Identification of protein markers of drug-induced nephrotoxicity by MALDI MS: in vivo discovery of ubiquitin-t

Abstract

This study demonstrated the utility of direct tissue MALDI MS in identifying early markers of antibiotic induced nephrotoxicity. Using a histology directed approach, twelve proteins were identified as having statistically significant changes as a result of gentamicin and kanamycin treatment. These proteins can be linked back to proposed mechanisms of toxicity thereby not only supplying additional diagnostic markers but providing mechanistic insight as well. One of the markers identified was a C-terminally truncated ubiquitin termed ubiquitin-t. Though this protein was originally described as an experimental artifact, this work demonstrates for the first time that the C-terminal cleavage of ubiquitin is created by cathepsin B in vivo. The activity of this cleavage was recapitulated and visualized in situ with exogenous ubiquitin and IMS directly from tissue. The addition of a CATB inhibitor on the tissue inhibited the reaction as evidenced by lack of a signal corresponding to the truncated ubiquitin in our MALDI images. Studies done to examine the effect of our sample preparation on the presence of this feature resulted in the determination that this cleavage was occurring in vivo. MALDI images comparing a control animal and one dosed with a CATB inhibitor supported our hypothesis that CATB was cleaving ubiquitin in vivo. Cathepsin B activity assays revealed that the decrease in ubiquitin-t seen with drug treatment was a result of decreased CATB activity, further providing a link between lysosomal enzyme activity and drug toxicity.