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Diagnostic Urinary Proteome Profile for Immunoglobulin a Nephropathy

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Diagnostic Urinary Proteome Profile for Immunoglobulin a Nephropathy Archive of SID KIDNEY DISEASES Diagnostic Urinary Proteome Profile for Immunoglobulin A Nephropathy Shiva Samavat,1,2 Shiva Kalantari,3 Mohsen Nafar,3 Dorothea Rutishauser,4,5 Mostafa Rezaei-Tavirani,6,7 Mahmud Parvin,8 Roman A Zubarev4,5 1Department of Nephrology, Introduction. Immunoglobulin A (IgA) nephropathy, the most Shahid Labbafinejad Medical common type of glomerulonephritis, is only diagnosed by invasive Center, Shahid Beheshti kidney biopsy. Urine proteome panel might help in noninvasive University of Medical Sciences, Tehran, Iran diagnosis and also better understanding of pathogenesis of IgA 2Urology and Nephrology nephropathy. Research Center, Shahid Materials and Methods. Second mid-stream urine samples of 13 Beheshti University of Medical patients with biopsy-proven IgA nephropathy and 8 healthy controls Sciences, Tehran, Iran were investigated by means of nanoscale liquid chromatography 3Chronic Kidney Diseases Research Center, Shahid tandem mass spectrometry. Multivariate analysis of quantified Beheshti University of Medical label-free proteins was performed by the principal component Sciences, Tehran, Iran analysis and partial least squares models. 4Department of Medical Results. A total number of 493 unique proteins were quantified Biochemistry and Biophysics, by nanoscale liquid chromatography tandem mass spectrometry, Karolinska Institute, Stockholm, of which 46 proteins were considered as putative biomarkers of Sweden 5SciLifeLab, Stockholm, Sweden IgA nephropathy, after multivariate analysis and additional filter 6Department of Basic Sciences, criterion and comparing the patients and the controls. Some of the Faculty of Paramedical Sciences, significant differentially expressed proteins were CD44, glycoprotein Shahid Beheshti University of 2, vasorin, epidermal growth factor, CLM9, protocadherin, Medical Sciences, Tehran, Iran utreoglobin, dipeptidyl peptidase IV, NHL repeat-containing protein 7Proteomics Research Center, Faculty of Paramedical Sciences, 3, and SLAM family member 5. These proteins were related to Original Paper Shahid Beheshti University of various involved pathogenic pathways of inflammatory response Medical Sciences, Tehran, Iran and complement system. 8Department of Pathology, Conclusions. This proteome profile could be utilized in the diagnosis Shahid Labbafinejad Medical of IgA nephropathy. In addition, providing a noninvasive diagnostic Center, Shahid Beheshti tool, it may shed light on the pathogenesis of IgA nephropathy. University of Medical Sciences, Tehran, Iran IJKD 2015;9:239-48 www.ijkd.org Keywords. IgA glomerulonephritis, urine, proteomics, mass spectrometry INTRODUCTION mesangial deposition of IgA immune complexes As the most common primary glomerulonepheritis with a spectrum of morphologic characteristics: worldwide, immunoglobulin A nephropathy (IgAN) from mesangial expansion and proliferation to is not as benign as it was thought.1 Nearly, 20% of crescent formation and sclerosis and fibrosis.3 patients with IgAN would reach end-stage renal Several factors affect pathophysiology of IgAN such disease over 20 years of follow-up.2 Renal biopsy is as complement factor C3, galactosyltransferase, required for definite diagnosis of IgAN. Diagnosis of and serum uric acid, the latter of which has been IgAN is based on histopathologic features, especially well reviewed by Nasri and colleagues.4 Iranian Journal of Kidney Diseases | Volume 9 | Number 3 | May 2015 239 www.SID.ir Archive of SID Urinary Biomarkers of Immunoglobulin A Nephropathy—Samavat et al Four decades after the first description of IgAN collected. Urine proteome was extracted, digested, by Berger,5 there is ambiguity in the pathogenesis of and prepared for liquid chromatography coupled the disease. Apart from the genetic predisposition, with mass spectrometry according to Kalantari and a wide range of immunologic processes (both innate colleagues’ protocol.15 Protein concentrations of and adaptive), cytokines, and growth factors are samples were determined using the bicinchoninic involved in pathogenesis of IgAN.6-8 Complement acid protein assay (Pierce, Thermo Scientific, systems,9 Toll-like receptors,10,11 and monocytes USA).16 are the main players of innate immunity, while Nanoscale liquid chromatography tandem mass B cells, T cells (regulated by vitamin D3),12 and spectrometry analyses were performed on an Easy- immune complex formation are components of nLC system coupled online to a Q Exactive mass adaptive immune system known to be involved.13 spectrometer (Thermo Scientific, Bremen, Germany). Urine proteomic analysis suggests a noninvasive A 10-cm fused silica tip column (SilicaTip, New test to evaluate changes in multiple proteins Objective Inc, Woburn MA, USA) in-house packed expression simultaneously, which could replace with Reprosil-Pur C18-AQ 3-µm resin (Dr Maisch renal biopsy in the diagnosis of IgAN. In addition GmbH, Ammerbuch-Entringen, Germany) used to its use in diagnosis, the urine proteome panel for peptide separation. Mobile phases consisted might offer means to assess pathways involved of 0.1% formic acid in water v/v (buffer A) and in the pathogenesis of IgAN.14 In this study, 0.1% formic acid in acetonitrile v/v (buffer B). we applied proteomic technic, nanoscale liquid The liquid chromatography gradient was set up chromatography tandem mass spectrometry, on as follows: 5% to 35% buffer B in 89 minutes, 48% urine samples of patients with IgAN to identify to 80% buffer B in 5 minutes, and 80% buffer B novel biomarkers for diagnosis and shed light on for 8 minutes, all at a flow rate of 300 nL/min. the pathogenesis pathways. Approximately 2.3 µg of total protein were injected via a temperature-controlled autosampler. MATERIALS AND METHODS Patients and Samples Mass Spectrometry Data Analysis At Shahid Labbafinejad Medical Center, 13 The mass spectrometry acquisition method was patients (11 men and 2 women) aged between 18 comprised of 1 survey full scan ranging from m/z and 52 years old (mean age, 33 years) with biopsy- 300 to m/z 1650 acquired with a resolution of proven IgAN and 8 healthy volunteers (6 men and R = 70 000 at m/z 400, followed by data-dependent 2 women) between 28 and 45 years old (mean age, high-energy collision dissociation tandem mass 34.5 years) were consecutively enrolled in this spectrometry of maximum 10 most abundant study. Patients with signs of systemic involvement precursor ions with a charge state ≥ 2. The tandem suggesting Schonlein-Henoch purpura, systemic mass spectrometry spectra were acquired with a lupus erythematosus, or chronic liver disease were resolution of R = 17 500. excluded. Age, sex, and demographic data of the Tandem mass spectra were extracted using patients were noted and patients with concurrent Raw2MGF (in-house-written program) and diseases such as diabetes mellitus were excluded. searched against a concatenated SwissProt protein None of the patients had gross hematuria at the database (Human taxonomy) using Mascot 2.3.0 time of sampling. At presentation, each patient search engine (Matrix Science Ltd, London, UK). was evaluated for serum creatinine level, estimated Carbamidomethylations of cysteins was set as a glomerular filtration rate (based on the CKD-EPI fixed modification, and deamidation of asparagine equation), presence of hypertension, and amount and glutamine as well as oxidation of methionine of proteinuria. All of the patients were from the were set as variable modifications. Up to 2 missed same ethnicity. tryptic cleavages were allowed and the mass tolerance was set to 10 ppm and to 0.05 Da for the Sample Preparation and Liquid precursor and fragment ions, respectively. Chromatography Tandem Mass Spectrometry Relative abundance of proteins identified with Approximately 20 mL to 40 mL of a second 2 or more unique peptides and a significance morning midstream urine from patients was threshold of E less than 0.05 was determined 240 Iranian Journal of Kidney Diseases | Volume 9 | Number 3 | May 2015 www.SID.ir Archive of SID Urinary Biomarkers of Immunoglobulin A Nephropathy—Samavat et al using Quanti (an in-house developed software RESULTS package)17 which performs accurate label-free Patients peptide and protein quantification with correction A cohort of 13 patients with biopsy-proven for instrumental response fluctuations. The list of IgAN and 8 healthy controls were enrolled in the quantified proteins was further filtered to 1% false study. The mean estimated glomerular filtration discovery rate, which corresponded to the protein rate level was 67.7 mL/min/1.73 m2 and the mean Mascot score of 20.49. 24-hour protein excretion was 3.01 g/d among the patients, with 10 having a urine protein excretion Statistical Analysis rate higher than 1 g/d. Clinical and laboratory The quantitative proteome data was subjected data on patients are provided in Table 1. to multivariate statistical analysis using SIMCA (SIMCA-p 13.0, Umetrics, Umeå, Sweden). Principal Component Analysis Unsupervised principal component analysis A total number of 493 unique proteins were (PCA)18,19 was performed without consideration identified and quantified by the nanoscale liquid of group information for observing the overview chromatography tandem mass spectrometry. The of the data structure, detecting the clusters of the proteins were considered as primary variables, data and recognizing the outliers if any. Partial while patients and controls (with 2 replicates for Least Squares (PLS) analysis20 was applied using each one) were set as observations. Score
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