EMCN knockout leads to increased monocyte infiltration in kidney and albuminuria in mice

Abstract

Endomucin (EMCN), a type I integral membrane glycoprotein, is specifically expressed in endothelium and enriched in kidney glomerular endothelium. We have previously reported that under resting conditions EMCN functions as an anti‐inflammatory molecule, preventing leukocyte‐endothelial cell adhesion and that EMCN plays an important role in modulating VEGFR2 signaling. The goal of this study is to characterize the phenotype of the kidney in the first‐reported EMCN knockout mouse model. EMCN−/−mice (with a EGFP tag for the knockout allele) were obtained by crossing EMCN‐floxed mice, generated in conjunction with Cyagen Biosciences, with the ROSA26‐Cre mice. The mRNA level of EMCN, CD31, CD34, E‐selection, VCAM‐1, and ICAM‐1 were quantified by reverse transcription PCR and semi‐quantitative PCR using total RNA isolated from the adult mouse kidneys. The mRNA level of EMCN in EMCN−/−mice were not detectable (n>4, p<0.0001) compared to EMCN+/+. Renal CD34 at mRNA level was decreased (fold change, 0.68±0.09 vs 1, n=4, p<0.05) and VCAM‐1 was increased (fold change, 2.2 ±0.45 vs 1, n=4, p<0.01) in EMCN−/− kidneys compared to wildtype (wt) controls. CD31, E‐selection, and ICAM‐1 mRNA levels were unchanged between EMCN−/−and wildtype kidneys. Kidneys from adult EMCN+/+ and EMCN−/− mice were collected, fixed, and sectioned. Expression and localization of EMCN, eGFP, CD45 and podocin in adult kidneys were examined by immunohistochemistry and quantified, which revealed a significant increase in CD45+ cell infiltration in EMCN−/− mice compared to EMCN+/+ mice (72.50±8.13 cells/mm2 vs 39.67±4.91 cells/ mm2, n=4, p<0.001). To characterize the renal inflammatory cell composition, freshly collected kidneys from EMCN+/+ and EMCN−/− were enzymatically digested and the leukocyte markers CD45, CD3, CD4, CD19, CD11b, Ly6G and Ly6C were examined by flow cytometry to identify the proportion of myeloid populations. Flow cytometry analysis suggested a trend of increased CD45+ cells in the kidney from EMCN−/−mice compared to wildtype (n=9, p=0.07), and revealed a significant increase in the percentage of Ly6GhighLy6Chighmyeloid cells in EMCN−/−compared to wildtype kidneys (1.6±0.23 vs 1.01±0.18, n=9, p<0.05). Sporadic albuminuria was detected through Coomassie staining in spot urine samples of EMCN−/− mice. Ultrastructural morphology of glomeruli, examined from both EMCN+/+ and EMCN−/− through transmission electron microscopy, showed the effacement of podocyte foot processes and disorganized endothelial cell fenestrations in glomerular capillaries of EMCN−/− mice compared to wt. The result indicates EMCN plays a key role in the prevention of inflammatory cells infiltration in kidney, presumably by blocking leukocyte adhesion, and is also involves in maintaining normal glomerular filtration barrier structure and function, potentially via its role in VEGFR2 signaling.