Increased urinary angiotensin-converting enzyme 2 in renal transplant patients with diabetes

Abstract

Angiotensin-converting enzyme 2 (ACE2) is expressed in the kidney and may be a renoprotective enzyme, since it converts angiotensin (Ang) II to Ang-(1-7). ACE2 has been detected in urine from patients with chronic kidney disease. We measured urinary ACE2 activity and protein levels in renal transplant patients (age 54 yrs, 65% male, 38% diabetes, n = 100) and healthy controls (age 45 yrs, 26% male, n = 50), and determined factors associated with elevated urinary ACE2 in the patients. Urine from transplant subjects was also assayed for ACE mRNA and protein. No subjects were taking inhibitors of the renin-angiotensin system. Urinary ACE2 levels were significantly higher in transplant patients compared to controls (p = 0.003 for ACE2 activity, and p≤0.001 for ACE2 protein by ELISA or western analysis). Transplant patients with diabetes mellitus had significantly increased urinary ACE2 activity and protein levels compared to non-diabetics (p<0.001), while ACE2 mRNA levels did not differ. Urinary ACE activity and protein were significantly increased in diabetic transplant subjects, while ACE mRNA levels did not differ from non-diabetic subjects. After adjusting for confounding variables, diabetes was significantly associated with urinary ACE2 activity (p = 0.003) and protein levels (p<0.001), while female gender was associated with urinary mRNA levels for both ACE2 and ACE. These data indicate that urinary ACE2 is increased in renal transplant recipients with diabetes, possibly due to increased shedding from tubular cells. Urinary ACE2 could be a marker of renal renin-angiotensin system activation in these patients.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Urinary ACE2 activity and protein in control subjects and renal transplant (Tx) recipients.

(A) Graph depicts box plots of urinary ACE2 activity in control subjects (n = 50) and Tx recipients (n = 100). For each box plot, median values are indicated by the line within the box, with value shown beside or above the line. The box represents 50% of the values (25th and 75th percentiles), with the upper bar representing the 90th percentile and the lower bar representing the 10th percentile. Open circles indicate outliers. * p = 0.003, Control vs Tx recipients. (B) Graph depicts box plots of urinary ACE2 protein by ELISA in control subjects and Tx recipients. ** p<0.001, Control vs Tx recipients. (C) Graph depicts box plots of urinary ACE2 protein by western analyses in control subjects and Tx recipients. Densitometry analysis was performed on both urinary ACE2 bands (120 kDa and 90 kDa), and the sum of the two bands was used for quantitative comparisons. **p = 0.001, Control vs Tx recipients.

Figure 2. Urinary ACE2 activity and protein in renal transplant recipients: Effect of diabetes.

(A) Graph depicts box plots of urinary ACE2 activity in transplant recipients without diabetes (No Diabetes), or with diabetes (Diabetes). For each box plot, median values are indicated by the line within the box, with value shown above the line. The box represents 50% of the values (25th and 75th percentiles), with the upper bar representing the 90th percentile and the lower bar representing the 10th percentile. Open circles indicate outliers. * p<0.001, Diabetes vs. No Diabetes; n = 62 (No Diabetes), n = 38 (Diabetes). (B) Graph depicts box plots of urinary ACE2 protein by ELISA in transplant recipients without diabetes (No Diabetes), or with diabetes (Diabetes). *p<0.001, Diabetes vs. No Diabetes. (C) Graph depicts box plots of urinary ACE2 protein by western analysis in transplant patients without diabetes (No Diabetes), or with diabetes (Diabetes). *p<0.001, Diabetes vs. No Diabetes. Above graph is representative immunoblot for ACE2 in urine, showing bands at 120 kDa and 90 kDa. Densitometry analysis was performed on both bands, and the sum of the two bands was used for quantitative comparisons. The protein bands for ACE2 in urine specimens were not observed when membranes were incubated with the secondary antibody alone, bypassing the primary antibody step. Lanes 1–3, No Diabetes. Lanes 4–6, Diabetes. Lane 7: recombinant human ACE2 protein (hACE2), used as a positive control. (D) Representative immunoblot for urinary ACE2 treated without (−) or with (+) the deglycosylase enzyme PNGase F. Lanes 1+, 2+, 3+, and 4+ show a reduction in the sizes of urinary ACE2 fragments to ∼85 kDa and ∼65 kDa in urine samples treated with PNGase F. Lanes 1 and 2, No Diabetes. Lanes 3 and 4, Diabetes. Lane 5: recombinant human ACE2 protein (hACE2).

Figure 3. Urinary ACE activity and protein in renal transplant recipients: Effect of diabetes.

(A) Graph depicts box plots of urinary ACE activity in transplant recipients without diabetes (No Diabetes) or with diabetes (Diabetes). For each box plot, median values are indicated by the line within the box, with value shown above the line. The box represents 50% of the values (25th and 75th percentiles), with the upper bar representing the 90th percentile and the lower bar representing the 10th percentile. Open circles indicate outliers. * p = 0.013, Diabetes vs No Diabetes. (B) Graph shows box plots for densitometric analysis of protein bands for urinary ACE, at 190 kDa, in transplant recipients without diabetes (No Diabetes) or with diabetes (Diabetes). * p = 0.019, Diabetes vs No Diabetes. Representative ACE immunoblot on urine specimens is shown above graph. In samples from mouse cortex, ACE appeared as a single band at 170 kDa, as expected (mACE, lane 7) (5). This blot was not used for densitometric quantitation of urinary ACE. Protein bands corresponding to ACE were absent when membranes were incubated with the secondary antibody alone. Lanes 1–3, No Diabetes. Lanes 4–6, Diabetes. Lane 7: mouse kidney cortex, used as a positive control.

Figure 4. Urinary Ang II and Ang-(1-7).

(A) Graph shows box plots of RIA for Ang II in urine specimens from transplant recipients without diabetes (no Diabetes) or with diabetes (Diabetes). For each box plot, median values are indicated by the line within the box, with value shown above the line. The box represents 50% of the values (25th and 75th percentiles), with the upper bar representing the 90th percentile and the lower bar representing the 10th percentile. Open circles indicate outliers. * p = 0.027, Diabetes vs. No Diabetes. (B) Graph shows box plots of EIA for Ang-(1-7) in urine specimens from transplant recipients without or with diabetes. There was no significant difference between the two groups (p = 0.126).