Date of Award

2017

Embargo Period

8-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Drug Discovery and Biomedical Sciences

College

College of Graduate Studies

First Advisor

Craig C. Beeson

Second Advisor

Rick G. Schnellmann

Third Advisor

C. James Chou

Fourth Advisor

DeAnna L. Adkins

Fifth Advisor

Jill Turner

Sixth Advisor

Joshua Lipschutz

Abstract

Mitochondrial dysfunction exacerbates cellular injury, impairs energy-dependent repair, and leads to kidney damage and failure following acute kidney injury (AKI). Mitochondrial dysfunction and impaired mitochondrial biogenesis correlate with decreased in peroxisome proliferator-activated receptor coactivator 1-α (PGC-1α), the reported master regulator of mitochondrial biogenesis, and its downstream targets following I/R-induced AKI. Furthermore, recovery of renal function and tubular injury is accelerated following pharmacological induction of mitochondrial biogenesis following I/R-induced AKI. These results suggest that recovery of mitochondrial number and function may be an effective therapeutic strategy in restoring renal function following AKI. We recently made the novel observation that agonists of 5-HT1F receptor induce mitochondrial biogenesis in vitro and in vivo. Specifically, following I/R-induced AKI, the 5-HT1F receptor selective agonist LY344864 enhanced the recovery of mitochondrial DNA (mtDNA) copy number and renal function, as indicated by decreased blood urea nitrogen (BUN). These findings demonstrate that 5-HT1F receptor stimulation promotes recovery from AKI and activates mitochondrial biogenesis pathways. The goal of the first aim was to determine the signaling pathways involved in mediating renal proximal tubule 5-HT1F receptor-induced mitochondrial biogenesis. Using pharmacological approaches, we identified Gβγ heterodimer-dependent activation of Akt/eNOS/cGMP/PKG/PGC-1α and inhibition of c-raf/ERK/FOXO3a pathways as the mechanism responsible for 5-HT1F receptor-induced mitochondrial biogenesis. We also identified Akt as the link between these stimulatory and inhibitory pathways, and that the stimulatory pathway is required for mitochondrial biogenesis. Elucidation of this pathway may facilitate the development of novel therapeutic approaches to enhance mitochondrial biogenesis for the treatment of diseases characterized by mitochondrial dysfunction. We then examined the role of the 5-HT1F receptor in renal mitochondrial homeostasis and biogenesis under physiological conditions. To complete this aim, we utilized young (10 weeks) and aged (26 weeks) 5-HT1F receptor knockout (KO) mice. In young 5-HT1F receptor KO mice, we observed increased expression of mtDNA copy number as well as of genes involved in renal mitochondrial biogenesis, oxidative phosphorylation, fission and autophagy compared to wild-type (WT) controls. Aged 5-HT1F receptor KO mice also exhibit increases in renal PGC-1α mRNA expression and mtDNA copy number. Interestingly, we detected a tissue-specific difference in renal cortical mitochondrial homeostasis compared to that of the heart. Specifically, cardiac left ventricular mitochondrial homeostasis markers were initially decreased in the absence of the 5-HT1F receptor KO mice compared to WT mice. However, as the mice aged, these markers returned to WT control levels and this rescue was associated with increased PGC-1α mRNA expression. To determine the potential mechanism responsible for tissue-specific differences in mitochondrial homeostasis markers and the compensatory effect displayed in the renal cortex of the 5-HT1F receptor KO mice, we assessed the gene expression of other 5-HT1 and 5-HT2 receptors. Interestingly, in the heart and kidney of 5-HT1F receptor KO mice, there is a tissue-dependent difference in the gene expression of 5-HT2A and 5-HT2B receptors, both of which have been linked to mitochondria. Further work may lead to the identification of compensatory mechanisms that are activated in the absence of the 5-HT1F receptor. Our final study tested the role of the 5-HT1F receptor in renal mitochondrial biogenesis in AKI and in the recovery of mitochondrial and renal function following I/R-induced AKI. The absence of the 5-HT1F receptor increased tubular injury as measured by KIM-1 and neutrophil gelatinase-associated lipocalin (NGAL) at 24 hr following I/R-induced AKI. Additionally, the 5-HT1F receptor KO mice exhibited reduced renal recovery at 144 hr following I/R-induced AKI as measured by serum creatinine and BUN levels. Impaired renal function and tubular injury recovery was also associated with a persistent suppression in mitochondrial biogenesis as evidenced by reduced PGC-1α and respiratory chain protein expression at 144 hr following renal I/R injury. Injured 5-HT1Freceptor KO mice also displayed sustained depletion in ATP generation and elevated oxidative protein damage at 144 hr. In summary, this study reveals that the 5-HT1F receptor 1) regulates mitochondrial biogenesis and homeostasis under physiological conditions in a tissue-dependent manner, 2) is renal protective in the setting of I/R-induced AKI and 3) promotes the recovery of mitochondrial homeostasis and renal function following I/R injury.

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