Deletion of the endothelial glycocalyx component endomucin leads to impaired glomerular structure and function.

Abstract

Endomucin (EMCN), an endothelial-specific glycocalyx component, was found to be highly expressed by the endothelium of the renal glomerulus. We reported an anti-inflammatory role of EMCN and its involvement in the regulation of vascular endothelial growth factor (VEGF) activity through modulating VEGF receptor 2 (VEGFR2) endocytosis. The goal of this study is to investigate the phenotypic and functional effects of EMCN deficiency using the first global EMCN knockout mouse model. Global EMCN knockout mice were generated by crossing EMCN-floxed mice with ROSA26-Cre mice. Flow cytometry was employed to analyze infiltrating myeloid cells in the kidneys. The ultrastructure of the glomerular filtration barrier was examined by transmission electron microscopy, while urinary albumin, creatinine, and total protein levels were analyzed from freshly collected urine samples. Expression and localization of EMCN, EGFP, CD45, CD31, CD34, podocin, albumin, and α-smooth muscle actin were examined by immunohistochemistry. Mice were weighed regularly, and their systemic blood pressure was measured using a non-invasive tail-cuff system. Glomerular endothelial cells and podocytes were isolated by fluorescence-activated cell sorting for RNA-seq. Transcriptional profiles were analyzed to identify differentially expressed genes in both endothelium and podocytes, followed by gene ontology analysis of up- and down-regulated genes. Protein levels of EMCN, albumin, and podocin were quantified by Western blot. EMCN -/- mice were viable with no gross anatomical defects in kidneys. The EMCN -/- mice exhibited increased infiltration of CD45 + cells, with an increased proportion of Ly6G high Ly6C high myeloid cells and higher VCAM-1 expression. EMCN -/- mice displayed albuminuria with increased albumin in the Bowman's space compared to the EMCN +/+ littermates. Glomeruli in EMCN -/- mice revealed fused and effaced podocyte foot processes and disorganized endothelial fenestrations. We found no significant difference in blood pressure between EMCN knockout mice and their wild-type littermates. RNA-seq of glomerular endothelial cells revealed downregulation of cell-cell adhesion and MAPK/ERK pathways, along with glycocalyx and extracellular matrix remodeling. In podocytes, we observed reduced VEGF signaling and alterations in cytoskeletal organization. Notably, there was a significant decrease in both mRNA and protein levels of podocin, a key component of the slit diaphragm. Our study demonstrates a critical role of the endothelial marker EMCN in supporting normal glomerular filtration barrier structure and function by maintaining glomerular endothelial tight junction and homeostasis and podocyte function through endothelial-podocyte crosstalk.