Glomerular endothelial cell heterogeneity in Alport syndrome

Hasmik Soloyan,Brendan Grubbs,Matthew Thornton,Patrick Khatchadourian,Laura Perin,Roger De Filippo,Paolo Cravedi,Sargis Sedrakyan,Valentina Villani,Andrea Angeletti

doi:10.1038/s41598-020-67588-0

Abstract

Glomerular endothelial cells (GEC) are a crucial component of the glomerular physiology and their damage contributes to the progression of chronic kidney diseases. How GEC affect the pathology of Alport syndrome (AS) however, is unclear. We characterized GEC from wild type (WT) and col4α5 knockout AS mice, a hereditary disorder characterized by progressive renal failure. We used endothelial-specific Tek-tdTomato reporter mice to isolate GEC by FACS and performed transcriptome analysis on them from WT and AS mice, followed by in vitro functional assays and confocal and intravital imaging studies. Biopsies from patients with chronic kidney disease, including AS were compared with our findings in mice. We identified two subpopulations of GEC (dimtdT and brighttdT) based on the fluorescence intensity of the TektdT signal. In AS mice, the brighttdT cell number increased and presented differential expression of endothelial markers compared to WT. RNA-seq analysis revealed differences in the immune and metabolic signaling pathways. In AS mice, dimtdT and brighttdT cells had different expression profiles of matrix-associated genes (Svep1, Itgβ6), metabolic activity (Apom, Pgc1α) and immune modulation (Apelin, Icam1) compared to WT mice. We confirmed a new pro-inflammatory role of Apelin in AS mice and in cultured human GEC. Gene modulations were identified comparable to the biopsies from patients with AS and focal segmental glomerulosclerosis, possibly indicating that the same mechanisms apply to humans. We report the presence of two GEC subpopulations that differ between AS and healthy mice or humans. This finding paves the way to a better understanding of the pathogenic role of GEC in AS progression and could lead to novel therapeutic targets.

Highlights

Glomerular endothelial cells (GEC) are a crucial component of the glomerular physiology and their damage contributes to the progression of chronic kidney diseases
Since no suitable anti-Tek antibodies are available to confirm co-staining of Tek and tdT, we used Cdh[5] and CD31 as a GEC specific marker and an anti-RFP antibody to demonstrate the endothelial specificity of the tdT signal
Of endothelial cells suggestive of their spatial position in the glomerular compartment by scRNA-seq in healthy mice, and their adaptation through differential gene expression when exposed to water d eprivation[6]

Summary

Introduction

Glomerular endothelial cells (GEC) are a crucial component of the glomerular physiology and their damage contributes to the progression of chronic kidney diseases. We report the presence of two GEC subpopulations that differ between AS and healthy mice or humans This finding paves the way to a better understanding of the pathogenic role of GEC in AS progression and could lead to novel therapeutic targets. Transcriptomic-profiling studies and single cell RNA-sequencing of isolated glomeruli have provided important insight into GEC heterogeneity and their potential role and adaptation to the changing microenvironment in kidney diseases[2,3,4,5,6]. Two recent studies based on sc-RNA sequencing of glomerular cells have identified diverse subclusters of GEC with distinct gene expression profiles. In Alport syndrome (AS), a progressive renal disease associated with mutations in the COL4α 3, α4 or α5 chains, the potential heterogeneity of GEC and their adaptive/pathologic role is poorly understood. We isolated labeled-GEC from 4-month old healthy and AS mice and

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Results

Conclusion