Mechanisms of tubular atrophy in renal disease

肾脏疾病中肾小管萎缩的机制

• 批准号: 8371038
• 负责人: JEFFREY R SCHELLING
• 金额: $ 19.63万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8371038
• 关键词: ASCL1 gene; Acyl Coenzyme A; Address; Affinity; Age; Albumins; Apical; Apoptosis; Atrophic; Binding; Binding Proteins; Biochemical Pathway; Biological Assay; Buffers; Carnitine; Cell Survival; Cell membrane; Cells; Chronic Kidney Failure; Cleaved cell; Coenzyme A; Coenzyme A Ligases; Complex; Coupled; Creatinine; Data; Diabetes Mellitus; Diabetic Nephropathy; Dialysis procedure; End stage renal failure; Energy-Generating Resources; Enzymes; Epithelial Cells; Esterified Fatty Acids; Excretory function; Failure; Family; Fatty Acids; Fibrosis; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Functional disorder; Genetic; Human; In Vitro; Injury; Kidney; Kidney Diseases; Kidney Failure; Knockout Mice; Label; Lead; Lipids; Measures; Mediating; Membrane; Methods; Mitochondria; Mus; NHE1; Oocytes; Patch-Clamp Techniques; Pathology; Peptides; Phenotype; Phosphatidylinositols; Phospholipids; Regulation; Renal function; Sepharose; Serum; Staging; Staining method; Stains; Streptozocin; Tail; Techniques; Testing; Tissues; Total Internal Reflection Fluorescent; Transferase; Transplantation; Triglycerides; Tubular formation; Urine; Xenopus oocyte; cytotoxic; db/db mouse; diabetic; in vivo; inhibitor/antagonist; interstitial; kidney cell; kidney epithelial cell; long chain fatty acid; loss of function; mouse model; prevent; research study; scaffold; triacsin C; uptake

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.

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。NHE1Na+/H+交换器通过与膜磷脂酰肌醇PI(4，5)P2结合来抑制肾小管上皮细胞(RTC)的凋亡。非酯化脂肪酸(NEFA)代谢产物在细胞内积累可刺激细胞凋亡。多种因素共同作用导致糖尿病肾病患者的RTC NEFA蓄积，包括与NEFA结合的过滤白蛋白的重吸收。NEFA进入细胞后被酯化，形成两亲性的长链酰基辅酶A(LC-CoA)中间体，其结构类似于PI(4，5)P2，优先用作能源。过量的LC-COA以细胞质甘油三酯的形式储存，以保护细胞免受脂肪毒性，尽管在RTC中NEFA的缓冲能力有限。初步数据显示，糖尿病小鼠模型的RTC中NEFA、LC-CoA和甘油三酯浓度升高。在体外，PI(4，5)P2和LC-COAS以相似的亲和力结合NHE1，PI(4，5)P2被刺激，LC-COAS抑制NHE1依赖的Na+/H+交换和细胞存活。我们假设，在糖尿病肾病中，与白蛋白结合的NEFA被近端小管重新吸收。LC-COAS的形成是代谢不良的，在RTC中积聚，导致细胞毒性。过剩的LC-COA与PI(4，5)P2竞争结合NHE1胞浆尾巴，导致NHE1依赖的Na+/H+交换失败和RTC凋亡，导致肾小管萎缩和进展性肾脏疾病。为了表征NEFA和LC-CoA对RTC功能障碍、对照和糖尿病肾病(eNOS-/-db/db)小鼠肾脏NEFA和LC-CoA浓度以及13C标记的NEFA摄取的调节作用。限速酶，NEFA代谢酶，酰辅酶A合成酶，酰辅酶A硫酯酶和肉碱棕榈酰转移酶的影响将使用药物抑制剂和基因敲除小鼠来解决。所有小鼠将通过三酰甘油、细胞凋亡和间质纤维化的组织化学染色进行表型鉴定；通过血清肌酐和尿白蛋白/肌酐比率对肾功能进行表型鉴定；通过荧光法对体外NHE1活性进行表型鉴定。在体外，药理学和遗传获得和功能丧失的方法将被用来改变野生型和NHE1缺失RTC中LC-CoA的浓度，这将被分析为细胞凋亡和NHE1活性。为了确定LC-CoA是否与PI(4，5)P2竞争NHE1的结合和调节，我们将在细胞、磷脂覆盖和固定在琼脂糖珠上的荧光标记PI(4，5)P2的置换的体外实验中检测LC-CoA对PI(4，5)P2与NHE1结合的竞争。在操纵LC-COAS和PI(4，5)P2含量之后，将在完整细胞和全细胞贴片中评估NHE1的活性。 与公共健康相关：美国有超过50万人患有肾衰竭，需要透析或移植才能存活。这个项目的目的是确定为什么 肾脏衰竭。特别是，我们计划研究防止肾脏细胞死亡的生化途径，这可能会导致阻止肾脏疾病进展的新疗法。