Chronic ethanol consumption induces mitochondrial protein acetylation and oxidative stress in the kidney

Abstract

In this study, we present the novel findings that chronic ethanol consumption induces mitochondrial protein hyperacetylation in the kidney and correlates with significantly increased renal oxidative stress. A major proteomic footprint of alcoholic liver disease (ALD) is an increase in hepatic mitochondrial protein acetylation. Protein hyperacetylation has been shown to alter enzymatic function of numerous proteins and plays a role in regulating metabolic processes. Renal mitochondrial targets of hyperacetylation include numerous metabolic and antioxidant pathways, such as lipid metabolism, oxidative phosphorylation, and amino acid metabolism, as well as glutathione and thioredoxin pathways. Disruption of protein lysine acetylation has the potential to impair renal function through metabolic dysregulation and decreased antioxidant capacity. Due to a significant elevation in ethanol-mediated renal oxidative stress, we highlight the acetylation of superoxide dismutase, peroxiredoxins, glutathione reductase, and glutathione transferase enzymes. Since oxidative stress is a known factor in ethanol-induced nephrotoxicity, we examined biochemical markers of protein hyperacetylation and oxidative stress. Our results demonstrate increased protein acetylation concurrent with depleted glutathione, altered Cys redox potential, and the presence of 4-HNE protein modifications in our 6-week model of early-stage alcoholic nephrotoxicity. These findings support the hypothesis that ethanol metabolism causes an influx of mitochondrial metabolic substrate, resulting in mitochondrial protein hyperacetylation with the potential to impact mitochondrial metabolic and antioxidant processes.

Keywords: 4-Hydroxynonenal; Acetylation; Ethanol; Glutathione; Kidney; Oxidative stress.

Graphical abstract

Fig. 1

Mitochondrial protein acetylation is increased in renal tissue of ethanol-fed mice. (A) Western blot (n=3 mice per group) and (B) Immunohistochemical analysis with anti-acetyl-lysine antibody demonstrate a significant increase in protein acetylation in renal tissue due to ethanol metabolism (*P<0.05) (size bar=50 μm).

Fig. 2

H&E staining (A and B) did not reveal any grossly abnormal structures, lipid accumulation, or inflammation. As observed by PAS staining, the glomerular basement membranes (blue arrows) and the muscular layer of the afferent arterioles (yellow arrows) of (C) WT and (D) EtOH fed mice were of similar thicknesses. Immunohistochemical analysis of kidney tissue demonstrates a major increase in Cyp2E1 protein levels between control (E) and ethanol feeding groups (F) and minor increase in 4-hydroxynonenal-protein adducts (G and H) as a result of ethanol metabolism (400× magnification). [P=proximal convoluted tubule, D=distal convoluted tubule, G=glomerulus, size bar=20 μm (C and D) and 50 μm (A, B, E, F, G and H).]

Fig. 3

Six weeks of ethanol consumption significantly alters renal cysteine and GSH redox status. Cys (A), CySS (B), GSH (D), and GSSG (E) were determined using dansyl chloride derivatization, HPLC, and fluorescence detection. Redox potential for cysteine (E_hCySS, C) and GSH (E_hGSSG, F) were calculated using the Nernst equation. N=5 per group; *P<0.05, **P<0.01.

Fig. 4

MS analysis reveals renal mitochondrial protein hyperacetylation during chronic ethanol consumption. A total of 215 unique acetylated proteins were identified by our acetylomics approach. 116 Proteins were found in both control and ethanol samples, with 16 and 83 found solely in control and ethanol, respectively. In total, 294 and 454 acetylated peptides were identified in control and ethanol samples, respectively, revealing a 55% increase in acetylated peptides found as a result of ethanol consumption.

Fig. 5

Ethanol metabolism induces renal mitochondrial protein hyperacetylation. Mounting evidence supports the conclusion ethanol metabolism results in an influx of carbon metabolic units into the mitochondria and stimulates the non-enzymatic acetylation of solvent accessible and electrostatic-amenable protein lysine residues. This hyperacetylation likely plays a role in altered protein function, contributing to renal dysfunction.