Spatial analysis of renal acetaminophen metabolism and its modulation by 4-methylpyrazole with DESI mass spectrometry imaging.

Acute kidney injury (AKI) is a common complication in acetaminophen (APAP) overdose patients and can negatively impact prognosis. Unfortunately, N-acetylcysteine, which is the standard of care for the treatment of APAP hepatotoxicity does not prevent APAP-induced AKI. We have previously demonstrated the renal metabolism of APAP and identified fomepizole (4-methylpyrazole, 4MP) as a therapeutic option to prevent APAP-induced nephrotoxicity. However, the kidney has several functionally distinct regions, and the dose-dependent effects of APAP on renal response and regional specificity of APAP metabolism are unknown. These aspects were examined in this study using C57BL/6J mice treated with 300-1200 mg/kg APAP and mass spectrometry imaging (MSI) to provide spatial cues relevant to APAP metabolism and the effects of 4MP. We find that renal APAP metabolism and generation of the nonoxidative (APAP-GLUC and APAP-SULF) and oxidative metabolites (APAP-GSH, APAP-CYS, and APAP-NAC) were dose-dependently increased in the kidney. This was recapitulated on MSI which revealed that APAP overdose causes an accumulation of APAP and APAP GLUC in the inner medulla and APAP-CYS in the outer medulla of the kidney. APAP-GSH, APAP-NAC, and APAP-SULF were localized mainly to the outer medulla and the cortex where CYP2E1 expression was evident. Interestingly, APAP also induced a redistribution of reduced GSH, with an increase in oxidized GSH within the kidney cortex. 4MP ameliorated these region-specific variations in the formation of APAP metabolites in renal tissue sections. In conclusion, APAP metabolism has a distinct regional distribution within the kidney, the understanding of which provides insight into downstream mechanisms of APAP-induced nephrotoxicity.

Desorption Electrospray Ionization Mass Spectrometry Imaging Allows Spatial Localization of Changes in Acetaminophen Metabolism in the Liver after Intervention with 4-Methylpyrazole.

Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the US, and hepatotoxicity is initiated by a reactive metabolite which induces characteristic centrilobular necrosis. The only clinically available antidote is N-acetylcysteine, which has limited efficacy, and we have identified 4-methylpyrazole (4MP, Fomepizole) as a strong alternate therapeutic option, protecting against generation and downstream effects of the cytotoxic reactive metabolite in the clinically relevant C57BL/6J mouse model and in humans. However, despite the regionally restricted necrosis after APAP, our earlier studies on APAP metabolites in biofluids or whole tissue homogenate lack the spatial information needed to understand region-specific consequences of reactive metabolite formation after APAP overdose. Thus, to gain insight into the regional variation in APAP metabolism and study the influence of 4MP, we established a desorption electrospray ionization mass spectrometry imaging (DESI-MSI) platform for generation of ion images for APAP and its metabolites under ambient air, without chemical labeling or a prior coating of tissue which reduces chemical interference and perturbation of small molecule tissue localization. The spatial intensity and distribution of both oxidative and nonoxidative APAP metabolites were determined from mouse liver sections after a range of APAP overdoses. Importantly, exclusive differential signal intensities in metabolite abundance were noted in the tissue microenvironment, and 4MP treatment substantially influenced this topographical distribution.