Wang Na, Lu Xifeng, Jan Danser A H
Division of Vascular Medicine and Pharmacology, Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands.
Clinical Research Center, the First Affiliated Hospital of Shantou University Medical College, Shantou, China.
J Cell Physiol. 2025 Jan;240(1):e31518. doi: 10.1002/jcp.31518.
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 Atp6va1 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 Atp6va1 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.
巨膜蛋白是一种多配体受体，有助于肾脏中的蛋白质重吸收。最近，发现巨膜蛋白可作为肾素原的新型内吞受体。内化作用依赖于（前）肾素受体。该受体是液泡H⁺-ATP酶（V-ATP酶）的辅助蛋白，V-ATP酶是一种由14个亚基和两种辅助蛋白组成的复合体。在此，我们探究了除（前）肾素受体外的V-ATP酶元件是否影响巨膜蛋白介导的肾素原摄取。我们使用RNA干扰技术在表达巨膜蛋白的BN16细胞中抑制每个单独的V-ATP酶亚基。随后，我们对巨膜蛋白表达和肾素原摄取进行了定量。为了阐明潜在的分子机制，我们研究了衔接蛋白常染色体隐性高胆固醇血症（ARH）和失能蛋白2（Dab2），它们对巨膜蛋白的内吞作用很重要，还研究了糖原合酶激酶3β（GSK3β），它是巨膜蛋白再循环的调节因子，以及内质网应激因子（ERSF）。沉默亚基Atp6va1可使肾素原摄取减少19%，而沉默辅助蛋白Atp6ap1则使其增加15%。沉默其他亚基的作用较为轻微或无影响。沉默Atp6va1可降低巨膜蛋白的表面密度，而不改变其mRNA和蛋白质水平，这与GSK3β磷酸化增加以及ARH、Dab2和ERSF无变化有关。沉默Atp6ap1可增加巨膜蛋白的mRNA和蛋白质表达，同时伴有ARH和ERSF上调，而Dab2表达未改变。总之，V-ATP酶亚基对巨膜蛋白介导的肾素原重吸收有不同影响，从而为不仅影响肾素-血管紧张素系统活性，而且治疗与蛋白质重吸收紊乱相关的肾脏疾病（如丹特病）提供了新的药理学靶点。
