Mechanisms of tubular atrophy in renal disease

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）更紧密地结合，这是由细胞凋亡引起的。NHE 1 Na+/H+交换器通过与膜磷酸肌醇PI（4，5）P2结合来抑制肾小管上皮细胞（RTC）凋亡。非酯化脂肪酸（NEFA）代谢产物的细胞内积累刺激细胞凋亡。多种因素共同导致糖尿病肾病（DN）中RTC NEFA蓄积，包括与NEFA结合的滤过白蛋白的重吸收。在转运到细胞中之后，NEFA被酯化以形成两亲性长链酰基辅酶A（LC-CoA）中间体，其在结构上类似于PI（4，5）P2，并且优先用作能量来源。过量的LC-CoA以细胞质甘油三酯的形式储存，以保护细胞免受脂毒性的影响，尽管在RTC中NEFA缓冲能力有限。初步数据表明，NEFA、LC-CoA和甘油三酯浓度在来自DN小鼠模型的RTC中升高。在体外，PI（4，5）P2和LC-CoA以相似的亲和力结合NHE 1; PI（4，5）P2刺激，LC-CoA抑制NHE 1依赖的Na+/H+交换和细胞存活。我们推测在DN中，NEFA与白蛋白结合被近端小管重吸收。LC-CoA的形成是代谢不良的，在RTC中积累并导致细胞毒性.过剩的LC-CoA与PI（4，5）P2竞争结合NHE 1胞质尾部，这导致NHE 1依赖性Na+/H+交换和RTC凋亡的失败，导致肾小管萎缩和进行性肾病。 为了表征NEFA和LC-CoA对RTC功能障碍的调节，将测定对照和糖尿病肾病（eNOS-/- db/db）小鼠的肾脏NEFA和LC-CoA浓度以及13 C标记的NEFA摄取。限速，NEFA代谢酶酰基辅酶A合成酶，酰基辅酶A硫酯酶和肉毒碱棕榈酰转移酶的影响将使用药理学抑制剂和基因敲除小鼠解决。通过组织化学染色对所有小鼠进行三酰甘油、细胞凋亡和间质纤维化的表型分型;通过血清肌酐和尿白蛋白：肌酐比率对肾功能进行分型;通过荧光方法对离体NHE 1活性进行分型。在体外，将应用药理学和遗传获得和功能丧失方法来改变野生型和NHE 1无效RTC中的LC-CoA浓度，将分析其细胞凋亡和NHE 1活性。为了确定LC-CoA是否与PI（4，5）P2竞争结合和调节NHE 1，将在体外使用细胞中的荧光显微镜技术、磷脂覆盖物和与固定在琼脂糖珠上的NHE 1结合的荧光标记的PI（4，5）P2的置换来确定LC-CoA与PI（4，5）P2结合NHE 1的竞争。在操作LC-CoA和PI（4，5）P2含量后，将在完整细胞和全细胞贴片中评估NHE 1活性。 公共卫生相关性：美国有超过50万人患有肾衰竭，需要透析或移植才能生存。这个项目的目的是确定为什么 肾衰竭特别是，我们计划研究防止肾脏细胞死亡的生物化学途径，这可能导致新的疗法来阻止肾脏疾病的进展。

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. The NHE1 Na+/H+ exchanger inhibits renal tubular epithelial cell (RTC) apoptosis by binding to the membrane phosphoinositide PI(4,5)P2. Intracellular accumulation of non-esterified fatty acid (NEFA) metabolites stimulates apoptosis. Multiple factors converge to cause RTC NEFA accumulation in diabetic nephropathy (DN), including reabsorption of filtered albumin bound to NEFA. Following transport into cells NEFA are esterified to form amphipathic long-chain acyl-Coenzyme A (LC-CoA) intermediates, which are structurally similar to PI(4,5)P2, and preferentially used as an energy source. Excess LC-CoAs are stored as cytoplasmic triglyceride to shield cells from lipotoxicity, though NEFA buffering capacity is limited in RTC. Preliminary data demonstrate that NEFA, LC-CoA and triglyceride concentrations are elevated in RTC from mouse models of DN. In vitro, PI(4,5)P2 and LC-CoAs bound NHE1 with similar affinities; PI(4,5)P2 stimulated, and LC-CoAs inhibited NHE1-dependent Na+/H+ exchange and cell survival. We hypothesize that in DN, NEFA bound to albumin are reabsorbed by proximal tubules. The formations of LC- CoAs, which are poorly metabolized, accumulate in RTC and lead to cytotoxity. Surplus LC-CoAs compete with PI(4,5)P2 for binding to the NHE1 cytosolic tail, which leads to failure of NHE1-dependent Na+/H+ exchange and RTC apoptosis, resulting in tubular atrophy and progressive kidney disease. To characterize NEFA and LC-CoA regulation of RTC dysfunction, control and diabetic nephropathy (eNOS-/- db/db) mice will be assayed for kidney NEFA and LC-CoA concentration, and 13C-labeled NEFA uptake. The impact of rate-limiting, NEFA-metabolizing enzymes acyl CoA synthetase, acyl CoA thioesterase and carnitine palmitoyl transferase will be addressed using pharmacologic inhibitors and knockout mice. All mice will be phenotyped by histochemical staining for triacylglycerol, apoptosis, and interstitial fibrosis; for renal function by serum creatinine and uine albumin: creatinine ratios; for ex vivo NHE1 activity by fluorescence methods. In vitro, pharmacologic and genetic gain and loss of function approaches will be applied to alter LC- CoA concentration in wild-type and NHE1-null RTC, which will be analyzed for apoptosis and NHE1 activity. To determine whether LC-CoAs compete with PI(4,5)P2 for binding and regulation of NHE1, competition of LC- CoA for PI(4,5)P2 binding to NHE1 will be determined in vitro using fluorescence microscopy techniques in cells, phospholipids overlays and displacement of fluorescently labeled PI(4,5)P2 bound to NHE1 immobilized on Sepharose beads. NHE1 activity will be assessed in intact cells and whole cells patches following manipulation of LC-CoAs and PI(4,5)P2 content. PUBLIC HEALTH RELEVANCE: Over 500,000 people in the U.S. have kidney failure that requires dialysis or transplantation to stay alive. The purpose of this project is to determine why kidneys fail. In particular, we plan to study biochemical pathways that prevent kidneys cells from dying, which may lead to new therapies to halt the progression of kidney diseases.