BASIC RESEARCH www.jasn.org Transcriptomic and Proteomic Profiling Provides Insight into Mesangial Cell Function in IgA Nephropathy † † ‡ Peidi Liu,* Emelie Lassén,* Viji Nair, Celine C. Berthier, Miyuki Suguro, Carina Sihlbom,§ † | † Matthias Kretzler, Christer Betsholtz, ¶ Börje Haraldsson,* Wenjun Ju, Kerstin Ebefors,* and Jenny Nyström* *Department of Physiology, Institute of Neuroscience and Physiology, §Proteomics Core Facility at University of Gothenburg, University of Gothenburg, Gothenburg, Sweden; †Division of Nephrology, Department of Internal Medicine and Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan; ‡Division of Molecular Medicine, Aichi Cancer Center Research Institute, Nagoya, Japan; |Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden; and ¶Integrated Cardio Metabolic Centre, Karolinska Institutet Novum, Huddinge, Sweden ABSTRACT IgA nephropathy (IgAN), the most common GN worldwide, is characterized by circulating galactose-deficient IgA (gd-IgA) that forms immune complexes. The immune complexes are deposited in the glomerular mesangium, leading to inflammation and loss of renal function, but the complete pathophysiology of the disease is not understood. Using an integrated global transcriptomic and proteomic profiling approach, we investigated the role of the mesangium in the onset and progression of IgAN. Global gene expression was investigated by microarray analysis of the glomerular compartment of renal biopsy specimens from patients with IgAN (n=19) and controls (n=22). Using curated glomerular cell type–specific genes from the published literature, we found differential expression of a much higher percentage of mesangial cell–positive standard genes than podocyte-positive standard genes in IgAN. Principal coordinate analysis of expression data revealed clear separation of patient and control samples on the basis of mesangial but not podocyte cell–positive standard genes. Additionally, patient clinical parameters (serum creatinine values and eGFRs) significantly correlated with Z scores derived from the expression profile of mesangial cell–positive standard genes. Among patients groupedaccordingtoOxfordMESTscore,patientswith segmental glomerulosclerosis had a significantly higher mesangial cell–positive standard gene Z score than patients without segmental glomerulosclerosis. By investi- gating mesangial cell proteomics and glomerular transcriptomics, we identified 22 common pathways induced in mesangial cells by gd-IgA, most of which mediate inflammation. The genes, proteins, and corresponding pathways identified provide novel insights into the pathophysiologic mechanisms leading to IgAN. J Am Soc Nephrol 28: ccc–ccc, 2017. doi: https://doi.org/10.1681/ASN.2016101103 The glomerular mesangium is a key element for a The diagnosis relies on histopathologic evaluation number of human kidney diseases, including dia- with findings of deposits of galactose-deficient IgA betic nephropathy, SLE, and IgA nephropathy (IgAN).1 However, knowledge of the role of Received October 18, 2016. Accepted May 5, 2017. mesangial cells in these diseases is not complete, Published online ahead of print. Publication date available at partly due to difficulties in developing appropriate www.jasn.org. 2 animal models and also because of the lack of Correspondence: Dr. Jenny Nyström, Institute of Neuroscience mesangial cell–specificmarkers.IgANisthemostcom- and Physiology, University of Gothenburg, Box 430, SE-405 30 monGNintheworld,and30%–40% of the patients Gothenburg, Sweden. Email: [email protected] develop ESRD within 20–30 years after diagnosis.3 Copyright © 2017 by the American Society of Nephrology J Am Soc Nephrol 28: ccc–ccc, 2017 ISSN : 1046-6673/2810-ccc 1 2 BASIC RESEARCH Journal of the American Society of Nephrology www.jasn.org Table 1. Clinical data for patients with IgAN Immuno Oxford Classification Urine Albumin-to-Creatinine Creatinine, BP Sex Age, yr eGFRa CKD Stage BP Steroids suppressive Progressionb Ratio mmol/L Medication Therapy MEST Woman 54 229 132 39.4 3 130/80 Yes No No 0 0 1 1 24.55 Man 46 323 155 45.6 3 150/90 Yes No No 1 1 1 1 0.60 Woman 45 155 132 40.5 3 140/80 Yes No No 1 0 1 1 23.05 Woman 41 189 165 33.0 3 130/80 Yes No No 0 1 1 1 25.37 Man 49 37 124 58.4 3 125/80 No No No 0 1 0 1 23.86 Man 48 65 139 51.3 3 125/85 No No No 0 0 1 1 218.99 Man 58 70 107 65.6 2 140/80 Yes No No 0 0 1 0 28.49 Man 35 14 103 80.7 2 120/80 Yes No No 0 0 1 0 2.40 Man 58 61 117 67.9 2 130/90 Yes No No 0 0 1 2 20.84 Man 34 5 124 64.9 2 126/85 Yes No No 0 0 1 1 1.18 Man 53 74 96 77.4 2 110/70 No Yesc No 0 1 0 0 227.74 Man 61 560d 84 86.0 2 150/95 Yes No No 0 0 1 0 23.94 Man 65 490d 93 74.0 2 155/95 Yes No No 0 0 1 1 21.88 Woman 17 15 71 108.2 1 130/80 No No No 1 1 1 0 22.24 Man 30 75 91 97.1 1 130/90 Yes No No 0 0 1 0 4.49 Man 20 1.2 76 124.8 1 100/60 No No No 0 0 0 0 26.94 Woman 23 0.6 68 109.3 1 110/65 No No No 0 0 0 0 3.92 Man 24 12 77 125.6 1 120/70 No No No 0 0 0 0 217.45 Woman 23 13 66 113.3 1 120/70 No No No 0 1 1 0 3.77 M, mesangial hypercellularity; E, endocapillary hypercellularity; S, segmental glomerulosclerosis; T, tubular atrophy/interstitial. aeGFR has been calculated using the CKD-EPI creatinine formula.bProgression is change in eGFR over time. c J Am Soc Nephrol Before the biopsy. dCalculated from total urinary protein. 28: ccc – ccc ,2017 www.jasn.org BASIC RESEARCH addition, by integration of the profiling information from both microarray and MS analyses, we have found several com- mon significantly differentially expressed pathways at both the transcriptomic and proteomic levels. Most of these are inflam- matory pathways that might be critical in the understanding of the underlying mechanisms of IgAN. RESULTS Statistics Reveal Important Transcriptomic Information about IgAN In Vivo After quality check, normalized microarray data from 19 pa- tients diagnosed with IgAN (one patient sample was run in duplicate in two different batches as an internal control) and 22 living donor controls were further analyzed (Table 1). A hierarchical clustering separated patient samples from the controls (Figure 1), indicating a distinct transcriptomic dif- ference between patients and controls. Using the Significant Figure 1. Hierarchical clustering of patient and control samples. Analysis of Microarray (SAM)11 method, we found 736 genes Clustering of the glomerular microarray data on the basis of whole- to be significantly differentially expressed between the groups genome expression values using Euclidean distance with the Ward (Supplemental Table 1). The significant genes were analyzed averaging method. Using whole-genome expression profiles, using the Ingenuity Pathway Analysis tool to investigate the samples from patients with IgAN were clearly distinguishable fi from living donor controls. corresponding signi cant pathways. 113 pathways were found to be significant with Fisher exact P,0.05, and most of the pathways were related to inflammation, cytokines, and growth factors. The (gd-IgA) in the glomerular mesangial region in addition to top 25 differentially expressed pathways are listed in Table 2. mesangial cell proliferation and mesangial matrix expansion.4 In an earlier study, it was shown that extracellular matrix Moreover, the amount of gd-IgA deposited is not correlated genes play an important role in IgAN in vitro.9 Toconfirm these with mesangial cell damage or kidney injury.5 However, data in patient material, we investigated Gene Ontology (GO) mesangialcellproliferationandinflammation induced by terms using the significantly differentially expressed genes. As gd-IgA deposits are important for disease development.6–8 seen in Table 3, the top three enriched GO terms for biologic In our previous study,9 we showed that mesangial cells de- processes are extracellular matrix disassembly (GO:0022617), rived from patients with IgAN have increased susceptibility to extracellular structure organization (GO:0043062), and extra- gd-IgA compared with mesangial cells from control patients. cellular matrix organization (GO:0030198). Additionally, Toincrease the understanding of the role of the mesangium in considering GO terms of cellular components as seen in Table IgAN, we investigated IgAN from a broader perspective using 3, extracellular matrix component (GO:0044420) and extra- both a transcriptomic and a proteomic approach. To achieve cellular matrix (GO:0031012) were also found to be highly this, we conducted a human genome microarray analysis of enriched along with basement membrane (GO:0005604) glomeruli from patients with IgAN and healthy controls. The and cell-cell adherens junction (GO:0005913). results were combined with proteomic data obtained from mass spectrometry (MS) analysis of mesangial cells treated Mesangial Cell–Positive Standard Genes Have a Strong with patient-derived gd-IgA. Effect in Patients with IgAN In 2013, Ju et al.10 developed an iterative algorithm “in silico On the basis of the algorithm presented in the study by Ju nanodissection” that predicts cell type–specifictranscriptsus- et al.,10 we investigated the expression profile of cell type– ing large-scale datasets. On the basis of literature curation, positive standard genes in the microarray data. In total, 35 they defined mesangial cell– or podocyte-positive standard positive standard genes were identified in mesangial cells, and genes as genes that are specifically expressed in mesangial cells 50 were identified in podocytes (for details of specific stan- or podocytes in the kidney. In this study, we have used these dard genes see Supplemental Table 2).