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

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.

A Novel Strategy to Increase the Yield of Exosomes (Extracellular Vesicles) for an Expansion of Basic Research

“…The suppressive effect of PEGylation might be considered a new benefit for using PEGylated liposomes in treating tumors, because it has been reported that exosomes play a controversial role in tumor progress by stimulating/suppressing the immune system. 2,4) Therefore, PEGylated liposomes may subside the bimodal role of exosomes, particularly in tumor metastasis. These observations suggest that our approach may be a new strategy to stimulate/inhibit the secretion of exosomes derived from cancer cells if the physicochemical properties of liposomes can be correctly manipulated.…”

“…45) Recently, many studies have indicated that exosomes have specialized functions and play a key role in processes such as intercellular signaling and waste management. 2) Consequently, there is growing interest in the clinical applications of exosomes. In the present study, it was shown that liposomes have the ability to upregulate and/or downregulate exosome secretion in response to the surface modification of liposomes.…”

“…Exosomes are extracellular nanoparticles (30-200 nm) secreted by most cells including normal and diseased cells such as tumor cells. 1) They are present in biological fluids such as serum 2) and urine. 3) These natural nanoparticles have several functions in the biological milieu that may be beneficial or harmful.…”

“…These functions include immune stimulation/tolerance, cell-cell communication, cellular resistance, and even tumor metastasis. 2,4) Exosomes are also known to act as a defensive mechanism against any changes in the extracellular environment triggered by disease or stress factors. 5) Cell-derived exosomes are well recognized as efficient carriers of small RNAs to neighboring or distant cells, which has resulted in the preponderance of exosomes as carriers for gene therapy and other therapies over other artificial delivery carriers.…”

Extracellular vesicles and their nucleic acids for biomarker discovery

“…In the absence of proper study design and normative controls it is impossible to partition biological variation from technical variation. It has thus been showed that demographic variables such as age, gender, and ethnicity can affect EV signatures [91][92][93] ; yet control groups over age, gender, and ethnicity common to other clinical research is largely lacking in EVtargeted studies 24,92 . Alternatively, pooled healthy subject samples can be used as a control 94 .…”