和药理学工具将被用来测试近端小管NEFA摄取是否调节eNOS-/-db/db小鼠的糖尿病肾病进展。在这些实验完成后，我们希望FATP2将成为治疗糖尿病肾病的可用药靶点。

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.