Reduction in renal ACE2 expression in subtotal nephrectomy in rats is ameliorated with ACE inhibition

Abstract

Alterations within the RAS (renin-angiotensin system) are pivotal for the development of renal disease. ACE2 (angiotensin-converting enzyme 2) is expressed in the kidney and converts the vasoconstrictor AngII (angiotensin II) into Ang-(1-7), a peptide with vasodilatory and anti-fibrotic actions. Although the expression of ACE2 in the diabetic kidney has been well studied, little is known about its expression in non-diabetic renal disease. In the present study, we assessed ACE2 in rats with acute kidney injury induced by STNx (subtotal nephrectomy). STNx and Control rats received vehicle or ramipril (1 mg. kg (-1) of body weight . day (-1), and renal ACE, ACE2 and mas receptor gene and protein expression were measured 10 days later. STNx rats were characterized by polyuria, proteinuria, hypertension and elevated plasma ACE2 activity (all P<0.01) and plasma Ang-(1-7) (P<0.05) compared with Control rats. There was increased cortical ACE binding and medullary mas receptor expression (P<0.05), but reduced cortical and medullary ACE2 activity in the remnant kidney (P<0.05 and P<0.001 respectively) compared with Control rats. In STNx rats, ramipril reduced blood pressure (P<0.01), polyuria (P<0.05)and plasma ACE2 (P<0.01), increased plasma Ang-(1-7) (P<0.001), and inhibited renal ACE(P<0.001). Ramipril increased both cortical and medullary ACE2 activity (P<0.01), but reduced medullary mas receptor expression (P<0.05). In conclusion, our results show that ACE2 activity is reduced in kidney injury and that ACE inhibition produced beneficial effects in association with increased renal ACE2 activity. As ACE2 both degrades AngII and generates the vasodilator Ang-(1-7), a decrease in renal ACE2 activity, as observed in the present study, has the potential to contribute to the progression of kidney disease.

Figure 1. Relative quantification of cortical (A and B) and medullary (C and D) ACE and ACE2 mRNA using QRT–PCR in control or STNx rats with vehicle or ramipril treatment

*P<0.05 compared with Control (n=7–10/group).

Figure 2. Localization and quantification of ACE radiolabelling in the rat kidney in Control or STNx rats with vehicle or ramipril treatment

(A) In vitro autoradiograph showing ACE radiolabelling in the rat kidney. The white colour denotes the highest density of labelling, whereas the black colour represents background labelling. Sections from Control (Sham) rat kidney and STNx kidney without and with ramipril treatment are shown. In the Control kidney, there is abundant ACE radiolabelling of the inner cortex and outer stripe of the outer medulla. STNx increases ACE radiolabelling, which was reduced by ramipril. (B) Relative quantification of ACE radiolabelling in the inner cortex of the rat kidney expressed as a percentage of that in Control rats. ***P<0.001 compared with Control vehicle ^###P<0.001 compared with appropriate vehicle-treated rats (n=5/group).

Figure 3. ACE2 activity in the cortex (A) and medulla (B) of rat kidney in Control or STNx rats with vehicle or ramipril treatment

*P<0.05 and ***P<0.001 compared with Control vehicle ^##P<0.01 compared with STNx vehicle (n=7–10/group).

Figure 4. Light microscopic micrographs showing ACE2 immunohistochemical labelling (brown staining) of the rat kidney cortex (A–E) and medulla (F–J) in Control or STNx rats with vehicle or ramipril treatment

Micrographs show staining of the tubules in the cortex from vehicle- and ramipril-treated Control (A and B), STNx (C) and ramipril-treated STNx (D) kidneys. Medullary collecting ducts (CD) also stain ACE2 in vehicle- and ramipril-treated Control (F and G), STNx (H) and ramipril-treated STNx (I) kidneys. Immunohistochemical negative (-ve) controls are shown in (E) and (J). DT, distal tubule; Gl, glomeruli; PT, proximal tubules.

Figure 5. Quantification and localization of the mas receptor in the cortex and medulla in Control or STNx rats with vehicle or ramipril treatment

Upper panels, relative quantification of cortical (A) and medullary (B) mas receptor mRNA using QRT–PCR in Control or STNx rats with vehicle or ramipril treatment. *P<0.05 compared with Control; ^#P<0.05 compared with STNx (n=7–10/group). Lower panels, light microscopic micrographs showing mas receptor immunohistochemical labelling (brown staining) in the rat kidney cortex (A–E) and medulla (F–J). Micrographs show staining of apical and basal membranes of distal tubules (DT) from Control (A), ramipril-treated Control (B), STNx (C) and ramipril-treated STNx (D) kidneys. Collecting ducts (CD), medullary thin limbs of Henle (TL) and cells between the tubules also stain for the mas receptor in Control (F), ramipril-treated Control (G), STNx (H) and ramipril-treated STNx (I) kidney medulla. Gl, glomeruli; PT, proximal tubules. Immunohistochemical negative (-ve) controls are shown in (E) and (J).