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Purpose: Since human urine is the most readily available biofluid whose proteome changes in response to disease, it is a logical sample for identifying protein biomarkers for kidney diseases., Experimental Design: Potential biomarkers were identified by using a multiproteomics workflow to compare urine proteomes of kidney transplant patients with immediate and delayed graft function. Differentially expressed proteins were identified, and corresponding stable isotope labeled internal peptide standards were synthesized for scheduled MRM., Results: The Targeted Urine Proteome Assay (TUPA) was then developed by identifying those peptides for which there were at least two transitions for which interference in a urine matrix across 156 MRM runs was <30%. This resulted in an assay that monitors 224 peptides from 167 quantifiable proteins., Conclusions and Clinical Relevance: TUPA opens the way for using a robust mass spectrometric technology, MRM, for quantifying and validating biomarkers from among 167 urinary proteins. This approach, while developed using differentially expressed urinary proteins from patients with delayed versus immediate graft function after kidney transplant, can be expanded to include differentially expressed urinary proteins in multiple kidney diseases. Thus, TUPA could provide a single assay to help diagnose, prognose, and manage many kidney diseases., Competing Interests: Statement The authors declare there are no conflicts of interest., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Extracellular vesicles (EVs) are bilayered lipid vesicles, 50-1000 nm in diameter and secreted by most types of cells. They contain many proteins, mRNAs, miRNAs, and lipids that reflect the pathophysiological state of the cells they originate from, and are therefore considered to be a rich source of potential biomarkers. In this issue (Pocsfalvi, G. et al., Proteomics Clin. Appl. 2015, 9, 552-567), Pocsfalvi et al. conducted pioneering investigations to determine whether changes in the protein content of EVs occur during progression of autosomal dominant polycystic kidney disease (ADPKD), a common genetic disorder that predominantly affects the kidneys. Most significantly, iTRAQ-based quantitative proteomics showed that cytoskeleton-regulating and Ca(2+) -binding proteins are differentially expressed in urinary EVs of ADPKD patients. Impressively, these proteins are involved in biological processes that are closely related to the pathogenic state of tubular epithelial cells in ADPKD, demonstrating the possibility to monitor the status of patients using urinary EVs., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathophysiological state of urinary system; and that EVs-induced ciliary signaling is a possible mechanism of intercellular communication within the tract. Here, we aimed to analyze the protein expression of urinary EVs during autosomal dominant polycystic kidney disease (ADPKD)., Experimental Design: EVs were isolated from pooled urine samples of healthy control and ADPKD patients at two different stages of the disease and under tolvaptan treatment using the double-cushion ultracentrifugation method. Proteins were identified and quantified by iTRAQ and multidimensional protein identification technology (MudPIT)-based quantitative proteomics., Results: Quantitative analyses identified 83 differentially expressed EV proteins. Many of these have apical membrane origin and are involved in signal transduction pathways of primary cilia, Ca(2+) -activated signaling, cell-cycle regulation, and cell differentiation., Conclusions and Clinical Relevance: The reduced AQP-2 and the increased APO-A1 levels observed in all ADPKD-affected groups may reflects the impaired renal concentrating capability of these patients and correlated with estimated glomerular filtration rate decline. The levels of some upregulated proteins involved in Ca(2+) -activated signaling declined upon tolvaptan treatment. The results obtained suggest that the quantitative proteomics of urinary EVs might be useful to monitor proteins difficult to access noninvasively, and thus advance our understanding of urinary tract physiology and pathology., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)