Date on Master's Thesis/Doctoral Dissertation

5-2020

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, PhD

Committee Chair

Lederer, Eleanor

Committee Co-Chair (if applicable)

Siskind, Leah

Committee Member

Siskind, Leah

Committee Member

Brier, Michael

Committee Member

Brier, Michael

Committee Member

Merchant, Michael

Author's Keywords

cisplatin nephrotoxicity; metabolic stress; cisplatin biotransformation

Abstract

Background. Acute kidney injury (AKI) develops in 30% of patients who receive cisplatin, a widely used chemotherapeutic agent. We previously showed that NHERF1 loss resulted in increased susceptibility to cisplatin nephrotoxicity. The overarching goal of this dissertation was to elucidate mechanisms of susceptibility to cisplatin-induced AKI, specifically the effects of NHERF1 loss on tubule cell metabolism, tubule cell mitochondrial function, and alterations in oxidative state and/or renal handling of cisplatin. Methods. 2-4 month male wild type (WT) and NHERF1 knock out (KO) mice were treated with either vehicle control or cisplatin (20 mg/kg dose IP) for 4, 24, and 72 hours. Urine was collected for NGAL and kidneys were harvested for histology and the following assays: Thiobarbituric Acid Reactive Substances (TBARS) for lipid peroxidation, γ-glutamyl transferase (GGT) activity, and Western Blot for GGT and cysteine S-conjugate beta lyase (CCBL). Mitochondrial respiration was conducted via the Seahorse XF24 analyzer on non-treated isolated kidney mitochondria. LC-MS analysis was used to evaluate ATP content in non-treated kidneys. Electron microscopy was utilized to evaluate mitochondrial morphology and number in non-treated kidneys. HPLC of the reduced and oxidized forms of the small molecular weight thiols (glutathione (GSH), glutathione disulfide (GSSG), cysteine (Cys), and cystine (CySS), and cysteine-glutathione disulfide (CySSG) on plasma and kidney cortex. Statistical analysis was completed using Student’s t-test for LC-MS, mitochondrial number, and mitochondrial respiration and Two-way ANOVA was used for all other analysis. P-values of Results. Chapter III demonstrates that WT and NHERF1 KO mice do not exhibit metabolic changes or changes in ATP content that would definitively sensitize the KO mice to cisplatin injury. Chapter IV shows that NHERF1 loss does not affect mitochondrial morphology or mitochondrial number, or oxidative phosphorylation capacity via Seahorse XF24 analysis. Thus, mitochondrial dysfunction does not appear to sensitize the KO mice to cisplatin injury. Lastly, Chapter V reveals that NHERF1 KO mouse kidneys do not exhibit changes in lipid peroxidation, oxidative stress, GGT or CCBL protein levels that would sensitize these animals to cisplatin. However, NHERF1 KO kidneys appear to respond differently to the cisplatin insult itself, characterized by differences in GGT activity in response to cisplatin. Conclusions. In conclusion, the work presented in this dissertation reveals that metabolic stress and mitochondrial dysfunction are not the mechanisms of susceptibility to cisplatin in NHERF1 KO mice. Furthermore, NHERF1 loss does not lead to changes in kidney GSH metabolism. In conclusion, these data do not support NHERF1 loss resulting in a fundamental metabolic defect that increases susceptibility to cisplatin injury. Instead NHERF1 loss appears to influence either the handling, the initial insult, or the response to injury resulting in exacerbated injury.

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