Histology-guided spatial metabolomics integrated with multi-omics and cellular validation maps renal metabolic heterogeneity in ochratoxin A-induced nephrotoxicity.
Abstract
Ochratoxin A (OTA) is a food contaminant with the kidney as a principal target. However, the regional distribution of OTA-associated metabolic alterations and their relationship to organ-wide molecular responses remain incompletely characterized. This study integrated histology-guided spatial metabolomics, whole-kidney metabolomics, SWATH proteomics, and in vitro cellular experiments to characterize renal responses to OTA exposure. Male C57BL/6 mice received OTA (0.5 mg/kg/day) or vehicle by oral gavage for four weeks (n = 10 per group). Spatial metabolomics was performed in a predefined subset of three animals per group, with renal regions delineated by histology-guided image analysis and evaluated at animal-level. OTA exposure impaired renal function and induced predominantly cortical tubular lesions. No individual spatial metabolic feature survived false discovery rate (FDR) correction. Module-level analyses, defined a priori and interpreted as exploratory, suggested region-specific shifts in antioxidant-redox and tryptophan-indole profiles in the cortex and medulla. Homogenate-based kidney metabolomics identified 48 FDR-significant metabolic features, while SWATH proteomics identified 550 FDR-significant proteins. Following batch-adjustment and animal matching, multi-omics integration further identified six significant metabolite-protein associations. In HK-2 cells, OTA exposure decreased cell viability, increased apoptosis and oxidative/lipid peroxidation-associated indices, while compromising NRF2-GPX4-associated antioxidant defenses. Ferrostatin-1 partially attenuated OTA-induced cellular injury, supporting the involvement of ferroptosis-associated responses in vitro. This study provides an integrated characterization of OTA-associated renal injury at tissue, molecular, and cellular levels, with organ-wide redox and metabolic disturbances, exploratory module-level spatial alterations, and ferroptosis-associated cellular responses.
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