Inflammatory and fibrotic proteins proteomically identified as key protein constituents in urine and stone matrix of patients with kidney calculi Chanchai Boonla a , Piyaratana Tosukhowong a , Björn Spittau b , Andreas Schlosser c , Chaowat Pimratana d , Kerstin Krieglstein b, ⁎ a Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330 Thailand b Department of Molecular Embryology, Institute for Anatomy and Cell Biology, University of Freiburg, 79104 Freiburg, Germany c Center for Biological Systems Analysis (ZBSA), Core Facility Proteomics, University of Freiburg, 79104 Freiburg, Germany d Division of Urological Surgery, Khon Kaen Hospital, Khon Kaen 40000 Thailand abstract article info Article history: Received 29 August 2013 Received in revised form 27 November 2013 Accepted 27 November 2013 Available online 9 December 2013 Keywords: Proteomics Kidney stone Inflammation Fibrosis S100A8 Fibronectin To uncover whether urinary proteins are incorporated into stones, the proteomic profiles of kidney stones and urine collected from the same patients have to be explored. We employed 1D-PAGE and nanoHPLC-ESI-MS/MS to analyze the proteomes of kidney stone matrix (n = 16), nephrolithiatic urine (n = 14) and healthy urine (n = 3). We identified 62, 66 and 22 proteins in stone matrix, nephrolithiatic urine and healthy urine, respec- tively. Inflammation- and fibrosis-associated proteins were frequently detected in the stone matrix and nephrolithiatic urine. Eighteen proteins were exclusively found in the stone matrix and nephrolithiatic urine, considered as candidate biomarkers for kidney stone formation. S100A8 and fibronectin, representatives of inflammation and fibrosis, respectively, were up-regulated in nephrolithiasis renal tissues. S100A8 was strongly expressed in infiltrated leukocytes. Fibronectin was over-expressed in renal tubular cells. S100A8 and fibronectin were immunologically confirmed to exist in nephrolithiatic urine and stone matrix, but in healthy urine they were undetectable. Conclusion, both kidney stones and urine obtained from the same patients greatly contained inflammatory and fibrotic proteins. S100A8 and fibronectin were up-regulated in stone-baring kidneys and nephrolithiatic urine. Therefore, inflammation and fibrosis are suggested to be involved in the formation of kidney calculi. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Boyce and colleagues firstly demonstrated an altered electrophoretic pattern of urinary proteins (particularly α-globulin fraction) from patients with urinary calculi, and a 3–13 times higher total excretion of urinary proteins in patients than in healthy controls [1]. Later, they reported that cellular elements were abundantly found in decalcified calculi, and this organic matrix comprised approximately 2.5% (w/w) of the dried-weight stone [2]. It is currently accepted as a rule that with- out organic matrix the stone is hardly formed. Also, it is well recognized that cellular biomolecules released into urine, particularly proteins, play a significant role in lithogenesis. Proteomic technology has been employed to identify protein constituents in urine and stone matrix of patients with urolithiasis. Cadieux et al. used surface-enhanced laser desorption/ionization- time-of-flight mass spectrometry (SELDI-TOF MS) for urinary protein profiling (midstream urine) and demonstrated that the ratio of p67 (albumin) to unidentified p24 proteins in urolithiasis patients (n = 25) was higher than in non-stone forming controls (n = 25) [3]. Chutipongtanate et al. discovered a new stone urinary stone inhibitor, trefoil factor 1, using anion exchange chromatography coupled with matrix-assisted laser desorption/ionization (MALDI)-TOF MS and electrospray ionization-quadrupole-time-of-flight (ESI-QTOF) MS [4]. Wai-Hoe et al. identified differential urinary proteins (midstream urine) from 50 healthy, 30 nephrolithiasis and 35 recurrent nephrolithiasis sub- jects using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography (LC)-MS/MS [5], and showed that albumin and immunoglobulins were the most abundant proteins in the patients' urine. The first quantitative proteomic comparison by Wright et al. [6] between urine from urolithiasis patients (n = 57) and non-stone forming patients with unrelated benign urological conditions (n = 57) showed that proteins involved in carbohydrate metabolism were uniquely found in the stone forming group. They also demonstrated that the level of urinary ceruloplasmin was significantly higher in the stone group than in the non-stone forming group, and that ceruloplasmin acted as a promoter of calcium oxalate (CaOx) crystallization [6]. Clinica Chimica Acta 429 (2014) 81–89 ⁎ Corresponding author. E-mail addresses: chanchai.b@chula.ac.th (C. Boonla), kerstin.krieglstein@anat.uni-freiburg.de (K. Krieglstein). 0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.11.036 Contents lists available at ScienceDirect Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim