Zheng Huaqing, Helms My N, Zou Changjiang, Zimmerman Elizabeth, Feng Ye, Yang Tianxin
George E. Wahlen Department of Veterans Affairs Medical Center, Salt Lake City, Utah, United States.
Division of Nephrology and Hypertension, Spencer Fox Eccles School of Medicine, University of Utah, Salt Lake City, Utah, United States.
Am J Physiol Renal Physiol. 2025 Jun 1;328(6):F766-F774. doi: 10.1152/ajprenal.00087.2024. Epub 2025 Jan 27.
(Pro)renin receptor (PRR) contains an overlapping cleavage site for site-1 protease (S1P) and furin for the generation of soluble PRR (sPRR). Although S1P-mediated cleavage mediates the release of sPRR, the functional implication of furin-mediated cleavage is unclear. Here, we tested whether furin-mediated cleavage was required for the activity of sPRR in activating epithelial Na channel (ENaC) in cultured M-1 cells. M-1 cells were transfected with pcDNA3.4 containing full-length PRR with (Furin-site Mut) or without (WT) mutagenesis of the furin cleavage site. As compared with empty vector (EM) control, Furin-site Mut showed the attenuation effect on WT-induced α-ENaC expression and amiloride-sensitive short-circuit current. In a separate experiment, M-1 cells were transfected with pcDNA3.4 containing cDNA for sPRR with S1P cleavage (AA 1-282) (sPRR-S1P) or with furin cleavage (AA 1-279) (sPRR-furin), indicating overexpression of the two types of sPRR induced a significant and comparable increase in the release of sPRR, but only sPRR-furin showed an increase of ENaC activity. Single-channel analysis of ENaC activity in Xenopus A6-2F3 cells confirms sPRR-furin activation of ENaC open probability. At last, HEK-293 cells were pretreated with furin inhibitor α-antitrypsin Portland (α-PDX) followed by transfection with EM, WT PRR. sPRR in the conditioned medium was enriched by using protein centrifugal filter devices and applied to M-1 cells followed by measurement of ENaC activity, demonstrating that pretreatment with α-PDX attenuated ENaC-acting activity induced by overexpression of WT PRR. In summary, we conclude that furin-mediated modification is required for the activity of sPRR to increase ENaC-mediated Na transport in the collecting duct cells. The present study for the first time examined the functional implication of furin-dependent cleavage in the activation of sPRR during ENaC regulation in cultured CD cells. We found that sPRR with the initial S1P-dependent cleavage remained silent and only became active following furin-dependent cleavage in terms of enhancement of ENaC activity and expression of α-ENaC. These results offer novel insight into the sPRR maturation process during ENaC regulation.
（前）肾素受体（PRR）含有一个与1型位点蛋白酶（S1P）和弗林蛋白酶重叠的切割位点，用于生成可溶性PRR（sPRR）。尽管S1P介导的切割介导了sPRR的释放，但弗林蛋白酶介导的切割的功能意义尚不清楚。在此，我们测试了在培养的M-1细胞中，sPRR激活上皮钠通道（ENaC）的活性是否需要弗林蛋白酶介导的切割。用含有全长PRR的pcDNA3.4转染M-1细胞，该全长PRR对弗林蛋白酶切割位点进行了（弗林蛋白酶位点突变）或未进行（野生型）诱变。与空载体（EM）对照相比，弗林蛋白酶位点突变对野生型诱导的α-ENaC表达和氨氯地平敏感的短路电流具有衰减作用。在另一项实验中，用含有S1P切割（氨基酸1-282）的sPRR（sPRR-S1P）或弗林蛋白酶切割（氨基酸1-279）的sPRR（sPRR-弗林蛋白酶）的cDNA的pcDNA3.4转染M-1细胞，这表明两种类型的sPRR的过表达诱导了sPRR释放的显著且相当的增加，但只有sPRR-弗林蛋白酶显示出ENaC活性的增加。非洲爪蟾A6-2F3细胞中ENaC活性的单通道分析证实了sPRR-弗林蛋白酶对ENaC开放概率的激活。最后，用弗林蛋白酶抑制剂α-抗胰蛋白酶波特兰（α-PDX）预处理HEK-293细胞，然后用EM、野生型PRR转染。使用蛋白质离心过滤装置富集条件培养基中的sPRR，并将其应用于M-1细胞，随后测量ENaC活性，表明用α-PDX预处理减弱了野生型PRR过表达诱导的ENaC活性。总之，我们得出结论，弗林蛋白酶介导的修饰是sPRR增加集合管细胞中ENaC介导的钠转运活性所必需的。本研究首次探讨了在培养的集合管细胞ENaC调节过程中，弗林蛋白酶依赖性切割在sPRR激活中的功能意义。我们发现，就增强ENaC活性和α-ENaC表达而言，最初依赖S1P切割的sPRR保持沉默，只有在依赖弗林蛋白酶切割后才变得活跃。这些结果为ENaC调节过程中sPRR成熟过程提供了新的见解。
