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

RamachandraRao SP, Matthias MA, Kokoy-Mondragon C, Mondrogon CK, Aghania E, Park C, Kong C, Ishaya M, Madrigal A, Horng J, Khoshaba R, Bounkhoun A, Basilico F, De Palma A, Agresta AM, Awdishu L, Naviaux RK, Vinetz JM, Mauri P - PLoS Negl Trop Dis (2015)

Bottom Line: In all, 842 proteins were identified by LC-MS/MS proteomics of total rat urine and 204 proteins associated specifically with exosomes.Whole urine analysis showed Tamm-Horsfall protein level reduction in the infected rat urine.We identified exosome-associated renal tubule-specific responses to Leptospira infection in a rat chronic colonization model.

View Article: PubMed Central - PubMed

Affiliation: University of California, San Diego Department of Medicine, Division of Nephrology-Hypertension, San Diego, California, United States of America; University of California, San Diego Department of Pediatrics, Center for Promotion of Maternal Health and Infant Development, La Jolla, California, United States of America.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

Two-dimensional (2D) partial least squares discriminant analysis separation using peptide count concentration-based proteomic measurements in the urine exosome of the rats infected with Leptospira vs control rats without Leptospira infection (n = 3).Although clear separation between control and infected rat urine exosomes is seen, a fraction of overlap between the infected male and infected female rat urine exosome proteins is observed.
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pntd.0003640.g003: Two-dimensional (2D) partial least squares discriminant analysis separation using peptide count concentration-based proteomic measurements in the urine exosome of the rats infected with Leptospira vs control rats without Leptospira infection (n = 3).Although clear separation between control and infected rat urine exosomes is seen, a fraction of overlap between the infected male and infected female rat urine exosome proteins is observed.

Mentions: Urinary exosome proteins in male vs. female infected rat urine were different as determined by PLS-discriminant analysis (Fig. 3). Moreover, the infected male rat urine exosome contents were far more different from those of uninfected rats compared to the infected female rat urine exosome contents. The VIP (Variable Importance in Projection) score of 25 proteins was higher than 1.5 (Fig. 4 and Table 1). Qualitatively, a total of 57 proteins were present among all the infected rats. Of these, only 3 were shared between infected males and females, while 37 were unique to infected males and 17 were unique to infected females. Further, we conducted separate analyses of proteins between proteins of proteins of male infected and female infected rats. Table 2 depicts male infected versus control rat urine exosome analysis. Accordingly, 11 proteins were significantly altered (p < 0.05). Table 3 depicts female infected versus control rat urine exoosme analysis, according to which the number of significantly dysregulated proteins was 9. In the male infected rat urine exosome, the alanyl (membrane) aminopeptidase upregulation not only reached the highest level of significance (p = 0.00019) but also had the lower FDR (3.22%). In the female infected rat however, although this upregulation was significant compared to the uninfected rat, the FDR did not reach the cutoff of <10% (14.37%). Thus both qualitatively and quantitatively, the protein content of exosomes showed gender specificity in infected rats.


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

RamachandraRao SP, Matthias MA, Kokoy-Mondragon C, Mondrogon CK, Aghania E, Park C, Kong C, Ishaya M, Madrigal A, Horng J, Khoshaba R, Bounkhoun A, Basilico F, De Palma A, Agresta AM, Awdishu L, Naviaux RK, Vinetz JM, Mauri P - PLoS Negl Trop Dis (2015)

Two-dimensional (2D) partial least squares discriminant analysis separation using peptide count concentration-based proteomic measurements in the urine exosome of the rats infected with Leptospira vs control rats without Leptospira infection (n = 3).Although clear separation between control and infected rat urine exosomes is seen, a fraction of overlap between the infected male and infected female rat urine exosome proteins is observed.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4368819&req=5

pntd.0003640.g003: Two-dimensional (2D) partial least squares discriminant analysis separation using peptide count concentration-based proteomic measurements in the urine exosome of the rats infected with Leptospira vs control rats without Leptospira infection (n = 3).Although clear separation between control and infected rat urine exosomes is seen, a fraction of overlap between the infected male and infected female rat urine exosome proteins is observed.
Mentions: Urinary exosome proteins in male vs. female infected rat urine were different as determined by PLS-discriminant analysis (Fig. 3). Moreover, the infected male rat urine exosome contents were far more different from those of uninfected rats compared to the infected female rat urine exosome contents. The VIP (Variable Importance in Projection) score of 25 proteins was higher than 1.5 (Fig. 4 and Table 1). Qualitatively, a total of 57 proteins were present among all the infected rats. Of these, only 3 were shared between infected males and females, while 37 were unique to infected males and 17 were unique to infected females. Further, we conducted separate analyses of proteins between proteins of proteins of male infected and female infected rats. Table 2 depicts male infected versus control rat urine exosome analysis. Accordingly, 11 proteins were significantly altered (p < 0.05). Table 3 depicts female infected versus control rat urine exoosme analysis, according to which the number of significantly dysregulated proteins was 9. In the male infected rat urine exosome, the alanyl (membrane) aminopeptidase upregulation not only reached the highest level of significance (p = 0.00019) but also had the lower FDR (3.22%). In the female infected rat however, although this upregulation was significant compared to the uninfected rat, the FDR did not reach the cutoff of <10% (14.37%). Thus both qualitatively and quantitatively, the protein content of exosomes showed gender specificity in infected rats.

Bottom Line: In all, 842 proteins were identified by LC-MS/MS proteomics of total rat urine and 204 proteins associated specifically with exosomes.Whole urine analysis showed Tamm-Horsfall protein level reduction in the infected rat urine.We identified exosome-associated renal tubule-specific responses to Leptospira infection in a rat chronic colonization model.

View Article: PubMed Central - PubMed

Affiliation: University of California, San Diego Department of Medicine, Division of Nephrology-Hypertension, San Diego, California, United States of America; University of California, San Diego Department of Pediatrics, Center for Promotion of Maternal Health and Infant Development, La Jolla, California, United States of America.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus