3.2.2. MRPEC purification and isolation from mouse renal cortices

MRPECs were initially isolated utilizing a modified method described previously (19). The following modifications were incorporated as we were unable to obtain the purity of MRPECs suitable for downstream physiological and pharmacological studies (Supplementary Figure 1). Briefly, we altered the renal enzymatic tissue digestion buffers’ composition by replacing collagenase type 4 with collagenase type 1 and trypsin was replaced by accutase, respectively. Collagenase type 1 was selected as collagenase type 4 enzymes (e.g., MMP-9) were previously reported to facilitate phenotypic loss in primary renal peritubular endothelial cells (18), while accutase was employed to aid in the retention of EC specific markers that are known to be trypsin sensitive (e.g., CD31, VE-cadherin, etc.) (19, 21, 22). Additionally, GentleMACS-C tubes with automated tissue dissociation (Miltenyi Biotec) were employed to reduce kidney processing times and improve overall cell integrity. Cell suspensions were then subjected to one cycle of EPCAM+ (CD326) magnetic microbead (Miltenyi Biotec) depletion, via negative selection, to remove contaminating epithelial cells, as described previously (23). MRPECs were then purified and isolated via two positive selection cycles with CD146+ (MCAM) magnetic microbeads (Miltenyi Biotec). Twice purified MRPEC were subsequently seeded onto either human plasma fibronectin (10 μg/ml/dish, PromoCell) precoated 35-mm μ-Dish-high cell culture imaging dishes (Ibidi), and/or 35-mm Primaria cell culture dishes (Corning). Fibronectin coating was chosen as it was previously reported to regulate PLVAP (PV-1) localization to endothelial fenestrae by stabilizing microtubules (24).