Date on Master's Thesis/Doctoral Dissertation
Pharmacology and Toxicology
Pharmacology and Toxicology, MS
Committee Co-Chair (if applicable)
alcohol-induced organ injury; alcoholic liver disease; lipopolysaccharide; extracellular matrix
Background. The association of chronic ethanol (EtOH) consumption with extracellular matrix (ECM) changes in alcoholic liver disease (ALD) is well established in the context of hepatic fibrosis. However, it is becoming increasingly understood that the hepatic ECM responds dynamically to stress well before fibrosis. The term “transitional tissue remodeling” describes qualitative and quantitative ECM changes in response to injury that do not alter the overall architecture of the organ; these ECM changes may contribute to early disease initiation and/or progression. The nature and magnitude of these changes to the ECM are currently poorly understood. In contrast to the liver, EtOH consumption is not considered a risk factor for disease in the kidney. However, the effects of ethanol on the kidney at a molecular level are largely unknown. Methods. The goal of this work was to characterize the impact of 6 weeks EtOH diet and/or acute lipopolysaccharide (LPS) on the hepatic ECM proteome (‘matrisome’) and the renal cortex proteome. Liver sections were processed in a series of increasingly rigorous extraction buffers to separate proteins by ‘age’ and crosslinking. Kidney cortex proteins were extracted in lysis buffer. Extracted proteins were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results. Both EtOH and LPS dramatically increased the number of hepatic matrisome proteins ~25%. The enhancement of LPS-induced liver damage by ethanol preexposure was associated with unique protein changes. In the renal cortex, 170 of 1863 proteins were significantly differentially abundant at FC ≥ 1.2 across groups. Ingenuity Pathways Analysis results show EtOH dysregulated the Nrf2-mediated pathway. Western blot and immunohistochemistry of Nrf2 target proteins validate proteomic findings. Transcriptomic analysis of the renal cortex reinforce proteomic findings and provide additional insight into Nrf2 pathway regulation. Conclusions. An extraction method to enrich the hepatic ECM was developed and characterized. The results demonstrate that the hepatic matrisome responds dynamically to both acute (LPS) and chronic (ethanol) stresses, long before more dramatic fibrotic changes to the liver. These changes may contribute, at least in part, to the pathologic responses to these stresses. It is also interesting that several ECM proteins responded similarly to both stresses, suggesting a common mechanism in both models. The study on the effects of EtOH and/or LPS on the renal transcriptome and proteome supported expected observations and revealed new changes in proteins, transcripts, and pathways. These changes provide insight into mechanisms by which EtOH affects the kidney and alters response to a second pathologic stimulus.
Dolin, Christine E., "The effects of moderate alcohol consumption and inflammation on the hepatic matrisome and the renal cortex proteome." (2017). Electronic Theses and Dissertations. Paper 2649.