An angiotensin-(1–7) peptidase in the kidney cortex, proximal tubules, and human HK-2 epithelial cells that is distinct from insulin-degrading enzyme

Abstract

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.

International Union of Basic and Clinical Pharmacology. XCIX. Angiotensin Receptors: Interpreters of Pathophysiological Angiotensinergic Stimuli

“…This peptide can be generated by either decarboxylation of the Asp residue of angiotensin- (1)(2)(3)(4)(5)(6)(7) or by catalytic action of angiotensin-converting enzyme-2 on decarboxylated AngII (also called angiotensin A) (Lautner et al, 2013). Two primary journal articles on alamandine, since its discovery, have paired this ligand with Masrelated G-protein-coupled receptor D; however, neither strong pharmacological nor direct radioligand binding data exist (Habiyakare et al, 2014;Wilson et al, 2015). Identification of alamandine and its putative receptor has attracted attention as a novel mediator of physiologic and pathophysiological actions of the RAS and may help to develop new therapeutic strategies for treating human cardiovascular diseases.…”

Biochemical evaluation of the renin-angiotensin system: the good, bad, and absolute?

“…In contrast, peptidase activities derived by different synthetic substrates are not directly comparable unless standardized to enzyme content. Moreover, use of endogenous peptides may reveal novel peptidase activities involved in angiotensin processing (62,144). Peptidase assays developed in the author's laboratory use 125 I-radiolabled peptides coupled to HPLC-based separation and in-line ␥-detection (129).…”

Peptidases and the Renin-Angiotensin System: The Alternative Angiotensin-(1-7) Cascade

“…Moreover, this activity was inversely correlated to CSF levels of Ang-(1-7) in control and betamethasone-exposed sheep, a model of fetal programming that exhibits elevated blood pressure and an attenuated baroreflex [7]. Subsequent studies found the Ang-(1-7)-degrading activity in sheep brain and kidney cortex, as well as in the human proximal tubule HK-2 cell line [57,98]. The enzyme activity exhibited unusual characteristics as Ang I and other peptides equal to or greater than 10 residues were not substrates for the peptidase [57,98].…”

“…Subsequent studies found the Ang-(1-7)-degrading activity in sheep brain and kidney cortex, as well as in the human proximal tubule HK-2 cell line [57,98]. The enzyme activity exhibited unusual characteristics as Ang I and other peptides equal to or greater than 10 residues were not substrates for the peptidase [57,98]. In addition, the peptidase was sensitive to both chelating agents such as o-phenanthroline and EDTA, and the sulfhydryl inhibitors APMA and PCMB [57,98].…”

“…We recently showed that both neprilysin and thimet oligopeptidase contributed to the processing of Ang I to Ang-(1-7) within isolated mitochondria [21]. In the human proximal tubule HK-2 cell line, cytosolic thimet oligopeptidase was the sole activity responsible for the generation of Ang-(1-7) from exogenous Ang I [57]. Moreover, thimet oligopeptidase activity in isolated nuclei processed Ang I exclusively to Ang-(1-7) and may be a potential candidate that contributes to the nuclear levels of Ang-(1-7) within the cell [22].…”