Date on Master's Thesis/Doctoral Dissertation

8-2017

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, PhD

Committee Chair

Siskind, Leah

Committee Member

Schnellmann, Rick

Committee Member

Beverly, Levi

Committee Member

Mitchell, Thomas

Committee Member

Arteel, Gavin

Author's Keywords

cisplatin; acute kidney injury; sphingolipids; ceramide; glucosylceramide; N-glycans

Abstract

Acute kidney injury (AKI), resulting from cisplatin, remains an obstacle in the treatment of cancer. Cisplatin-induced AKI involves cell death pathways regulated by sphingolipids such as ceramide and glucosylceramide. Results indicate that cisplatin-treated mice had increased levels of ceramide and hexosylceramide. Pre-treatment of mice with inhibitors of ceramide synthesis prevented accumulation of ceramide and hexosylceramide in the renal cortex and attenuated cisplatin-induced AKI. To determine the role of ceramide metabolism to hexosylceramides in kidney injury, we treated mice with an inhibitor of glycosphingolipid synthesis. Inhibition of glycosphingolipid synthesis attenuated the accumulation of the hexosylceramide and exacerbated ceramide accumulation in the renal cortex of cisplatin-treated mice and exacerbated cisplatin-induced AKI. Under conditions of high ceramide generation, data suggest that metabolism of ceramides to glucosylceramides buffers kidney ceramides and attenuate kidney injury. Neutral ceramidase (nCDase) is an enzyme responsible for the breakdown of ceramide. Loss of nCDase is protective in models of injury. Thus, we hypothesized that loss of nCDase would protect mice from cisplatin-induced AKI. In this study, we utilized a nCDase knockout mouse in combination with a mouse model of cisplatin-induced AKI. Data indicate that loss of nCDase protects the kidney from cisplatin-induced AKI as evidenced by improved markers of kidney function, reduced markers of kidney injury, and improved kidney pathology. Data presented indicate that loss of nCDase protects the kidney from the nephrotoxic effects of cisplatin treatment, which suggests that inhibition of this enzyme has potential for development as a renoprotective agent against cisplatin-induced AKI. To study the role of glycosphingolipids in kidney disease, we generated mice containing a doxycyline inducible shRNA that targets expression of UGCG (Tet-O-shUgCg). UCGC is the gene that encodes for glucosylceramide synthase, the enzyme that catalyzes synthesis of glucosylceramide from ceramide. Tet-O-shUgCg mice were bred with CAG+/-rtTA3 mice (CAG-rtTA3/Tet-O-shUgCg) for universal expression of the shRNA. Following UGCG knockdown (KD) renal tertiary lymphoid organ (TLOs) development was observed. TLOs are ectopic accumulations of lymphoid cells that can arise in areas of chronic inflammation via lymphoid neogenesis. UGCG KD mice were also shown to have altered renal N-glycan signatures, suggesting interplay between glycosphingolipid and N-glycan metabolism.

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