Urine proteomic analysis in cystinuric children with renal stones

Abstract

Summary Introduction The gene mutations responsible for cystinuria do not fully explain kidney stone activity, suggesting that specific proteins may serve as promoters of cystine precipitation, aggregation or epithelial adherence. In this study we assessed (1) the differences in the urinary proteins between children with cystinuria and kidney stones (CYS) and healthy controls (HC), with particular attention to the fibrosis-related proteins, and (2) the presence of diagnostic biomarkers for CYS. Material and methods We conducted a pilot study comparing individual urinary proteomes of 2 newly diagnosed children with CYS and 2 age- and gender-matched HC, using liquid chromatography-mass spectrometry. Relative protein abundance was estimated using spectral counting. Proteins of interest in both CYS and HC were selected using the following criteria: i) ≥5 spectral counts; ii) ≥2-fold difference in spectral counts; and iii) ≤0.05 p-value for the Fisher's Exact Test. Discussion This study demonstrates a different urinary polypeptide profile in two children with CYS compared to two HC. Of the 623 proteins identified by proteomic analysis, 180 exhibited at least a 2-fold increased relative abundance in CYS compared to HC. Of these, 39 were involved in response to stress, 26 in response to wounding, 21 in inflammatory response, 18 in immune response, and 4 in cellular response to oxidative stress. 133 proteins were found only in children with CYS, 33 of which met the selection criteria. Of these 33 unique proteins, six are known to be associated with fibrosis pathways (Table). Accession NumberaProteinMW (kDa)No. of assigned peptidesbNo. of unique peptidesbSequence coverage(%)ACTGActin42202/11752/3770/51VIMEVimentin5410/98/521/14HSP70Heat shock 70 kDa protein7024/187/514/15ITIH2Inter-alpha-trypsin inhibitor10633/8112/1127/16MMP9Matrix metalloproteinase 97826/159/718/12PON1Serum paraoxonase4023/749/1448/55Human.Values presented in each patient (Patient 1/Patient 2). The major limitation of this study is the small number of samples that were analyzed. Validation using highly specific methods such as ELISA is needed. Conclusion We provide proteomic evidence of oxidative injury, inflammation, wound healing and fibrosis in two children with CYS. We speculate that oxidative stress and inflammation may cause remodeling via actin and vimentin pathways, leading to fibrosis. Additionally, we identified ITIH and MMP-9 as potential diagnostic biomarkers and novel therapeutic targets in CYS. These proteins merit further investigation.