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.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-04022009-103327 |
| Date | 09 April 2009 |
| Creators | Herring, Kristen |
| Contributors | Richard Caprioli, Richard Armstrong, Larry Marnett, Billy Hudson, Dan Liebler |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu//available/etd-04022009-103327/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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