Proteomic Analysis of Urinary Extracellular Vesicles Reveals a Role for the Complement System in Medullary Sponge Kidney Disease.

Bruschi Bruschi,Granata Granata,Fabris Fabris,Zaza Zaza,Candiano Candiano,Gambaro Gambaro,Petretto Petretto,Ghiggeri Ghiggeri

doi:10.3390/ijms20215517

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 co-expression 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.

Highlights

In the last decade, great efforts have been undertaken to characterize the biological networking associated with medullary sponge kidney (MSK) disease, a rare clinical condition often associated with nephrocalcinosis and nephrolithiasis, urinary acidification and concentration defects, and cystic anomalies in the precalyceal ducts [1]
To generate a more concise picture of the biochemical process associated with the three proteins that maximize the discrimination between the exosomes of idiopathic calcium nephrolithiasis (ICN) and MSK samples [Mannan-binding lectin serine protease 2 (MASP2), Ficolin 1 (FCN1), and Complement component 4-binding protein β (C4BPB)], we explored the enrichment of Gene Ontology (GO) annotations in greater detail
We found that MASP2 was expressed more strongly in ICN patients compared to MSK patients (Figure 6A, black circle)

Summary

Introduction

Great efforts have been undertaken to characterize the biological networking associated with medullary sponge kidney (MSK) disease, a rare clinical condition (prevalence of approximately 5 cases per 100,000 in the general population) often associated with nephrocalcinosis and nephrolithiasis, urinary acidification and concentration defects, and cystic anomalies in the precalyceal ducts [1]. The most abundant protein found in MSK urinary extracellular vesicles was laminin subunit α2 (LAMA2, Merosin), a well-characterized member of the laminin family of at least 15 αβG heterotrimeric proteins, which contributes to the extracellular matrix and is a major component of the basement membrane [12,13] This protein is thought to promote cyst formation [14,15,16]. It regulates extracellular laminin assembly, and the laminin determines the orientation of the apical pole [17]

Methods

Results

Conclusion