Vacuolar H⁺-ATPase and Megalin-Mediated Prorenin Uptake: Focus on Elements Beyond the (Pro)Renin Receptor

Megalin is a multiple-ligand receptor that contributes to protein reabsorption in the kidney. Recently, megalin was found to act as a novel endocytic receptor for prorenin. Internalization depended on the (pro)renin receptor. This receptor is an accessory protein of vacuolar H⁺-ATPase (V-ATPase), a complex consisting of 14 subunits and two accessory proteins. Here we explored whether V-ATPase elements other than the (P)RR affect megalin-mediated prorenin uptake. Using RNAi technology, we inhibited each individual V-ATPase subunit in megalin-expressing BN16 cells. Subsequently, we quantified megalin expression and the uptake of prorenin. To unravel the underlying molecular mechanisms, we investigated the adaptor proteins autosomal recessive hypercholesterolemia (ARH) and Disabled-2 (Dab2), which are important for the endocytosis of megalin, glycogen synthase kinase 3β (GSK3β), a regulatory factor of megalin recycling, and endoplasmic reticulum stress factors (ERSF). Silencing subunit Atp6v_oa1 reduced prorenin uptake by 19%, while silencing accessory protein Atp6ap1 increased it by 15%. Silencing other subunits exerted a more modest or no effect. Silencing Atp6v_oa1 reduced surface megalin density, without altering its mRNA and protein levels, and this was associated with increased GSK3β phosphorylation and no change in ARH, Dab2, and ERSF. Silencing Atp6ap1 increased megalin mRNA and protein expression and this was accompanied by upregulation of ARH and ERSF, while Dab2 expression was unaltered. In conclusion, V-ATPase units differently affect megalin-mediated reabsorption of prorenin, thereby offering novel pharmacological targets to not only affect renal renin-angiotensin system activity, but also to treat renal diseases that are associated with disturbed protein reabsorption, like Dent's disease.