Evidence for a mitochondrial angiotensin-(1-7) system in the kidney

Abstract

Evidence for an intracellular renin-angiotensin system (RAS) in various cell organelles now includes the endoplasmic reticulum, nucleus, and mitochondria (Mito). Indeed, angiotensin (ANG) AT₁ and AT₂ receptor subtypes were functionally linked to Mito respiration and nitric oxide production, respectively, in previous studies. We undertook a biochemical analysis of the Mito RAS from male and female sheep kidney cortex. Mito were isolated by differential centrifugation followed by a discontinuous Percoll gradient and were coenriched in Mito membrane markers VDAC and ATP synthase, but not β-actin or cathepsin B. Two distinct renin antibodies identified a 37-kDa protein band in Mito; angiotensinogen (Aogen) conversion was abolished by the inhibitor aliskiren. Mito Aogen was detected by an Aogen antibody to an internal sequence of the protein, but not with an antibody directed against the ANG I N terminus. ANG peptides were quantified by three direct RIAs; mitochondrial ANG II and ANG-(1-7) contents were higher compared with ANG I (23 ± 8 and 58 ± 17 vs. 2 ± 1 fmol/mg protein; P < 0.01, n = 3). ¹²⁵I-ANG I metabolism primarily revealed the formation of ¹²⁵I-ANG-(1-7) in Mito that reflects the endopeptidases neprilysin and thimet oligopeptidase. Last, immunoblot studies utilizing the ANG-(1-7)/Mas receptor antibody revealed the protein in isolated Mito from sheep renal cortex. Collectively, the current data demonstrate that Mito actively metabolize the RAS precursor protein Aogen, suggesting that ANG-(1-7) may be generated within Mito to establish an intramitochondrial RAS tone and contribute to renal mitochondrial function.

Keywords: ANG-(1–7); angiotensin-(1–7); kidney; mitochondria; renin-angiotensin system.

Fig. 1.

Purification and enrichment of mitochondria from sheep renal cortex. Whole renal cortex homogenate (lane 1), crude mitochondrial fraction (lane 2), and Percoll-purified mitochondrial fraction (lane 3) were subjected to 10% SDS gel and immunoblotting with anti-VDAC (50 μg total protein; A) as well as ATP5A, UQCRC2, SDHB (5 μg total protein; B), and cathepsin B (50 μg total protein; C), as well as β-actin. Purified mitochondria exhibited greater expression of outer mitochondria marker (VDAC) and inner mitochondrial markers (ATP5A, UQCRC2, and SDHB), while cathepsin B and β-actin expression were diminished.

Fig. 2.

Renin and prorenin receptor expression in isolated mitochondria from sheep renal cortex. A: immunoblot of rat renal proximal tubular (NRK-52E) cells (lane 2) and sheep renal mitochondria preparations (lanes 3–5) with the Inagami renin antibody revealed 2 distinct protein bands (37 and 55 kDa). Lanes 3–4 represent female mitochondrial preparations, while lane 5 represents a male preparation. B: immunoblot of renal cortex homogenate (lane 2) and sheep renal mitochondria preparations from 3 male sheep (lanes 3–5) with the Aviva renin antibody revealed 2 distinct protein bands (37 and 55 kDa). C: immunoblot of prorenin receptor (ATP6IP2) revealed a predominant protein band at 35 kDa in renal cortex homogenate (lane 2), but not in the mitochondrial preparations from 3 male sheep (lanes 3–5). Lane 1 (A–C) is the protein molecular weight marker.

Fig. 3.

Renin activity in the mitochondria. Renin activity in the mitochondria of a male (A, C, E, and G) and female (B, D, F, and H) sheep. Non-trypsin (A and B)- or trypsin (C and D)-treated mitochondrial homogenates with (+) or without (−) the renin inhibitor aliskiren was detected by immunoblot analysis using an ANG I (AI)-angiotensinogen (Aogen)-directed antibody. Relative quantification of the ANG I-Aogen band without (E and F) or containing aliskiren (G and H) was performed. The disappearance or half-life (t½) of AI-Aogen was determined from each experiment.

Fig. 4.

Angiotensinogen (Aogen) and angiotensin peptide expression in isolated mitochondria from sheep renal cortex. A: immunoblot of Aogen in nephrectomized sheep plasma (NSP; lane 1) and isolated mitochondria from the renal cortex of 3 male sheep (lanes 2–4). A protein band of 55 kDa was detected for total Aogen in all lanes (top), but AI-Aogen was not detected in the mitochondria preparations (bottom). B: angiotensin peptide expression was quantified in isolated mitochondria from sheep renal cortex using distinct radioimmunoassays. ANG I, ANG II, and ANG-(1–7) were detected; however, there was a significant difference between the levels of ANG I compared with ANG II and ANG-(1–7). Values are means ± SE; n = 3. *P < 0.01 ANG I vs. ANG II, ANG I vs. ANG-(1–7).

Fig. 5.

Mitochondrial processing of ¹²⁵I-ANG I. Chromatograph reveals ¹²⁵I-ANG I (¹²⁵I-AI) was metabolized to ¹²⁵I-ANG-(1–7) (¹²⁵I-A7) in renal mitochondrial homogenate fractions (A). ¹²⁵I-AI metabolism to ¹²⁵I-A7 was reduced by 10 μM SCH (B), and ¹²⁵I-AI metabolism to ¹²⁵I-A7 was reduced by 10 μM SCH in combination with 10 μM CPP (C). Immunoblots for neprilysin (NEP) and thimet oligopeptidase (TOP) in mitochondrial homogenates (lanes 2–4, n = 3) along with renal cortex homogenate as a positive control (lane 1; D). ¹²⁵I-labeled products were separated by HPLC under gradient conditions. Statistical quantification of ¹²⁵I-AI metabolism demonstrated SCH (10 μM) and SCH in combination with CPP (10 μM) preserved ¹²⁵I-ANG I, while SCH (10 μM) and CPP (10 μM) reduced ¹²⁵I-ANG-(1–7) formation (E and F). The addition of SCH and CPP did not significantly further influence ¹²⁵I-ANG-(1–7) formation or preservation of ¹²⁵I-ANG I (E and F). Values are means ± SE; n = 3. *P < 0.05 vs. control (CON).

Fig. 6.

Immunodetection of receptor expression in sheep renal mitochondria. A: whole renal cortex homogenate (lane 2), crude mitochondrial fraction (lane 3), and Percoll-purified mitochondrial fraction (lane 4) were subjected to 10% SDS gel and immunoblotting with an antibody against the MAS receptor protein. B: whole renal cortex homogenate (lane 2) and purified mitochondria (lane 3–5) from 3 distinct male sheep exhibited protein expression of the MAS receptor. Lane 1 (A and B) is the protein molecular weight marker.