Alternate and classical pathways for angiotensin peptides uptake in the proximal tubule

Megalin and the Angiotensin type 1 receptor (AT1R) are coexpressed in several polarized epithelia including the renal proximal tubule and the yolk sac where megalin is heavily involved in receptor-mediated protein endocytosis. Because angiotensin II uptake by proximal tubule cells and yolk sac cells was only partially inhibited by AT1R specific blockers, we tested to see if megalin interacts with angiotensin peptides. Angiotensin II and Angiotensin-(1-7) were chosen on the basis of their physiological relevance and their dissimilar affinity for the AT1R. Cell experiments were conducted on several proximal tubule cell lines (HRCEs, IRPPT cells and NRK-52E cells) and BN/MSV cells. Uptake experiments were performed in cell monolayers exposed to fluorescent-labeled angiotensin II or angiotensin-(1-7) +/- different competitors, and the amount of cell-associated fluorescence (an indication of internalized angiotensin) was determined by flow cytometry. Anti-megalin antisera and AT1R blockers (olmesartan or candesartan) were used to interfere with uptake via megalin and the AT1R respectively. Anti-megalin antisera universally interfered with angiotensin II uptake by proximal tubule and BN/MSV cells, but the degree of reduction was cell type-dependent (ranging from 80 to 30% in reduction, p < 0.05 in all cases vs. positive control). Angiotensin-(1-7) uptake by BN/MSV cells was also prevented by anti-megalin antisera (63%, p < 0.001). Angiotensin II interfered with uptake of metallothionein (a known ligand for megalin) by BN/MSV cells. Flow cytometry analysis of binding experiments performed in brush border membrane vesicles freshly isolated from kidney cortices of CD-1 mice showed that anti-megalin antisera interfered with angiotensin II and Angiotensin-(1-7) binding (27% and 30% respectively, p < 0.05 vs. positive control). Molecular interactions of megalin with angiotensin II and angiotensin-(1-7) were studied by surface plasmon resonance. Angiotensin II and angiotensin-(1-7) bind megalin dose and time-dependently and with a similar affinity (∼3 mM). Collectively, the data from these studies demonstrate that megalin binds and internalizes angiotensin II and angiotensin-(1-7). These results also indicate that angiotensin II or angiotensin-(1-7) internalization in the proximal tubule might be megalin-dependent and that the scavenger receptor megalin may play a role in regulating proximal tubule and therefore intrarenal angiotensin peptide levels.