Matthieu Bourderioux1, Thao Nguyen-Khoa1-3, Cerina Chhuon2, ¶, Danielle Tondelier1, Mario Ollero5, Marie Courbebaisse4, Soumeya Bekri6, Bertrand Knebelmann 7, Aleksander Edelman1, Ida Chiara Guerrera 1-2.
1INSERM U1151 ; 2Proteomics Platform Necker, PPN-3P5, SFR Necker, INSERM US24, Université Paris Descartes, Sorbonne Paris Cité; 3Assistance Publique-Hôpitaux de Paris, Laboratory of General Biochemistry ; 4Assistance Publique-Hôpitaux de Paris, Department of Renal Physiology, Hôpital Européen Georges Pompidou ; 5INSERM U955, Université Paris−Est Créteil Val-de-Marne ; 6Department of Biochemistry, CHU Charles Nicolle ; 7Assistance Publique-Hôpitaux de Paris, Nephrology Department, Necker Hospital.
Cystinuria is a purely renal, rare genetic disease caused by mutations in cystine transporter genes and characterized by defective cystine reabsorption leading to kidney stones. In 14% of cases, patients undergo nephrectomy, but it is currently difficult to predict the evolution of the disease. The identification of predictive markers of kidney damage would improve the follow-up of patients with a higher risk. Exosomes are nanovescicles secreted by various cell types and circulating in all body fluids. A growing number of studies points out the role of exosomes in cell signaling and in cell-to-cell communication, raising the question of their importance in physiology and physiopathology. Urinary exosomes have captured the attention of the nephrology community as they may reflect renal pathophysiology and therefore being a precious source of biomarkers. We performed a clinical pilot study on 8 cystinuria patients and compared their exosomal proteins profiles to those of 10 controls, each individually. We developed a robust, reproducible, and noninvasive methodology for proteomic analysis of urinary exosomes using high resolution mass spectrometry (MS) of urinary exosomal proteins. This allowed the identification of ≈1200 proteins, including proteins related to biogenesis of the exosomes (Alix, CD9, TSG101), and membrane renal proteins (AQP2, rBAT). Statistical analysis highlighted 165 proteins differentially expressed in cystinuria compared to healthy subjects, of which 38 were up-regulated. These proteins include known markers of kidney injury and circulating proteins. Interestingly, a third of the up-regulated proteins in cystinuria patients are typical of a neutrophilic signature, namely: azurocidin, cathelicidin antimicrobial peptide, coronin-1A, cathepsin G, neutrophil defensin 3, neutrophil elastase(ELANE), histidine-rich glycoprotein, integrin alpha-M, neutrophil gelatinase-associated lipocalin(LCN2), plastin-2, matrix metalloproteinase-9(MMP9), myeloperoxidase(MPO), myeloblastin and leukocyte elastase inhibitor. The increase of MPO, MMP9, LCN2 and ELANE was validated by immunobloting, on six additional cystinuria patients. To our knowledge, this is the first successful proteomic study in cystinuria suggesting a subclinical intrarenal inflammatory reaction to microscopic crystals. We are currently developing a multiplexed method to dosage the up-regulated proteins by MS in order to evaluate the predictive power and the specificity of these markers.