Role of KLF15 in proximal tubule metabolism

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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.