Circulating angiotensin-converting enzyme 2 activity in patients with chronic kidney disease without previous history of cardiovascular disease

Abstract

Background: Patients with cardiovascular (CV) disease have an increased circulating angiotensin-converting enzyme 2 (ACE2) activity, but there is little information about changes in ACE2 in chronic kidney disease (CKD) patients without history of CV disease. We examined circulating ACE2 activity in CKD patients at stages 3-5 (CKD3-5) and in dialysis (CKD5D) without any history of CV disease.

Methods: Circulating ACE2 activity was measured in human ethylenediamine-tetraacetic acid (EDTA)-plasma samples from the NEFRONA study (n = 2572): control group (CONT) (n = 568), CKD3-5 (n = 1458) and CKD5D (n = 546). Different clinical and analytical variables such as gender; age; history of diabetes mellitus (DM), dyslipidemia and hypertension; glycaemic, renal, lipid and anaemia profiles; vitamin D analogues treatment and antihypertensive treatments (angiotensin-converting enzyme inhibitor and angiotensin receptor blockade) were analysed. Circulating ACE2 and ACE activities were measured using modified fluorimetric assay for EDTA-plasma samples, where zinc chloride was added to recover enzymatic activity.

Results: In CKD3-5 and CKD5D, significant decrease in circulating ACE2 activity was observed when compared with CONT, but no differences were found between CKD3-5 and CKD5 when performing paired case-control studies. By multivariate linear regression analysis, male gender and advanced age were identified as independent predictors of ACE2 activity in all groups. Diabetes was identified as independent predictor of ACE2 activity in CKD3-5. Significant increase in the activity of circulating ACE was found in CKD3-5 and CKD5D when compared with CONT and in CKD5D when compared with CKD3-5. By multiple regression analysis, female gender and younger age were identified as independent predictors of ACE activity in CONT and CKD3-5. Diabetes was also identified as an independent predictor of ACE activity in CKD3-5 patients.

Conclusions: Circulating ACE2 and ACE activities can be measured in human EDTA-plasma samples with zinc added to recover enzymatic activity. In a CKD population without previous history of CV disease, ACE2 activity from human EDTA-plasma samples directly correlated with the classical CV risk factors namely older age, diabetes and male gender. Our data suggest that circulating ACE2 is altered in CKD patients at risk for CV event.

Keywords: ACE2; biomarkers; cardiovascular disease; chronic kidney disease; diabetes; renin–angiotensin system.

FIGURE 1:

Circulating ACE2 (A) and ACE (B) enzymatic activities at increased concentrations of zinc chloride (ZnCl₂). (C) Standard curve of recombinant human ACE2 (rhACE2). (D) ACE2 activity in EDTA-plasma samples with the rhACE2 and ZnCl₂. (A) ACE2 activity was assessed in six different EDTA-plasma samples (1–6) using the following concentrations of ZnCl₂: 0, 0.5, 1 and 3 mM. (B) ACE activity was assessed in four different EDTA-plasma samples (A–D) using the following concentrations of ZnCl₂: 0, 7.81, 15.63 and 31.25 mM. (C) ACE2 activity was linearly increased with increasing the rhACE2 concentrations. (D) ACE2 activity was assessed in four EDTA-plasma samples with the following conditions: EDTA-plasma alone, EDTA-plasma adding ZnCl₂ at a concentration of 0.5 mM, EDTA-plasma adding 1 ng of rhACE2 and EDTA-plasma adding both ZnCl₂ at 0.5 mM and 1 ng of rhACE2.

FIGURE 2:

Circulating ACE2 activity between studied groups. (A) ACE2 activity significantly decreased in CKD3-5 (grey bars) and CKD5D (white bars) patients as compared with CONT (black bars) (*P < 0.001). CKD3-5 showed an increase in plasma ACE2 activity as compared with CKD5D (^$P < 0.001). When matching samples with an equal distribution of gender, diabetes, hypertension, dyslipidemia, smoking habits, weight and age (B), ACE2 activity significantly decreased in CKD3-5 and CKD5D as compared with CONT, but no differences were found between CKD3-5 and CKD5D (P = 0.27). CONT, control patients; CKD3-5, non-dialysis patients with chronic kidney disease stage 3–5; CKD5D, dialysis.

FIGURE 3:

Circulating ACE activity between studied groups. (A) ACE activity was increased in CKD3-5 (grey bars) (*P = 0.035) and in CKD5D (white bars) (*<0.001) as compared with CONT (black bars). CKD5D showed an increase in plasma ACE activity as compared with CKD3-5 (^$P = 0.001). When assessing a paired case-control study (B), same results were obtained. CONT, control patients; CKD3-5, non-dialysis patients with chronic kidney disease stage 3–5; CKD5D, dialysis.