International Urology and Nephrology (2019) 51:593–599 https://doi.org/10.1007/s11255-018-2044-1 UROLOGY - ORIGINAL PAPER Urine proteomic profiling in patients with nephrolithiasis and cystinuria Larisa Kovacevic1 · Joseph A. Caruso2 · Hong Lu1 · Natalija Kovacevic1,3 · Yegappan Lakshmanan1 · Nicholas J. Carruthers2 · David S. Goldfarb4 Received: 11 September 2018 / Accepted: 23 November 2018 / Published online: 5 December 2018 © Springer Nature B.V. 2018 Abstract Purpose The purpose of the study was to assess the differences in the concentration and function of urinary proteins between patients with cystine stones (CYS) and healthy controls (HC). We postulated that CYS and HC groups would demonstrate different proteomic profiles. Methods A pilot study was performed comparing urinary proteomes of 10 patients with CYS and 10 age- and gender- matched HC, using liquid chromatography-mass spectrometry. Proteins which met the selection criteria (i) ≥ 2 unique pep- tide identifications; (ii) ≥ twofold difference in protein abundance; and (iii) ≤ 0.05 p value for the Fisher’s Exact Test were analyzed using Gene Ontology classifications. Results Of the 2097 proteins identified by proteomic analysis, 398 proteins were significantly different between CYS and HC. Of those, 191 were involved in transport processes and 61 in inflammatory responses. The majority were vesicle- mediated transport proteins (78.5%), and 1/3 of them were down-regulated; of those, 12 proteins were involved in endosomal transport (including 6 charged multivesicular body proteins (CHMP) and 3 vacuolar sorting-associated proteins) and 9 in transmembrane transport. Myosin-2 and two actin-related proteins were significantly up-regulated in the vesicle-mediated transport group. Conclusion We provide proteomic evidence of impaired endocytosis, dysregulation of actin and myosin cytoskeleton, and inflammation in CYS. Endosomal transport proteins were down-regulated mainly through defective CHMP. These findings may contribute to further understanding of the pathogenesis of CYS, potentially affecting its management. Keywords Nephrolithiasis · Cystinuria · Urine · Proteomics Introduction Cystinuria is a rare genetic disease that is characterized by impaired transport of cystine and dibasic amino acids in the Electronic supplementary material The online version of this proximal renal tubule [1]. About half of all cases are trans- article (https ://doi.org/10.1007/s1125 5-018-2044-1) contains mitted in an autosomal recessive fashion, caused by muta- supplementary material, which is available to authorized users. tions in the SLC3A1 gene. This gene encodes rBAT, one of * Larisa Kovacevic the two protein components of the neutral and basic amino [email protected] acid transporter [2]. The other half are autosomal dominant with incomplete penetrance, caused by mutations in SLC7A9 1 Department of Pediatric Urology, Children’s Hospital gene, which encodes the light component, the catalytic pro- of Michigan, 3901 Beaubien Blvd, Detroit, MI 48201, USA tein, of the transporter, b0,+ AT. 2 Institute of Environmental Health Sciences, Wayne State Patients with cystinuria often present with kidney stones University, Detroit, MI, USA owing to the low solubility of cystine in urine. Their likeli- 3 Vattikuti Urology Institute, Henri Ford Hospital, Detroit, MI, hood of forming stones is about 50% [3]. Cystine stones are USA large and bilateral in the majority of cases. Even though 4 Nephrology Division, New York University Langone cystine stones represent only 1–2% of all cases with stones, Medical Center, New York, NY, USA Vol.:(0123456789)1 3 594 International Urology and Nephrology (2019) 51:593–599 the morbidity is high due to increased recurrence rate, need HC group consisted of 10 age- and gender-matched vol- for multiple surgeries and the risk of developing chronic unteer individuals. Random mid-stream fresh urine samples kidney disease [4]. Renal function in patients with cystine were collected after a detailed history and physical examina- kidney stones is reduced compared to those with non-cystine tion was performed. The HC participants denied history of stones [5], but the cause of this decline is not entirely clear. kidney stones, had normal urinalysis by dipstick and were The gene mutations identified in cystinuria cannot fully not taking any medications. The urine was obtained for rou- explain kidney stone activity. It has been shown that not all tine urinalysis for clinical care, and would have then been patients with two mutated SLC3A1 genes will form a stone. discarded; it was sent to the proteomics laboratory without Additionally, patients with identical SLC3A1 or SLC7A9 any personal health identification information. Under these genotypes may have onset of stone disease at different ages circumstances, analysis of the urine is not considered human and with different severity. Therefore, other unidentified fac- subjects research. tors seem to contribute to kidney stone formation. These factors may include endogenous proteins that could serve Sample collection and preparation as promoters or inhibitors of cystine precipitation, aggrega- tion or epithelial adherence. Proteomics allows simultaneous Random mid-stream fresh urine samples were obtained in examination of the patterns of multiple urinary proteins and sterile cups, prepared within 3 h of collection (centrifuged has become one of the most promising tools in nephrology at 2500 rpm for 15 min) at both institutions and stored as and urology [6, 7]. Using a proteomic approach, we aimed recommended by standardized protocols (developed by the to assess the differences in the concentration and function of Human Urine and Kidney Proteome Project, HUKPP, and urinary proteins between cystinuria and renal stones (CYS) the European Urine and Kidney Proteomics, EuroKUP Initi- and healthy controls (HC). We postulated that CYS and HC atives) until use [8]. Following preparation at NYU, de-iden- groups would demonstrate different proteomic profiles. We tified urine specimens and data were sent to our institution. also presumed that CYS patients would have a significant Aliquots were stored at − 80 °C until all urine specimens number of proteins involved in abnormal cellular processes were received and processed for proteomic analysis. Each including stress, inflammation and immune response, as well aliquot was used just once. Proteins in each sample were as others yet to be identified. concentrated in a Centricon-type filter. Albumin and IgG were removed by anti-HAS/IgG resin (Sartorius). Protein concentration in each sample was measured by the BCA Methods protein assay (Pierce). Protein digestion and tandem mass tag (TMT) We compared urinary proteomes of 10 patients with CYS labeling and 10 age- and gender-matched HC, using liquid chroma- tography-mass spectrometry (LC–MS/MS). For HC and CYS groups, 10 µg of depleted urine protein from 10 individual samples were pooled together for 100 µg Patient selection total, and then dried. Samples were resuspended in 10 µl of 1% SDS, and denatured for 5 min at 95 °C. Samples were We prospectively enrolled 10 consecutive patients with CYS reduced with 2 mM DTT in 50 mM TEAB for 30 min at (35.4 ± 11.2 years, 5 males and 5 females) who presented 65 °C, followed by alkylation with 6 mM iodoacetamide for routine follow-up visit at New York University Langone for 30 min at RT. Proteins were digested with 2 µg trypsin Medical Center 9 patients), and at Children’s Hospital of overnight at 37 °C. Each sample was uniquely labeled using Michigan (1 patient). All were established patients with an isobaric TMT tag (Pierce, 1.2 mg) in 50% acetonitrile cystinuria who had a history of nephrolithiasis. The diagno- for 1 h at RT, followed by quenching with 0.5% hydroxy- sis of cystinuria was made when a stone composed of cystine lamine. Samples were purified over detergent-removal col- was obtained, and confirmed when urine cystine levels were umns (Pierce) and dried. found to be higher than 75 mg in a 24-h urine collection analyzed by Litholink Corporation (LabCorp, Chicago, IL). 2D LC–MS/MS Random mid-stream fresh urine samples were collected from all participants. At the time of urine collection, patients Peptides were resuspended in 5% acetonitrile/0.1% formic were asymptomatic (i.e., were not experiencing episodes of acid/0.005% trifluoroacetic acid and desalted/separated urinary tract obstruction and/or urinary tract infections), and over a 5-µm reverse phase PLRP-S column (Agilent) using were not taking antibiotics. The patients were taking citrate mobile phases A (2% acetonitrile) and B (98% acetonitrile), (9/10), tiopronin (5/10), or d-penicillamine (2/10). with both buffers adjusted to pH 10.0 using ammonium 1 3 International Urology and Nephrology (2019) 51:593–599 595 hydroxide. Thirty first dimension fractions were collected Results using a TriVersa NanoMate (Advion) system paired to a LTQ-XL mass spectrometer (Thermo). Second dimension Patient’s characteristics peptide separation was performed using reverse phase chro- matography (Acclaim PepMap RSLC column, Thermo) Serum creatinine values were available in 6 out of the 10 under acidic conditions (0.1% formic acid) with an EASY CYS patients, with a mean of 1.09 ± 0.31 mg/dl (range nLC-1000 uHPLC system (Thermo). Peptides were sepa- 0.71–1.59 mg/dl). Mean eGFR for these patients was rated over a 90-min gradient and analyzed with an OrbiTrap 92 ± 38.1 ml/min/1.73 m2 (range 46–146 ml/min/1.73 m2). Fusion mass spectrometer (Thermo). MS1 scans were per- The data regarding comorbidities were available only in formed in profile mode in the orbitrap at 120,000 resolution. 1 patient and included obesity. However, CYS was repre- Data-dependent MS2 acquisitions were triggered on the top sented by a relatively young group of people who, besides 10 most abundant ions (charge states 2–7) and fragmented having cystinuria, are relatively healthy. None of the HC using CID at 35% collision energy in the linear ion trap. had any comorbidities and their serum creatinine concen- Dynamic exclusion was turned on (40 s).