Date of Award

2015

Embargo Period

8-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

College

College of Graduate Studies

First Advisor

Rick G. Schnellmann

Second Advisor

Kenneth D. Chavin

Third Advisor

C. James Chou

Fourth Advisor

Zhi Zhong

Fifth Advisor

Jun Zhu

Abstract

Mitochondrial dysfunction is a well-characterized pathophysiological feature of acute kidney injury (AKI). Our laboratory has previously implicated suppression of mitochondrial biogenesis, the process by which cells generate new and functional mitochondria, as an important contributor to development of mitochondrial dysfunction in multiple experimental models of AKI. However, relatively little is known about the molecular mechanisms responsible for disruption of biogenesis in renal cells. The primary goals of this project were to define signaling pathways mediating acute suppression of renal cortical mitochondrial biogenesis and to characterize changes in glycolytic metabolism that might support both cellular and organ function following sepsis-induced AKI. Mitochondrial biogenesis is primarily regulated by peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which has been termed the master regulator of this process. In an endotoxin model of septic AKI in mice, we noted rapid suppression of both renal cortical PGC-1α mRNA and protein levels at 3 and 18 hours post-LPS, respectively. Endotoxin-induced loss of PGC-1α led to reduced expression of downstream regulators of mitochondrial biogenesis and electron transport chain proteins along with a decrease in mitochondrial DNA content. Using genetic and pharmacological approaches, we identified an essential role for TLR4-mediated activation of MEK/ERK signaling in acute (< 3 hr) disruption of PGC-1α expression and subsequent mitochondrial biogenesis in the renal cortex. Elucidation of this pathway may facilitate development of novel therapeutic approaches to reverse mitochondrial dysfunction and enhance renal recovery after AKI. We next examined changes in glycolytic metabolism that might serve as an adaptive mechanism to generate ATP and support renal function in sepsis-induced AKI. We observed a specific and rapid (< 3 hr) increase in activity of renal cortical hexokinase (HK), the first committed step of glycolysis, that was maintained up to 18 h after systemic LPS exposure. LPS-mediated HK activation was not sufficient to increase glucose flux through the glycolytic pathway as indicated by reduced or unchanged pyruvate and lactate levels in the renal cortex. Surprisingly, HK activation was closely associated with increased activity of glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme of the pentose phosphate pathway (PPP), suggesting that glucose is selectively utilized via this pathway following sepsis-induced AKI. We also demonstrated that LPS-induced HK activation occurs in an EGFR/PI3K/Akt-dependent manner in this model. Further work may lead to identification of increased glucose metabolism through the PPP as an adaptive mechanism to counteract oxidative stress in the septic kidney. In our final study, we tested the efficacy of a potent and specific MEK/ERK inhibitor GSK1120212 in a clinically relevant model of sepsis induced by cecal ligation and puncture (CLP) in mice. Pharmacological blockade of MEK/ERK signaling partially attenuated the systemic response to CLP as indicated by reduced levels of circulating proinflammatory cytokines (TNF-α, IL-1β, IL-6, and GM-CSF) and restoration of core body temperature in GSK1120212-treated mice. In the kidney, GSK1120212 post-treatment reversed CLP-induced microvascular perfusion deficits and reduced expression of well-characterized markers of proximal tubular injury. Despite these effects, MEK/ERK inhibition was not sufficient to prevent renal dysfunction as measured by BUN. Taken together, these findings suggest that MEK/ERK inhibition represents a novel approach to partially limit sepsis-induced inflammatory responses and organ injury.

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