Wilson Bryan A, Cruz-Diaz Nildris, Marshall Allyson C, Pirro Nancy T, Su Yixin, Gwathmey TanYa M, Rose James C, Chappell Mark C
Hypertension and Vascular Research Center, Wake Forest University School of Medicine, Winston-Salem, North Cartolina; and.
Department of Obstetrics and Gynecology, Wake Forest University School of Medicine, Winston-Salem, North Cartolina.
Am J Physiol Renal Physiol. 2015 Mar 15;308(6):F594-601. doi: 10.1152/ajprenal.00609.2014. Epub 2015 Jan 7.
Angiotensin 1-7 [ANG-(1-7)] is expressed within the kidney and exhibits renoprotective actions that antagonize the inflammatory, fibrotic, and pro-oxidant effects of ANG II. We previously identified an peptidase that preferentially metabolized ANG-(1-7) to ANG-(1-4) in the brain medulla and cerebrospinal fluid (CSF) of sheep (Marshall AC, Pirro NT, Rose JC, Diz DI, Chappell MC. J Neurochem 130: 313-323, 2014); thus the present study established the expression of the peptidase in the kidney. Utilizing a sensitive HPLC-based approach, we demonstrate a peptidase activity that hydrolyzed ANG-(1-7) to ANG-(1-4) in the sheep cortex, isolated tubules, and human HK-2 renal epithelial cells. The peptidase was markedly sensitive to the metallopeptidase inhibitor JMV-390; human HK-2 cells expressed subnanomolar sensitivity (IC50 = 0.5 nM) and the highest specific activity (123 ± 5 fmol·min(-1)·mg(-1)) compared with the tubules (96 ± 12 fmol·min(-1)·mg(-1)) and cortex (107 ± 9 fmol·min(-1)·mg(-1)). The peptidase was purified 41-fold from HK-2 cells; the activity was sensitive to JMV-390, the chelator o-phenanthroline, and the mercury-containing compound p-chloromercuribenzoic acid (PCMB), but not to selective inhibitors against neprilysin, neurolysin and thimet oligopeptidase. Both ANG-(1-7) and its endogenous analog [Ala(1)]-ANG-(1-7) (alamandine) were preferentially hydrolyzed by the peptidase compared with ANG II, [Asp(1)]-ANG II, ANG I, and ANG-(1-12). Although the ANG-(1-7) peptidase and insulin-degrading enzyme (IDE) share similar inhibitor characteristics of a metallothiolendopeptidase, we demonstrate marked differences in substrate specificity, which suggest these peptidases are distinct. We conclude that an ANG-(1-7) peptidase is expressed within the renal proximal tubule and may play a potential role in the renal renin-angiotensin system to regulate ANG-(1-7) tone.
血管紧张素1-7 [ANG-(1-7)] 在肾脏中表达，并表现出肾脏保护作用，可拮抗血管紧张素II的炎症、纤维化和促氧化作用。我们之前在绵羊的脑髓质和脑脊液（CSF）中鉴定出一种肽酶，该肽酶可优先将ANG-(1-7) 代谢为ANG-(1-4)（Marshall AC, Pirro NT, Rose JC, Diz DI, Chappell MC. J Neurochem 130: 313-323, 2014）；因此，本研究确定了该肽酶在肾脏中的表达。利用一种基于高效液相色谱的灵敏方法，我们在绵羊皮质、分离的肾小管和人HK-2肾上皮细胞中证实了一种将ANG-(1-7) 水解为ANG-(1-4) 的肽酶活性。该肽酶对金属肽酶抑制剂JMV-390极为敏感；与肾小管（96 ± 12 fmol·min(-1)·mg(-1)）和皮质（107 ± 9 fmol·min(-1)·mg(-1)）相比，人HK-2细胞表现出亚纳摩尔敏感性（IC50 = 0.5 nM）和最高的比活性（123 ± 5 fmol·min(-1)·mg(-1)）。该肽酶从HK-2细胞中纯化了41倍；其活性对JMV-390、螯合剂邻菲罗啉和含汞化合物对氯汞苯甲酸（PCMB）敏感，但对中性内肽酶、神经溶素和硫醚内肽酶的选择性抑制剂不敏感。与血管紧张素II、[天冬氨酸(1)]-血管紧张素II、血管紧张素I和血管紧张素-(1-12) 相比，ANG-(1-7) 及其内源性类似物 [丙氨酸(1)]-ANG-(1-7)（阿兰曼丁）均优先被该肽酶水解。尽管ANG-(1-7) 肽酶和胰岛素降解酶（IDE）具有金属硫醇内肽酶相似的抑制剂特征，但我们证实了它们在底物特异性上存在显著差异，这表明这些肽酶是不同的。我们得出结论，一种ANG-(1-7) 肽酶在肾近端小管中表达，可能在肾脏肾素-血管紧张素系统中发挥潜在作用以调节ANG-(1-7) 的水平。
