A New Workflow for Proteomic Analysis of Urinary Exosomes and Assessment in Cystinuria Patients

Abstract

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 given the difficulty to predict the evolution of the disease, the identification of markers of kidney damage would improve the follow-up of patients with a higher risk. The aim of the present study is to develop a robust, reproducible, and noninvasive methodology for proteomic analysis of urinary exosomes using high resolution mass spectrometry. A clinical pilot study conducted on eight cystinuria patients versus 10 controls highlighted 165 proteins, of which 38 were up-regulated, that separate cystinuria patients from controls and further discriminate between severe and moderate forms of the disease. These proteins include markers of kidney injury, circulating proteins, and a neutrophil signature. Analysis of selected proteins by immunobloting, performed on six additional cystinuria patients, validated the mass spectrometry data. To our knowledge, this is the first successful proteomic study in cystinuria unmasking the potential role of inflammation in this disease. The workflow we have developed is applicable to investigate urinary exosomes in different renal diseases and to search for diagnostic/prognostic markers. Data are available via ProteomeXchange with identifier PXD001430.

Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery

“…The largest fraction of differentially abundant urinary extracellular vesicle phospholipids identified were lysophospholipids, phosphatidic acid, phosphatidylcholine and phosphatidylethanolamine. In a second study 119 , a combined proteomics and lipidomics analysis of urinary extracellular vesicles from healthy individuals and patients with cystinuria was carried out. Whereas the proteomics analysis detected a pattern of robust differences in the abundance of proteins between the healthy individuals and the patients with cystinuria, the lipidomics analysis was less conclusive.…”

The human urinary exosome as a potential metabolic effector cargo

“…In fact, exosomes have come to the forefront in the nephrology community due to their potential for accessible biomarker discovery [18,24] of renal disease and injury. A recent high-resolution mass spectrometry proteomic analysis of urinary exosomes in cystinuria revealed the role of inflammation in this disease [25]. In-depth proteomic analysis and EM of individual urinary exosomal samples showed that exosomes are innate immune effectors that contribute to host defense within the urinary tract.…”

Comprehensive Metaproteomic Analyses of Urine in the Presence and Absence of Neutrophil-Associated Inflammation in the Urinary Tract

“…Lacroix et al studied the urine supernatant proteome of the newborns with obstructive nephropathy and quantified 970 proteins 14. Bourderioux et al studied urinary exosomes derived from patients with cystinuria and identified 1,794 proteins 49. The protein ID overlaps were 48% (compared to Santucci's study) and 78% (compared to the other studies).…”