Caveolae May Enable Albumin to Enter Human Renal Glomerular Endothelial Cells

ABSTRACT Caveolae on human renal glomerular endothelial cells (HRGECs) are increased in glomerular disease and correlate with the degree of albuminuria. To assess the mechanism by which caveolae contribute to albuminuria, we investigated whether albumin enters into HRGECs through caveolae. HRGECs were incubated with Alexa Fluor 488 labeled BSA or transferrin, followed by immunofluorescence localization with antibody to caveolin‐1 (Cav‐1), the main structural protein of caveolae, or clathrin, the major structural protein of clathrin coated pits, to assess whether BSA colocalized with Cav‐1. HRGECs were also incubated with albumin and caveolae disrupting agents, including methyl beta cyclodextrin (MBCD) and nystatin, to determine whether disrupting caveolae interfered with albumin endocytosis into HRGECs. HRGECs were also incubated with albumin after transfection with Cav‐1 small interfering RNAs (siRNAs). Labeled BSA colocalized with Cav‐1, but not with clathrin. In contrast, labeled transferrin colocalized with clathrin, but not with Cav‐1. Incubation of HRGECs with MBCD or nystatin, or transfection with Cav‐1 siRNA, significantly reduced the intracellular amounts of albumin and Cav‐1, relative to normal HRGECs, as shown by western blotting and immunofluorescence. These findings indicate that albumin enters HRGECs through the caveolae, suggesting that caveolae play an important role in the pathogenesis of albuminuria by providing a pathway through which albumin can enter glomerular endothelial cells. J. Cell. Biochem. 116: 1060–1069, 2015. © 2015 Wiley Periodicals, Inc.