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Perturbations in the urinary exosome in transplant rejection.

Sigdel TK, Ng YW, Lee S, Nicora CD, Qian WJ, Smith RD, Camp DG, Sarwal MM - Front Med (Lausanne) (2015)

Bottom Line: The proteome data were analyzed for significant differential protein abundances in AR.Ue AR-specific biomarkers (1) were also detected in Uw, but since they were observed at significantly lower abundances in Uw, they were not significant for AR in Uw.Ue-specific protein alterations in renal disease provide potential mechanistic insights and offer a unique panel of sensitive biomarkers for monitoring AR.

View Article: PubMed Central - PubMed

Affiliation: Division of Transplant Surgery, Department of Surgery, University of California San Francisco , San Francisco, CA , USA.

ABSTRACT

Background: Urine exosomes are small vesicles exocytosed into the urine by all renal epithelial cell types under normal physiologic and disease states. Urine exosomal proteins may mirror disease specific proteome perturbations in kidney injury. Analysis methodologies for the exosomal fraction of the urinary proteome were developed for comparing the urinary exosomal fraction versus unfractionated proteome for biomarker discovery.

Methods: Urine exosomes were isolated by centrifugal filtration of urine samples collected from kidney transplant patients with and without acute rejection (AR), which were biopsy matched. The proteomes of unfractionated whole urine (Uw) and urine exosomes (Ue) underwent mass spectroscopy-based quantitative proteomics analysis. The proteome data were analyzed for significant differential protein abundances in AR.

Results: A total of 1018 proteins were identified in Uw and 349 proteins in Ue. Two hundred seventy-nine overlapped between the two urinary compartments and 70 proteins were unique to the Ue compartment. Of 349 exosomal proteins identified from transplant patients, 220 had not been previously identified in the normal Ue fraction. Eleven Ue proteins, functionally involved in an inflammatory and stress response, were more abundant in urine samples from patients with AR, three of which are exclusive to the Ue fraction. Ue AR-specific biomarkers (1) were also detected in Uw, but since they were observed at significantly lower abundances in Uw, they were not significant for AR in Uw.

Conclusion: A rapid urinary exosome isolation method and quantitative measurement of enriched Ue proteins was applied. Perturbed proteins in the exosomal compartment of urine collected from kidney transplant patients were specific to inflammatory responses, and were not observed in the Ue fraction from normal healthy subjects. Ue-specific protein alterations in renal disease provide potential mechanistic insights and offer a unique panel of sensitive biomarkers for monitoring AR.

No MeSH data available.


Related in: MedlinePlus

Common proteins (e.g., albumin and tubulin) are not found in abundance in the exosomal fraction. Our findings show that the fold change of albumin and tubulin are much lower in the exosomal fraction than soluble fraction of urine.
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Figure 3: Common proteins (e.g., albumin and tubulin) are not found in abundance in the exosomal fraction. Our findings show that the fold change of albumin and tubulin are much lower in the exosomal fraction than soluble fraction of urine.

Mentions: 2D-LC-MS/MS analyses of urine exosome samples resulted in the identification of a total of 349 proteins in the exosomal extract of which 343 proteins were identified in all injury phenotypes (Table S1 in Supplementary Material). We performed gene ontology (GO) analysis which revealed that these proteins were enriched in a number of biological pathways such as platelet degranulation (p-value = 8.07e-4) and activation (p-value = 7.39e-3), humoral immune response (p-value = 1.099e-9), complement activation (p-value = 2.410–9), and response to stress (p-value = 1.06e-4). GO molecular functions enriched are lipid transport (p-value = 1.979e-3), lipoprotein binding (p-value = 3.579e-4), and antioxidant activity (p-value = 5.049e-3). The cellular components enriched included blood extracellular components (p-value = 2.820e-21), cell surface components (p-value = 1.578e-8), blood mircoparticle (p-value = 2.769e-8), secretory granule lumen (p-value = 4.520e-8), and cytoplasmic membrane-bounded vesicle lumen (p-value = 7.849e-8). Evaluation of public domain data for urine proteins previously described in the Ue fraction of healthy subject control urine in both the ExoCarta database and published literature (14, 23) revealed that 220/349 Ue proteins in kidney transplant patients had not been previously identified in the healthy Ue fraction, indicative of unique Ue proteins in kidney transplant recipients. When we compared the 349 proteins to those previously identified in the whole urine proteome data set (13), 70 are unique to the exosomal fraction and 59 of these exclusive Ue proteins had not been previously identified (Figure 2). They mainly enriched for biological pathways related to cell mediated immunity and response to stress. The most significant molecular function was structural molecule activity and their cellular components were mainly extracellular or vesicle components. Twenty-five percent (18) of the proteins exclusive to the exosomal fraction were enriched for their higher expression in the renal cortex, based on our previous studies (24). High abundance urinary proteins, such as albumin and tubulin, were not enriched in high abundance in the Ue fraction (Figure 3).


Perturbations in the urinary exosome in transplant rejection.

Sigdel TK, Ng YW, Lee S, Nicora CD, Qian WJ, Smith RD, Camp DG, Sarwal MM - Front Med (Lausanne) (2015)

Common proteins (e.g., albumin and tubulin) are not found in abundance in the exosomal fraction. Our findings show that the fold change of albumin and tubulin are much lower in the exosomal fraction than soluble fraction of urine.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4292055&req=5

Figure 3: Common proteins (e.g., albumin and tubulin) are not found in abundance in the exosomal fraction. Our findings show that the fold change of albumin and tubulin are much lower in the exosomal fraction than soluble fraction of urine.
Mentions: 2D-LC-MS/MS analyses of urine exosome samples resulted in the identification of a total of 349 proteins in the exosomal extract of which 343 proteins were identified in all injury phenotypes (Table S1 in Supplementary Material). We performed gene ontology (GO) analysis which revealed that these proteins were enriched in a number of biological pathways such as platelet degranulation (p-value = 8.07e-4) and activation (p-value = 7.39e-3), humoral immune response (p-value = 1.099e-9), complement activation (p-value = 2.410–9), and response to stress (p-value = 1.06e-4). GO molecular functions enriched are lipid transport (p-value = 1.979e-3), lipoprotein binding (p-value = 3.579e-4), and antioxidant activity (p-value = 5.049e-3). The cellular components enriched included blood extracellular components (p-value = 2.820e-21), cell surface components (p-value = 1.578e-8), blood mircoparticle (p-value = 2.769e-8), secretory granule lumen (p-value = 4.520e-8), and cytoplasmic membrane-bounded vesicle lumen (p-value = 7.849e-8). Evaluation of public domain data for urine proteins previously described in the Ue fraction of healthy subject control urine in both the ExoCarta database and published literature (14, 23) revealed that 220/349 Ue proteins in kidney transplant patients had not been previously identified in the healthy Ue fraction, indicative of unique Ue proteins in kidney transplant recipients. When we compared the 349 proteins to those previously identified in the whole urine proteome data set (13), 70 are unique to the exosomal fraction and 59 of these exclusive Ue proteins had not been previously identified (Figure 2). They mainly enriched for biological pathways related to cell mediated immunity and response to stress. The most significant molecular function was structural molecule activity and their cellular components were mainly extracellular or vesicle components. Twenty-five percent (18) of the proteins exclusive to the exosomal fraction were enriched for their higher expression in the renal cortex, based on our previous studies (24). High abundance urinary proteins, such as albumin and tubulin, were not enriched in high abundance in the Ue fraction (Figure 3).

Bottom Line: The proteome data were analyzed for significant differential protein abundances in AR.Ue AR-specific biomarkers (1) were also detected in Uw, but since they were observed at significantly lower abundances in Uw, they were not significant for AR in Uw.Ue-specific protein alterations in renal disease provide potential mechanistic insights and offer a unique panel of sensitive biomarkers for monitoring AR.

View Article: PubMed Central - PubMed

Affiliation: Division of Transplant Surgery, Department of Surgery, University of California San Francisco , San Francisco, CA , USA.

ABSTRACT

Background: Urine exosomes are small vesicles exocytosed into the urine by all renal epithelial cell types under normal physiologic and disease states. Urine exosomal proteins may mirror disease specific proteome perturbations in kidney injury. Analysis methodologies for the exosomal fraction of the urinary proteome were developed for comparing the urinary exosomal fraction versus unfractionated proteome for biomarker discovery.

Methods: Urine exosomes were isolated by centrifugal filtration of urine samples collected from kidney transplant patients with and without acute rejection (AR), which were biopsy matched. The proteomes of unfractionated whole urine (Uw) and urine exosomes (Ue) underwent mass spectroscopy-based quantitative proteomics analysis. The proteome data were analyzed for significant differential protein abundances in AR.

Results: A total of 1018 proteins were identified in Uw and 349 proteins in Ue. Two hundred seventy-nine overlapped between the two urinary compartments and 70 proteins were unique to the Ue compartment. Of 349 exosomal proteins identified from transplant patients, 220 had not been previously identified in the normal Ue fraction. Eleven Ue proteins, functionally involved in an inflammatory and stress response, were more abundant in urine samples from patients with AR, three of which are exclusive to the Ue fraction. Ue AR-specific biomarkers (1) were also detected in Uw, but since they were observed at significantly lower abundances in Uw, they were not significant for AR in Uw.

Conclusion: A rapid urinary exosome isolation method and quantitative measurement of enriched Ue proteins was applied. Perturbed proteins in the exosomal compartment of urine collected from kidney transplant patients were specific to inflammatory responses, and were not observed in the Ue fraction from normal healthy subjects. Ue-specific protein alterations in renal disease provide potential mechanistic insights and offer a unique panel of sensitive biomarkers for monitoring AR.

No MeSH data available.


Related in: MedlinePlus