From the Division of Pharmacology and Vascular Medicine, Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands (Y.S., A.G.M., I.M.G., A.H.J.D.).
Department of Physiology, Shenzhen University Health Science Center, Shenzhen University, China (Y.S., X.L.).
Hypertension. 2020 May;75(5):1242-1250. doi: 10.1161/HYPERTENSIONAHA.120.14845. Epub 2020 Mar 23.
Megalin is an endocytic receptor contributing to protein reabsorption. Impaired expression or trafficking of megalin increases urinary renin and allowed the detection of prorenin, which normally is absent in urine. Here, we investigated (pro)renin uptake by megalin, using both conditionally immortalized proximal tubule epithelial cells and Brown Norway Rat yolk sac cells (BN16). To distinguish binding and internalization, cells were incubated with recombinant human (pro)renin at 4°C and 37°C, respectively. (Pro)renin levels were assessed by immunoradiometric assay. At 4°C, BN16 cells bound 3× more prorenin than renin, suggestive for a higher affinity of prorenin. Similarly, at 37°C, prorenin accumulated at 3- to 4-fold higher levels than renin in BN16 cells. Consequently, depletion of medium prorenin (but not renin) content occurred after 24 hours. No such differences were observed in conditionally immortalized proximal tubule epithelial cells, and M6P (mannose-6-phosphate) greatly reduced conditionally immortalized proximal tubule epithelial cells (pro)renin uptake, suggesting that these cells accumulate (pro)renin largely via M6P receptors. M6P did not affect (pro)renin uptake in BN16 cells. Yet, inhibiting megalin expression with siRNA greatly reduced (pro)renin binding and internalization by BN16 cells. Furthermore, treating BN16 cells with albumin, an endogenous ligand of megalin, also decreased binding and internalization of (pro)renin, while deleting the (pro)renin receptor affected the latter only. Exposing prorenin's prosegment with the renin inhibitor aliskiren dramatically increased prorenin binding, while after prosegment cleavage with trypsin prorenin binding was identical to that of renin. In conclusion, megalin might function as an endocytic receptor for (pro)renin and displays a preference for prorenin. Megalin-mediated endocytosis requires the (pro)renin receptor.
巨胞饮受体是一种参与蛋白重吸收的内吞受体。巨胞饮受体表达或转运受损会增加尿肾素，从而允许检测到通常不存在于尿液中的原肾素。在这里，我们使用条件永生化的近端肾小管上皮细胞和棕褐色挪威鼠卵黄囊细胞（BN16）研究了巨胞饮受体对（pro）肾素的摄取。为了区分结合和内化，细胞分别在 4°C 和 37°C 下用重组人（pro）肾素孵育。通过免疫放射测定法评估（pro）肾素水平。在 4°C 下，BN16 细胞结合的原肾素比肾素多 3 倍，提示原肾素的亲和力更高。同样，在 37°C 下，BN16 细胞中积聚的原肾素比肾素高 3-4 倍。因此，在 24 小时后，培养基中（pro）肾素（而非肾素）含量下降。在条件永生化的近端肾小管上皮细胞中未观察到这种差异，并且 M6P（甘露糖-6-磷酸）大大降低了条件永生化的近端肾小管上皮细胞的（pro）肾素摄取，表明这些细胞主要通过 M6P 受体积累（pro）肾素。M6P 不影响 BN16 细胞中（pro）肾素的摄取。然而，用 siRNA 抑制巨胞饮受体的表达大大降低了 BN16 细胞对（pro）肾素的结合和内化。此外，用白蛋白（巨胞饮受体的内源性配体）处理 BN16 细胞也会降低（pro）肾素的结合和内化，而删除（pro）肾素受体仅影响后者。用肾素抑制剂阿利克仑暴露原肾素的前肽段会显著增加原肾素的结合，而用胰蛋白酶切割前肽段后，原肾素的结合与肾素相同。总之，巨胞饮受体可能作为（pro）肾素的内吞受体发挥作用，并显示出对原肾素的偏好。巨胞饮受体介导的内吞作用需要（pro）肾素受体。
