Date on Master's Thesis/Doctoral Dissertation

5-2019

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, PhD

Committee Chair

Merchant, Michael

Committee Co-Chair (if applicable)

Arteel, Gavin

Committee Member

Barati, Michelle

Committee Member

Beier, Juliane

Committee Member

Freedman, Jonathan

Committee Member

Hoyle, Gary

Author's Keywords

liver disease; alcoholic liver disease; inflammation; fibrosis; hepatorenal syndrome; oxidative stress

Abstract

Background. Ethanol (EtOH) consumption is known to affect multiple organs; this is unsurprising, as the concentration of EtOH in the blood at relevant doses reaches the millimolar range. The overarching goal of this dissertation was to elucidate mechanisms of alcohol-induced organ injury, specifically the effects of alcohol on the hepatic extracellular matrix (ECM) proteome, the alcoholic hepatitis (AH) plasma peptidome, and the effects of alcohol on the renal cortex proteome and transcriptome. Methods. Mice were pair-fed ethanol-containing liquid diet chronically, and then some mice were administered lipopolysaccharide (LPS). Liver sections from these mice were processed in a series of increasingly rigorous extraction buffers to separate proteins by ‘age’ and crosslinking. Extracted proteins were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). For the AH study, a workflow was developed for the peptidomic analysis of plasma from healthy participants or AH patients. AH severity was stratified by MELD score as mild (19; n=37). The peptidome in AH and control plasma was analyzed with LC-MS/MS. For the kidney study, renal cortex proteins were extracted in lysis buffer, and RNA was also isolated. Extracted proteins were identified using LC-MS/MS, and RNA sequencing (i.e. transcriptomics) identified transcripts. Results. Chapter III introduced a new proteomic approach for characterizing the hepatic matrisome, which demonstrated that the hepatic matrisome responds dynamically to both acute (LPS) and chronic (ethanol) stresses, long before more dramatic fibrotic changes to the liver. Chapter IV demonstrated that AH causes detectable changes in the plasma ECM degradome/peptidome of patients, and that the LC-MS/MS analysis of the plasma peptidome is a novel, minimally-invasive method for prognosis stratification in patients with AH. Finally, Chapter V revealed that chronic, moderate ethanol consumption affects renal cortical oxidant response pathways at the protein and transcript level. Conclusions. The work presented in this dissertation has, in conclusion, revealed that the hepatic ECM responds dynamically to stress, plasma peptides, including ECM peptides, change with AH severity, and chronic ethanol consumption affects renal cortical oxidant response pathways.

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