Urinary exosomes and diabetic nephropathy: a proteomic approach

Abstract

Urinary exosomes (UE) are nanovesicles released by every epithelial cell facing the urinary space and they are considered a promising source of molecular markers for renal dysfunction and structural injury. Exosomal proteomics has emerged as a powerful tool for understanding the molecular composition of exosomes and has potential to accelerate biomarker discovery. We employed this strategy in the study of diabetic nephropathy (DN) and the consequent end stage renal disease, which represent the dramatic evolution of diabetes, often leading the patients to dialysis or kidney transplantation. The identification of DN biomarkers is likely to help monitoring the disease onset and progression. A label free LC-MS/MS approach was applied to investigate the alteration of the proteome of urinary exosomes isolated from the Zucker diabetic fatty rats (ZDF), as a model of type 2 DN. We collected 24 hour urine samples from 7 ZDF and from 7 control rats at different ages (6, 12 and 20 weeks old) to monitor the development of DN. Exosomes were isolated by ultracentrifugation and their purity assessed by immunoblotting for known exosomal markers. Exosomal proteins from urine samples of 20 week old rats were pooled and analyzed by nLC-ESI-UHR-QToF-MS/MS after pre-filtration and tryptic digestion, leading to the identification and label free quantification of 286 proteins. Subcellular localization and molecular functions were assigned to each protein by UniprotKB, showing that the majority of identified proteins were membrane-associated or cytoplasmic and involved in transport, signalling and cellular adhesion, typical functions of exosomal proteins. We further validated label free mass spectrometry results by immunoblotting, as exemplified by: Xaa-Pro dipeptidase, Major Urinary Protein 1 and Neprilysin, which resulted increased, decreased and not different, respectively, in exosomes isolated from diabetic urine samples compared to controls, by both techniques. In conclusion we show the potential of exosome proteomics for DN biomarker discovery.

Urinary Fetuin-A Is a Novel Marker for Diabetic Nephropathy in Type 2 Diabetes Identified by Lectin Microarray

“…Recently, the urinary proteome analyses have been performed using 2-dementional gel electrophoresis and subsequent mass spectrometry to identify the novel urinary markers [8]–[10]; however, the identification of new markers may be suffered from contamination of urinary major proteins such as albumin, immunoglobulins, α1-antitrypsin, transferrin, and haptoglobin. In the line of considerations, we focused on the alterations of glycochains to identify useful urinary biomarkers.…”

The comparison of CHCA solvent compositions for improving LC-MALDI performance and its application to study the impact of aflatoxin B1 on the liver proteome of diabetes mellitus type 1 mice

“…Overexpression of MUP1 can lower blood glucose levels and cause glucose intolerance with enhanced insulin sensitivity [34]. Recently, MUP1 was found to have decreased expression level in urinary exosomes of diabetic nephropathy [35]. As such, MUP1 is considered a systemic glucose and lipid metabolism regulator in the liver.…”

Development of an Automated, High‐throughput Sample Preparation Protocol for Proteomics Analysis

“…Moreover, it allows higher peptide recovery after digestion, leading to better coverage of the proteome . At present, FASP is increasingly used in proteomics studies, indicating that it is the gold standard for sample preparation . However, the use of FASP for handling high‐throughput data in parallel is limited because it takes longer time and requires highly skilled man power .…”