Fig. 1. Caveolae and caveolin-1 expression. a In electron micrographs depicting AL-amyloidosis, caveolae in glomerular endothelial cells are present in the cytoplasm and on both cell surfaces, facing the capillary lumen and glomerular basement membrane (black arrows); fenestrae are also present (arrow heads). Caveolae are present in the cytoplasm of glomerular epithelial cells (white arrows), and foot processes are fused. b In an immunofluorescence study of membranoproliferative glomerulonephritis, Caveolin-1 was highly expressed on the capillaries in the glomeruli. It was also detected on the arterioles.
Albumin Transportation through Caveolae
Glomerular Endothelial Cells
In our previous reports, we showed that Cav-1 is expressed very weakly in the glomeruli in 0-hour renal biopsy specimens of renal transplant donors as healthy control subjects; however, upon renal biopsy we found that Cav-1 expression significantly increased in glomeruli in patients suffering from glomerular diseases, such as IgA nephropathy, crescentic glomerulonephritis, minimal change disease, focal segmental glomerular disease, membranous nephritis, membranoproliferative glomerulonephritis, and diabetic nephropathy (Fig. 1b). Interestingly, in cases of glomerulonephritis that were treated with immunosuppressive agents and showed decreases in urinary protein excretion, the expression of Cav-1 decreased to a similar extent as it did in healthy controls. In this cohort of patients and healthy subjects, Cav-1 expression was positively correlated with urinary albumin excretion levels; further, Cav-1 co-localized with the pathologische anatomie leiden-endothelium (typically known as PAL-E) endothelial marker on histologic analysis. These results suggest that caveolae may play a pivotal role in the etiology of albuminuria, allowing albumin to pass through glomerular endothelial cells by a caveolae-dependent pathway [19]. To test this hypothesis, we utilized an in vitro study that employed human renal glomerular endothelial cells and found that Alexa Fluor 488-labeled albumin particles were highly co-localized with Cav-1, but not with clathrin, which was present in other invaginations on the cell surface. The caveolae-disrupting agents, methyl-beta-cyclodextrin (MBCD) and nystatin significantly decreased albumin internalization into glomerular endothelial cells, demonstrated by western blotting and immunofluorescence analyses. Upon siRNA knockdown of Cav-1 expression in glomerular endothelial cells, their uptake of albumin also significantly decreased. These results indicate that albumin enters glomerular endothelial cells through caveolae [20]. After the endocytosis of albumin through caveolae, albumin particles co-localized with early endosomes, but not with actin, microtubules, lysosomes, proteasomes, endoplasmic reticulum (ER), and Golgi apparatus (GA). We also showed that albumin particles were excreted to the other side of cells in a study using transwell plates. These results suggested that, after the endocytosis of albumin through caveolae, albumin was transported to early endosomes without moving along cytoskeletal components such as actin and microtubules, and that albumin particles were sorted at early endosomes for transport directly to the other side of the cells without undergoing either degradation in lysosomes and proteasomes, or modification in the ER or GA. In vivo analysis using the puromycin aminonucleoside (PAN) mouse model demonstrated that the amount of albuminuria significantly decreased following treatment with MBCD, as indicated by a decrease in Cav-1 expression in the capillaries [21]. Taken together, these analyses indicate that albumin is endocytosed, transcytosed, and exocytosed through glomerular endothelial cells via a caveolae-dependent pathway, and suggest that this caveolae-dependent pathway might provide an alternative etiology of albuminuria in addition to the classical fenestrae pathway.
Glomerular Epithelial Cells
Albumin particles have been shown to pass through abnormal slit membrane in cases of decreased nephrin expression between the foot processes of glomerular epithelial cells (podocytes). However, in cases of nephrotic syndrome, foot processes are dramatically effaced and the number of slit membranes in the gap is drastically decreased; this provides a useful model to study the intracellular trafficking pathways by which albumin traverses through glomerular epithelial cells.
Tojo et al. [22] reported that in the induced-nephrotic syndrome rat model (using intraperitoneal injection of PAN), a high density of gold-labeled albumin particles were detected in the endosomes of glomerular epithelial cells, and larger FITC-labeled albumin particles were also detected in the bodies of glomerular epithelial cells [22]. Tojo et al. [22] also reported that the Fc receptor (FcRn) functions as a receptor for albumin transportation; in PAN-treated rats, they showed an increase in FcRn-bound albumin in glomerular epithelial cells and a decrease in urinary protein excretion and albumin uptake in podocytes by treatment with anti-FcRn antibody [23]. Recently, in an in vitro study using human urine-derived podocyte-like epithelial cells, FITC-labeled albumin was co-localized with FcRn and endosomes [24], supporting the hypothesis that albumin uses intracellular trafficking pathways to traffic through glomerular epithelial cells. Moreover, Dobrinskikh et al. [24] reported that FITC-labeled albumin particles co-localized with Cav-1 but not with clathrin; in the same study, nystatin (an inhibitor of caveolae-dependent endocytosis) interfered with the internalization of albumin into glomerular epithelial cells, but pitstop2 (an inhibitor of clathrin-mediated endocytosis) did not [24]. The cholesterol-lowering agents, statins, also interfered with the endocytosis of albumin into glomerular epithelial cells during in vitro and in vivo assays [25]. Additionally, caveolae are enriched in cholesterol and sphingolipids, and statins have been reported to interfere with caveolae-dependent BK virus internalization into renal proximal tubular epithelial cells [26], further supporting the hypothesis of the endocytosis of albumin through caveolae. Taken together, these results suggest that FcRn-bound albumin particles enter glomerular epithelial cells through caveolae and are transported to early endosomes. In another study, it was demonstrated that, after the endocytosis of albumin through caveolae, albumin particles were transported to early endosomes that move along actin; further, several albumin particles were degraded in lysosomes [27]. Albumin exposure also induced cell apoptosis through combined activation of caspase 3/7 and nuclear factor kappa B and release of interleukin-1β, tumor necrosis factor, and interleukin-6 [28]. In a recent report, Schießl et al. [29] used an intravital multiphoton microscope to show transcellular transport of albumin – through caveolae – across the glomerular epithelial cells, and noted that this transcytosis increased upon stimulation with angiotensin II [30]. These data imply that there may be a transcellular pathway for albumin transport across glomerular epithelial cells.