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A Modified Precipitation Method to Isolate Urinary Exosomes
Published on: January 16, 2015
Proteomic Analysis of Urinary Extracellular Vesicles Reveals a Role for the Complement System in Medullary Sponge
Maurizio Bruschi1, Simona Granata2, Giovanni Candiano1
1Laboratory of Molecular Nephrology, IRCCS Istituto Giannina Gaslini, 16147 Genova, Italy.
Abstract:
Medullary sponge kidney (MSK) disease is a rare and neglected kidney condition often associated with nephrocalcinosis/nephrolithiasis and cystic anomalies in the precalyceal ducts. Little is known about the pathogenesis of this disease, so we addressed the knowledge gap using a proteomics approach. The protein content of microvesicles/exosomes isolated from urine of 15 MSK and 15 idiopathic calcium nephrolithiasis (ICN) patients was investigated by mass spectrometry, followed by weighted gene coexpression network analysis, support vector machine (SVM) learning, and partial least squares discriminant analysis (PLS-DA) to select the most discriminative proteins. Proteomic data were verified by ELISA. We identified 2998 proteins in total, 1764 (58.9%) of which were present in both vesicle types in both diseases. Among the MSK samples, only 65 (2.2%) and 137 (4.6%) proteins were exclusively found in the microvesicles and exosomes, respectively. Similarly, among the ICN samples, only 75 (2.5%) and 94 (3.1%) proteins were exclusively found in the microvesicles and exosomes, respectively. SVM learning and PLS-DA revealed a core panel of 20 proteins that distinguished extracellular vesicles representing each clinical condition with an accuracy of 100%. Among them, three exosome proteins involved in the lectin complement pathway maximized the discrimination between MSK and ICN: Ficolin 1, Mannan-binding lectin serine protease 2, and Complement component 4-binding protein β. ELISA confirmed the proteomic results. Our data show that the complement pathway is involved in the MSK, revealing a new range of potential therapeutic targets and early diagnostic biomarkers.
Insights
This study investigated urinary microvesicles in Medullary Sponge Kidney (MSK) disease using proteomics. Researchers identified key proteins in the complement pathway, offering potential diagnostic biomarkers and therapeutic targets for MSK.
Area of Science:
- Nephrology
- Proteomics
- Biochemistry
Background:
- Medullary Sponge Kidney (MSK) disease is a rare kidney condition linked to nephrocalcinosis and cystic anomalies.
- The pathogenesis of MSK remains poorly understood, necessitating advanced research approaches.
Purpose of the Study:
- To elucidate the pathogenesis of Medullary Sponge Kidney (MSK) disease by analyzing urinary microvesicle and exosome proteins.
- To identify novel diagnostic biomarkers and potential therapeutic targets for MSK.
Main Methods:
- Urine samples from 15 MSK patients and 15 idiopathic calcium nephrolithiasis (ICN) patients were analyzed using mass spectrometry-based proteomics.
- Proteomic data were subjected to weighted gene coexpression network analysis, Support Vector Machine (SVM) learning, and Partial Least Squares Discriminant Analysis (PLS-DA).
- Key protein findings were validated using Enzyme-Linked Immunosorbent Assay (ELISA).
Main Results:
- A total of 2998 proteins were identified, with a significant overlap between MSK and ICN samples.
- Machine learning algorithms identified a core panel of 20 proteins that distinguished MSK from ICN with 100% accuracy.
- Three exosome proteins—Ficolin 1, Mannan-binding lectin serine protease 2, and Complement component 4-binding protein β—involved in the lectin complement pathway, were crucial for discrimination.
Conclusions:
- The study reveals the involvement of the complement pathway in Medullary Sponge Kidney (MSK) disease.
- Identified proteins, particularly those in the lectin complement pathway, serve as promising early diagnostic biomarkers for MSK.
- These findings open avenues for developing targeted therapies for Medullary Sponge Kidney (MSK) disease.
