Date of Award

2015

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

Rick G. Schnellmann

Second Advisor

James Chou

Third Advisor

Craig Beeson

Fourth Advisor

Robin C. Muise-Helmericks

Fifth Advisor

Doug Pittman

Abstract

Mitochondrial damage and dysfunction are major pathophysiological mechanisms underlying acute kidney injury (AKI). Following various forms of AKI, mitochondrial biogenesis, the de novo generation of new, functional mitochondria, is suppressed. Pharmacological stimulation of PPARγ-coactivator-1α (PGC-1α), the master regulator of mitochondrial biogenesis, promotes recovery of mitochondrial and renal function after AKI. The primary goals of this project were the evaluation of renal cGMP as a modulator of mitochondrial biogenesis in AKI, and the assessment of phosphodiesterase (PDE) inhibitors and guanylyl cyclase (GC) activators as novel agents to induce mitochondrial biogenesis and promote renal recovery. cGMP has been demonstrated to stimulate mitochondrial biogenesis and function. Both cGMP generation through guanylyl cyclase and degradation through PDEs are highly regulated processes. Compounds that regulate cGMP levels, including the PDE3 inhibitors cilostamide and trequinsin, and the PDE5 inhibitor sildenafil, increased mitochondrial gene expression, and elevated mitochondrial respiration in renal proximal tubule cell (RPTC) cultures. Furthermore, these compounds increased renal cortical mitochondrial gene expression and mtDNA copy number in naïve mice. PDE4 inhibitors, which regulate only cAMP levels had no effect. Treatment of mice with sildenafil in a folic acid model of AKI promoted recovery of mitochondrial gene expression, increased mtDNA content, and reduced expression of the tubular injury marker, kidney injury molecule-1 (KIM-1). An occurrence of AKI is a strong risk factor for the development of chronic kidney disease (CKD) and end stage renal disease (ESRD). Following folic acid-induced AKI, mice developed renal fibrosis and early signs of CKD. These changes were associated with a chronic suppression of mitochondrial gene expression and mtDNA copy number. Treatment of mice with trequinsin or sildenafil failed to restore mitochondrial gene expression, and failed to prevent the progression of fibrosis. Induction of cGMP through activation of guanylyl cyclase by the compound BAY 58-2667 increased mitochondrial respiration in RPTC, and additionally, increased mitochondrial gene and protein expression, and mtDNA copy number in mouse renal cortex. Daily treatment of mice with BAY 58-2667 beginning 24 h after renal ischemia-reperfusion (I/R) injury promoted renal functional and morphological recovery at 144 h after reperfusion. Renal inflammation and oxidative stress were also reduced. Renal recovery was associated with recovery of mitochondrial gene and protein expression, and mtDNA content in the renal cortex. Taken together, we have demonstrated that cGMP is a regulator of mitochondrial biogenesis in the kidney, and activation of mitochondrial biogenesis through modulators of cGMP can promote recovery of renal function following AKI. PDE inhibitors and guanylyl cyclase activators represent novel therapeutics that warrant further evaluation as potential interventions to treat AKI and other diseases characterized by mitochondrial dysfunction. To provide a platform for clinical evaluation of mitochondrial biogenics in renal disease in humans, we examined urinary ATP synthase subunit beta and urinary mtDNA as non-invasive biomarkers of renal mitochondrial dysfunction. These markers were shown to be efficacious as diagnostic and/or prognostic biomarkers of renal damage and mitochondrial dysfunction, and will aid in future development of mitochondrial-targeted renal therapies.

Rights

All rights reserved. Copyright is held by the author.

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