Role of KLF15 in proximal tubule metabolism

慢性肾脏疾病(CKD)是心血管疾病的主要危险因素，具有 给美国退伍军人带来不成比例的负担。最近的数据表明，尽管急性肾损伤(AKI) 早期肾功能恢复是CKD的主要危险因素。近端小管(PT)是AKI的主要靶点，原因是 它对缺血和DNA损伤的肾毒素(如化疗药物)高度敏感。 受损的PT细胞去分化，最初经历细胞周期停滞，主要发生在G2/M检查点。 这种细胞周期停滞可能允许修复由继发于 线粒体损伤或直接由破坏DNA的毒素所致。持续的细胞周期停滞与 转而分泌促纤维化信号分子，诱导驻留的成纤维细胞增殖和 分化为肌成纤维细胞，开始向纤维性损伤过渡。PT细胞也经历了代谢 重新编程，严重下调脂肪酸b氧化(粮农组织)，并限制补偿 厌氧糖酵解。同时通过过表达PparA或使用过氧化物酶增殖物来恢复FAO 激活的受体α(PPARa)激动剂减弱小鼠模型中的AKI和CKD，这尚未转化为 在临床AKI中的应用，表明需要额外的因素来缓解从AKI到CKD的进展。 KRüppel-like factor15(KLF15)是一种肾脏富集型转录因子，参与多种不同的 细胞过程，包括细胞分化和粮农组织。在退伍军人奖励计划的初始资助期，我们 证明了KLF15在肾小球疾病中的有益作用，导致KLF15上的物质组成IP 退伍军人事务部的激动剂。在最初的阶段，我们还发现KLF15在 分化的PT细胞，但在PT损伤的小鼠模型中显著减少。利用小鼠模型 PT特异性损伤继发于DNA损伤，我们观察到PT特异性基因敲除Klf15加剧了 Aki以及CKD。PT特异性的Klf15基因敲除也增加了涉及细胞周期停滞的途径， 氧化应激，促纤维化信号转导和利用FA生成乙酰辅酶A的途径减少， 在大分子生物合成和能量生产中的中心代谢中间体。我们还观察到了一个 利用推测的和近端的KLF15和PPARa结合位点丰富对FA利用至关重要的基因， 提示KLF15-PPARa在调节FA利用方面具有潜在的协作性。此外，我们 在环境中甘油脂合成途径和脂滴形成显著增加 这可能是一种潜在的DNA损伤后的补偿机制。KLF15表达式 在患有和不患有慢性肾脏病的人肾活检组织中，PPARA的表达也与此相关。此外, 多元回归分析表明，KLF15表达的降低是独立的 与EGFR下降相关，提示KLF15的缺失可能是PT损伤的关键驱动因素。 基于这些初步数据和先前研究的强大科学严谨性，我们假设 KLF15-PPARa协同作用驱动过量游离脂肪酸对乙酰辅酶A和甘油脂的利用 合成以防止不良适应的PT修复后DNA损伤。我们建议通过(1)来检验这一假设 KLF15-PPARa协同作用恢复甲状旁腺损伤后PT代谢的机制探讨 继发于DNA损伤；(2)探讨KLF15-PPARa在继发性甲状旁腺损伤中的作用 DNA损伤。这项提议将通过调查调解机制来解决目前该领域的一个空白 DNA损伤后PT细胞对FA利用的转录调控。我们项目的长期目标是 是为了证明KLF15和PPARa激动剂的组合是一种新的治疗策略 减轻DNA损伤后的PT损伤。寻找治疗AKI的新靶点具有重要意义 退伍军人管理局，考虑到美国退伍军人中CKD的沉重负担。

Chronic kidney disease (CKD) is a leading risk factor for cardiovascular disease, with a disproportionate burden on U.S. Veterans. Recent data demonstrates that acute kidney injury (AKI), despite initial renal recovery, is a major risk factor for CKD. The proximal tubule (PT) is the primary target in AKI due to its high susceptibility to ischemia and DNA-damaging nephrotoxins such as chemotherapeutic agents. Damaged PT cells dedifferentiate, and initially undergo cell cycle arrest, predominantly at the G2/M checkpoint. This cell cycle arrest may allow repair of DNA damage caused by reactive oxygen species secondary to mitochondrial damage or directly by DNA-damaging toxins. Sustained cell cycle arrest is associated with a switch to secretion of pro-fibrotic signaling molecules, inducing resident fibroblasts to proliferate and differentiate to myofibroblasts, beginning the transition to a fibrotic injury. PT cells also undergo metabolic reprograming, with severe downregulation of fatty acid b-oxidation (FAO), and limited compensation by anerobic glycolysis. While restoring FAO either by overexpressing Ppara or by using a peroxisome proliferator activated receptor alpha (PPARa) agonist attenuates AKI and CKD in murine models, this has not translated to use in clinical AKI, suggesting additional factors are required to mitigate the progression from AKI to CKD. Krüppel-Like Factor 15 (KLF15) is a kidney-enriched transcription factor, involved in a diverse range of cellular processes, including cell differentiation and FAO. In the initial funding period of the VA Merit, we demonstrated the salutary role of KLF15 in glomerular disease leading to a composition-of-matter IP on KLF15 agonists by the Veterans Affairs. During this initial period, we also identified that KLF15 is highly expressed in differentiated PT cells, but is significantly reduced in murine models of PT injury. Utilizing a murine model of PT-specific injury secondary to DNA damage, we observed that PT-specific knockdown of Klf15 exacerbated AKI as well as CKD. PT-specific knockdown of Klf15 also increased pathways involving cell cycle arrest, oxidative stress, pro-fibrotic signaling and a decrease in pathways utilizing FA for the generation of acetyl-CoA, a central metabolic intermediate in macromolecule biosynthesis and energy production. We also observed an enrichment of genes critical for FA utilization with putative and proximal KLF15- and PPARa-binding sites, suggesting potential KLF15-PPARa co-operativity in the regulation of FA utilization. In addition, we demonstrated a significant increase in glycerolipid synthesis pathways and lipid droplet formation in the setting of suppressed FAO, suggesting a potential compensatory mechanism post-DNA damage. KLF15 expression was also associated with PPARA expression in human kidney biopsies with and without CKD. In addition, multivariate regression analysis demonstrated that a decrease in KLF15 expression was independently associated with eGFR decline, suggesting that the loss of KLF15 might be a key driver of PT injury. Based on these preliminary data and strong scientific rigor of prior research, we hypothesize that KLF15-PPARa co-operativity drives the utilization of excess free fatty acids for acetyl-CoA and glycerolipid synthesis to prevent maladaptive PT repair post-DNA damage. We propose to test this hypothesis by (1) determining the mechanism by which KLF15-PPARa co-operativity restores PT metabolism after PT injury secondary to DNA-damage and (2) to investigate the requisite role of KLF15-PPARa in PT injury secondary to DNA-damage. This proposal will address a current gap in the field by investigating the mechanisms mediating transcriptional regulation of FA utilization in the PT cells post-DNA damage. The long-term goal of our project is to demonstrate that the combination of KLF15 and PPARa agonists is a novel therapeutic strategy to mitigate PT injury post-DNA damage. Identification of novel targets for the treatment of AKI is of major interest to the VA, given the high burden of CKD among U.S. Veterans.