Diminazene Aceturate Improves Cardiac Fibrosis and Diastolic Dysfunction in Rats with Kidney Disease

Abstract

Angiotensin converting enzyme (ACE) 2 is a negative regulator of the renin angiotensin system (RAS) through its role to degrade angiotensin II. In rats with subtotal nephrectomy (STNx), adverse cardiac remodelling occurs despite elevated cardiac ACE2 activity. We hypothesised that diminazene aceturate (DIZE), which has been described as having an off-target effect to activate ACE2, would have beneficial cardiac effects in STNx rats. STNx led to hypertension, diastolic dysfunction, left ventricular hypertrophy, cardiac fibrosis, and increased cardiac ACE, ACE2, Ang II and Ang 1-7 levels. Cardiac gene expression of ADAM17 was also increased. In STNx, two-weeks of subcutaneous DIZE (15mg/kg/d) had no effect on blood pressure but improved diastolic dysfunction and cardiac fibrosis, reduced ADAM17 mRNA and shifted the cardiac RAS balance to a cardioprotective profile with reduced ACE and Ang II. There was no change in cardiac ACE2 activity or in cardiac Ang 1-7 levels with DIZE. In conclusion, our results suggest that DIZE exerts a protective effect on the heart under the pathological condition of kidney injury. This effect was not due to improved kidney function, a fall in blood pressure or a reduction in LVH but was associated with a reduction in cardiac ACE and cardiac Ang II levels. As in vitro studies showed no direct effect of DIZE on ACE2 or ACE activity, the precise mechanism of action of DIZE remains to be determined.

Fig 1. DIZE improved diastolic dysfunction in STNx.

Systolic blood pressure (A), ventricular contractility (B), ventricular relaxation (C) and time constant of isovolumic relaxation (Tau; D) in Control (Cont) and subtotal nephrectomy (STNx) rats (n = 8/group). Data expressed as mean±SEM. *P<0.05, ***P<0.001 disease effect (Control vs. STNx) and # P<0.05 treatment effect (Vehicle vs. DIZE)

Fig 2. DIZE was associated with a reduction in cardiac fibrosis in STNx.

Left ventricular hypertrophy (A), interstitial collagen (B) and perivascular collagen (C) in Control (Cont) and subtotal nephrectomy (STNx) rats (n = 8/group). Right hand panel consists of representative photomicrographs of left ventricular total collagen content (red staining) (magnification x200). Data expressed as mean±SEM. **P<0.01, ***P<0.001 disease effect (Control vs. STNx) and # P<0.05 treatment effect (Vehicle vs. DIZE)

Fig 3. DIZE shifts the cardiac RAS balance to a cardioprotective profile in STNx.

Left ventricular (LV) ACE (A) and ACE2 activity (B), ACE/ACE2 activity ratio (C), LV Ang II (D) and Ang 1–7 (E) peptide content in Control (Cont) and subtotal nephrectomy (STNx) rats (n = 8/group). Data expressed as mean ± SEM. *P<0.05 disease effect (Control vs. STNx) and # P<0.05 treatment effect (Vehicle vs. DIZE)

Fig 4. Cardiac ACE2 activity is increased to counteract elevated cardiac ACE and is shed into the circulation.

Correlation analysis shows increased left ventricular (LV) ACE2 activity is associated with increased LV ACE activity (A) and plasma ACE2 activity (B), with no correlation between LV tissue and plasma ACE. Control and subtotal nephrectomy (STNx) rats without active treatment were used for the correlation analysis. Open squares represent Control rats; closed squares represent STNx rats.

Fig 5. DIZE does no effect cardiac ACE and ACE2 under ex vivo conditions.

Ex vivo DIZE had no effect on endogenous ACE2 (A) and ACE (B) activity in LV membrane preps (100μg per well) from Control (n = 4) and subtotal nephrectomy (STNx, n = 4) rats. The ACE inhibitor, ramiprilat caused a concentration-dependent displacement of specific ¹²⁵I-MK351A binding from rat LV ACE and DIZE had no effect on ACE binding (C).