Research Article: Proteomic Analysis of Urine Exosomes Reveals Renal Tubule Response to Leptospiral Colonization in Experimentally Infected Rats

Date Published: March 20, 2015

Publisher: Public Library of Science

Author(s): Satish P. RamachandraRao, Michael A. Matthias, Chanthel-Kokoy Mondrogon, Eamon Aghania, Cathleen Park, Casey Kong, Michelle Ishaya, Assael Madrigal, Jennifer Horng, Roni Khoshaba, Anousone Bounkhoun, Fabrizio Basilico, Antonella De Palma, Anna Maria Agresta, Linda Awdishu, Robert K. Naviaux, Joseph M. Vinetz, Pierluigi Mauri, Pamela L. C. Small.

Abstract: BackgroundInfectious 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 findingsExosome 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.ConclusionsWe 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.

Partial Text: Leptospirosis is among the world’s most important zoonotic infectious diseases [1,2], characterized by variable manifestations ranging from asymptomatic or self-resolving acute febrile illness to severe disease with a combination of fever, acute kidney injury, jaundice, severe pulmonary hemorrhage syndrome, refractory shock, and aseptic meningitis [3]. Important advances have been made in diverse aspects of leptospirosis including the differential host responses to leptospiral infection [4–9]. Despite these advances, mechanistic details by which end organ damage develops in some individuals but not in others remain to be elucidated. Further, factors that govern host susceptibility to leptospiral infection are not well understood. A recent study by our collaborative group found that approximately 6% of randomly sampled individuals in a highly endemic rural Amazonian village were chronically colonized by Leptospira without any recent clinical evidence of infection [10]. Although renal colonization by leptospires may occur in humans without serological or clinical evidence of infection, the clinical relevance and functional consequences of leptospiral colonization in humans remain to be characterized. New, less invasive, less expensive and more practical tools (compared to kidney biopsy) are needed to study pathologic changes in the kidney in order to understand clinically relevant sequelae of infection. In this report, we use proteomic analysis of urine exosomes in a rat chronic colonization model as a non-invasive window to kidney function in asymptomatic leptospiral infection.

This proteomic analysis of urinary exosomes in a rat leptospiral colonization model identified important biomarkers of infection, including differentially regulated alanine (membrane) aminopeptidase (CD13) and Tamm Horsfall Protein. Further, there were important sex differences of these biomarkers of leptospiral renal tubular colonization, which is particularly provocative given the male predominance of symptomatic and severe leptospirosis universally found in human clinical studies.



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