Proteomic analysis of urine exosomes reveals renal tubule response to leptospiral colonization in experimentally infected rats

Background Infectious Leptospira colonize the kidneys of reservoir (e.g. rats) and accidental hosts such as humans. The renal response to persistent leptospiral colonization, as measured by urinary protein biosignatures, has not been systematically studied. Urinary exosomes--bioactive membrane-bound nanovesicles--contain cell-state specific cargo that additively reflect formation all along the nephron. We hypothesized that Leptospira-infection will alter the content of urine exosomes, and further, that these Leptospira-induced alterations will hold clues to unravel novel pathways related to bacterial-host interactions. Methodology/Principal findings Exosome protein content from 24 hour urine samples of Leptospira-infected rats was compared with that of uninfected rats using SDS-PAGE and liquid chromatography/tandem mass spectrometry (LC-MS/MS). Statistical models were used to identify significantly dysregulated proteins in Leptospira-infected and uninfected rat urine exosomes. In all, 842 proteins were identified by LC-MS/MS proteomics of total rat urine and 204 proteins associated specifically with exosomes. Multivariate analysis showed that 25 proteins significantly discriminated between uninfected control and infected rats. Alanyl (membrane) aminopeptidase, also known as CD13 topped this list with the highest score, a finding we validated by Western immunoblotting. Whole urine analysis showed Tamm-Horsfall protein level reduction in the infected rat urine. Total urine and exosome proteins were significantly different in male vs. female infected rats. Conclusions We identified exosome-associated renal tubule-specific responses to Leptospira infection in a rat chronic colonization model. Quantitative differences in infected male and female rat urine exosome proteins vs. uninfected controls suggest that urine exosome analysis identifies important differences in kidney function that may be of clinical and pathological significance.
Author Summary Leptospirosis is a bacterial disease commonly transmitted from animals to humans. Though this disease affects more than three quarters of a million people every year and takes a disproportionate toll on the poor in in tropical regions, few virulence factors have been identified and very little is known regarding the pathogenesis of leptospirosis. Symptoms vary from fever and fatigue to severe pulmonary hemorrhage and death. Approximately 5–10% of Leptospira infections in humans are chronic (>1 year) and asymptomatic (no overt signs of disease). Nonetheless, very little is known about the clinical significance of these infections. In this report, we show that non-invasive tools namely proteomic analysis of urinary exosomes can be used to identify differences between healthy and Leptospira-infected rat kidney and between Leptospira-infected male and female rat kidney. In future studies, these analyses will be extended to determine clinical significance and extent of renal dysfunction in the asymptomatic human.