Article Proteomic Analysis of Urinary Microvesicles and Exosomes in Medullary Sponge Kidney Disease and Autosomal Dominant Polycystic Kidney Disease Maurizio Bruschi,1 Simona Granata,2 Laura Santucci,1 Giovanni Candiano,1 Antonia Fabris,2 Nadia Antonucci,2 Andrea Petretto,3 Martina Bartolucci,3 Genny Del Zotto ,4 Francesca Antonini,4 Gian Marco Ghiggeri ,5 Antonio Lupo,2 Giovanni Gambaro,6 and Gianluigi Zaza 2 1Division of Nephrology, Dialysis, Abstract and Transplantation, Background and objectives Microvesicles and exosomes are involved in the pathogenesis of autosomal dominant Laboratory of polycystic kidney disease. However, it is unclear whether they also contribute to medullary sponge kidney, a Molecular sporadic kidney malformation featuring cysts, nephrocalcinosis, and recurrent kidney stones. We addressed this Nephrology, 3Laboratory of Mass knowledge gap by comparative proteomic analysis. Spectrometry—Core Facilities, Design, setting, participants, & measurements The protein content of microvesicles and exosomes isolated from 4Department of the urine of 15 patients with medullary sponge kidney and 15 patients with autosomal dominant polycystic kidney Research and Diagnostics, and disease was determined by mass spectrometryfollowedby weightedgenecoexpression network analysis,support 5 fi Division of vector machine learning, and partial least squares discriminant analysis to compare the pro les and select the Nephrology, Dialysis most discriminative proteins. The proteomic data were verified by ELISA. and Transplantation, Istituto di Ricovero e Results A total of 2950 proteins were isolated from microvesicles and exosomes, including 1579 (54%) identified in Cura a Carattere fi Scientifico, Istituto all samples but only 178 (6%) and 88 (3%) speci c for medullary sponge kidney microvesicles and exosomes, and Giannina Gaslini, 183 (6%) and 98 (3%) specific for autosomal dominant polycystic kidney disease microvesicles and exosomes, Genoa, Italy; 2Renal respectively. The weighted gene coexpression network analysis revealed ten modules comprising proteins with Unit, Department of similar expression profiles. Support vector machine learning and partial least squares discriminant analysis Medicine, University identified 34 proteins that were highly discriminative between the diseases. Among these, CD133 was upregulated HospitalofVerona, Verona, Italy; and in exosomes from autosomal dominant polycystic kidney disease and validated by ELISA. 6Division of Nephrology and Conclusions Our data indicate a different proteomic profile of urinary microvesicles and exosomes in patients Dialysis, School of with medullary sponge kidney compared with patients with autosomal dominant polycystic kidney disease. The Medicine, Columbus- fi Gemelli University urine proteomic pro le of patients with autosomal dominant polycystic kidney disease was enriched of proteins Hospital Catholic involved in cell proliferation and matrix remodeling. Instead, proteins identified in patients with medullary University, Rome, Italy sponge kidney were associated with parenchymal calcium deposition/nephrolithiasis and systemic metabolic derangements associated with stones formation and bone mineralization defects. Correspondence: Prof. CJASN 14: 834–843, 2019. doi: https://doi.org/10.2215/CJN.12191018 Gianluigi Zaza, Renal Unit, Department of Medicine, University HospitalofVerona, Introduction The hypothesis that extracellular vesicles are present Piazzale A Stefani 1, fi 37126 Verona, Italy. Extracellular vesicles, such as microvesicles (diameter in human urine (8) was con rmed by the proteomic Email: gianluigi. of 100–1000 nm) and exosomes (diameter of 30–100 identification of membrane proteins in a pellet isolated [email protected] nm), are membrane-enclosed particles released by by the ultracentrifugation of urine samples (9). Such most cells under normal and pathologic conditions urinary extracellular vesicles contain cell-specific (1–5). Microvesicles are shed directly from the plasma marker proteins from every segment of the nephron membrane, whereas exosomes are formed by the (9,10), and they offer a source of potentially valuable fusion of intracellular multivesicular bodies (also urinary biomarkers (10). The intrinsic characteristics of known as late endosomes) with the plasma membrane, extracellular vesicles also suggest that they may play leading to the release of their vesicular contents into an important role in kidney development and kidney the extracellular space. These vesicles can mobilize a disease. Accordingly, extracellular vesicles seem to be large number of biologic factors, including receptors, involved in the mechanism of cystogenesis in autoso- other proteins, nucleic acids, and lipids, thus shuttling mal polycystic kidney disease, a common hereditary information to other cells (6). The transfer of RNA and kidney disorder with a prevalence of 0.1%–0.25%. miRNA can reprogram recipient cells and modify their Autosomal polycystic kidney disease gives rise to phenotype (7). predominantly kidney symptoms, including cysts that 834 Copyright © 2019 by the American Society of Nephrology www.cjasn.org Vol 14 June, 2019 CJASN 14: 834–843, June, 2019 Urinary Proteome Analysis Differentiated MSK versus ADPKD, Bruschi et al. 835 progressively disrupt the kidney parenchyma, leading to autosomal dominant polycystic kidney disease was de- interstitial fibrosis, cellular infiltration, and the loss of pendent on the revised Ravine criteria (20). The study functional nephrons. was carried out in accordance with the Declaration of The proteomic analysis of urinary exosome-like vesicles Helsinki and approved by the institutional ethical board (particularly those containing polycystin) revealed ap- of the University Hospital of Verona (Verona, Italy; proximately 500 autosomal dominant polycystic kidney code 1312CESC) and the Independent Ethics Committee disease–associated proteins, many with signaling func- (Comitato Etico Regione Liguria) on October 14, 2014 tions (11). Furthermore, the quantitative proteomic analysis (study number 408REG2014). of urinary extracellular vesicles from patients affected by a complete spectrum of chronic kidney functional damage Isolation of Microvesicles and Exosomes highlighted 30 proteins strongly associated with the autoso- Second morning urine samples were obtained from mal dominant polycystic kidney disease phenotype, includ- patients and healthy donors. Extracellular vesicles were ing periplakin, envoplakin, villin-1, and complement C3 (12). isolated by centrifugation. Briefly, aliquots of 16 ml were In contrast to the wealth of information available for centrifuged at 16,0003g for 30 minutes at 16°C to remove autosomal dominant polycystic kidney disease, little is cells, debris, and organelles, such as mitochondria. To known about the role of extracellular vesicles in the onset obtain the microvesicle fraction, the supernatant was of medullary sponge kidney, a sporadic cystic kidney centrifuged at 22,0003g for 120 minutes at 16°C (21). The malformation that involves nephrocalcinosis and recurrent microvesicle pellet was rinsed in PBS and centrifuged again kidney stones (13). The detailed analysis of extracellular at 22,0003g; this rinse/centrifugation cycle was carried out vesicles could provide insight into the pathogenesis of this five times in total to obtain a clean microvesicle fraction. The rare disease. Despite sporadic genetic associations (14,15) 3g – supernatant was then centrifuged at 100,000 for 120 and the dysregulation of a few biologic factors (16 18), the minutes at 16°C to pellet the exosomes. The pellet was systemic and kidney biologic/cellular network underlying resuspended in 1 ml 0.25 M sucrose, loaded on a 1-ml this disease is poorly characterized, and its relationship with 30% sucrose cushion, and centrifuged at 100,0003g for other cystic diseases is unclear. 120 minutes at 16°C. The pellet was rinsed in PBS and To address this knowledge gap, we carried out a com- centrifuged again at 100,0003g for 10 minutes at 4°C, and prehensive comparative proteomic analysis of urinary this rinse/centrifugation cycle was carried out five times microvesicles and exosomes to identify differences between in total to obtain a clean exosome fraction. For each assay, medullary sponge kidney and autosomal dominant poly- we have performed the same purification procedure. Each cystic kidney disease in terms of the mechanism of cysto- pellet fraction was stored at 280°C until use. The size and genesis and identify putative diagnostic biomarkers that purity of microvesicles and exosomes isolated by ultracen- distinguish these diseases. In fact, at the moment, no trifugation were confirmed by dynamic light scattering, diagnostic biomarkers are available for both diseases. whereas the antigen profile of exosomes and microvesicles Although some urinary biomarkers for autosomal dominant was performed by Western blot as described in Supple- polycystic kidney disease (NGAL, M-CSF, and MCP-1) (19) mental Material. have been proposed, none of them have been used in clinical practice (19). Additionally, most of them are only effective in the advanced stage of the disease. Identification of Mass Spectrometry both diseases at early stages could help clinicians start The samples were processed by the in-StageTip method prevention, diet adjustment, and for selected patients, with two poly(styrene divinylbenzene) reverse phase m pharmacologic treatment. Finally, they could potentiate sulfonate disks (22). Each pellet was solubilized in 25 l diagnostic