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Neprilysin is a Mediator of Alternative Renin-Angiotensin-System Activation in the Murine and Human Kidney - PubMed

  • ️Fri Jan 01 2016

Neprilysin is a Mediator of Alternative Renin-Angiotensin-System Activation in the Murine and Human Kidney

Oliver Domenig et al. Sci Rep. 2016.

Abstract

Cardiovascular and renal pathologies are frequently associated with an activated renin-angiotensin-system (RAS) and increased levels of its main effector and vasoconstrictor hormone angiotensin II (Ang II). Angiotensin-converting-enzyme-2 (ACE2) has been described as a crucial enzymatic player in shifting the RAS towards its so-called alternative vasodilative and reno-protective axis by enzymatically converting Ang II to angiotensin-(1-7) (Ang-(1-7)). Yet, the relative contribution of ACE2 to Ang-(1-7) formation in vivo has not been elucidated. Mass spectrometry based quantification of angiotensin metabolites in the kidney and plasma of ACE2 KO mice surprisingly revealed an increase in Ang-(1-7), suggesting additional pathways to be responsible for alternative RAS activation in vivo. Following assessment of angiotensin metabolism in kidney homogenates, we identified neprilysin (NEP) to be a major source of renal Ang-(1-7) in mice and humans. These findings were supported by MALDI imaging, showing NEP mediated Ang-(1-7) formation in whole kidney cryo-sections in mice. Finally, pharmacologic inhibition of NEP resulted in strongly decreased Ang-(1-7) levels in murine kidneys. This unexpected new role of NEP may have implications for the combination therapy with NEP-inhibitors and angiotensin-receptor-blockade, which has been shown being a promising therapeutic approach for heart failure therapy.

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Figures

Figure 1
Figure 1. Elevated Ang-(1-7) levels in kidneys of ACE2 KO mice.

(a) Kidney angiotensin concentrations of wild-type (C57BL/6) and ACE2 knockout (ACE2 KO) mice (pg/g gram net weight). n = 8 mice per group. Data presented as mean ± s.d. Two-tailed Student’s t-test. *P < 0.05 **P < 0.01 ***P < 0.001 vs. wild-type. (b) Relative mRNA abundance (to beta actin) of renin in murine renal medulla (dark grey) and cortex (light grey) of wild-type and ACE2 KO. n = 8 mice per group. Data presented as mean ± s.d. One-way analysis of variance (ANOVA). *P < 0.05 vs. medulla. (c) Renal renin activity of wild-type (black) ACE2 KO (white) assayed by Ang I formation determination in homogenates following recombinant murine angiotensinogen spiking. n = 4 mice per group. Data presented as mean ± s.d. Two-tailed Student’s t-test. not significant (NS) vs. wild-type.

Figure 2
Figure 2

Analysis of angiotensin metabolism in kidney homogenates of mice (a–c) and human (d–f ). (a) Renal Ang II turnover to Ang-(1-7) of wild-type (black) and ACE2 KO mice (white) in presence and absence of specific inhibitors. n = 4 per group. Data presented as mean ± s.d. One-way analysis of variance (ANOVA). *P < 0.001 within wild-type group vs. solvent control. (b) Ang I turnover to Ang-(1-7) (left) and Ang II (right) of wild-type (black) and ACE2 KO (white). n = 4 per group. Data presented as mean ± s.d. One-way analysis of variance (ANOVA). *P < 0.001 vs. solvent control. (c) Enzymatic contribution to Ang II or Ang-(1-7) formation in mice was calculated on the inhibitor sensitive angiotensin formation rate of fig. 2a,b. Data presented as mean ± s.d. (d) Human renal Ang II turnover to Ang-(1-7) in presence and absence of specific inhibitors. n = 5 per group. Data presented as mean ± s.d. One-way analysis of variance (ANOVA). *P < 0.001 vs. solvent control. (e) Human Ang I turnover to Ang-(1-7) (left) and Ang II (right) in kidney homogenates. n = 5 per group. Data presented as mean ± s.d. One-way analysis of variance (ANOVA). *P < 0.001 vs. solvent control. (f) Enzymatic contribution to Ang II or Ang-(1-7) formation in human was calculated on the inhibitor sensitive angiotensin formation rate of fig. 2d,e. Data presented as mean ± s.d.

Figure 3
Figure 3. MALDI-Imaging reveals DL-thiorphan-sensitive Ang-(1-7) formation in renal cortex of mice.

(a) Diminishing effect of increasing DL-thiophan concentrations (0 μM – 100 μM) on local turnover of Ang I to Ang-(1-7) is shown by MALDI-Imaging. Brighter colors of the murine kidney section indicate pronounced Ang-(1-7) formation in the renal cortex. (b) Increasing Ang-(1-7) signals depending on lisinopril concentration (0 μM–10 μM). (c) Inhibitory capacity of 100 μM DL-thiorphan on Ang I to Ang-(1-7) turnover in presence of 10 μM lisinopril (d) Intensity of Ang-(1-7) formation (Fig. 3c) was calculated and is given in bars. n = 4 per group. Data presented as mean ± s.d. Two-tailed Student’s t-test. *P < 0.05 vs. 10 μM lisinopril. (e) Schematic graph highlights the targeted angiotensin pathways of the used inhibitors.

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