Regulation of urinary ACE2 in diabetic mice

Abstract

Angiotensin-converting enzyme-2 (ACE2) enhances the degradation of ANG II and its expression is altered in diabetic kidneys, but the regulation of this enzyme in the urine is unknown. Urinary ACE2 was studied in the db/db model of type 2 diabetes and stretozotocin (STZ)-induced type 1 diabetes during several physiological and pharmacological interventions. ACE2 activity in db/db mice was increased in the serum and to a much greater extent in the urine compared with db/m controls. Neither a specific ANG II blocker, telmisartan, nor an ACE inhibitor, captopril, altered the levels of urinary ACE2 in db/db or db/m control mice. High-salt diet (8%) increased whereas low-salt diet (0.1%) decreased urinary ACE2 activity in the urine of db/db mice. In STZ mice, urinary ACE2 was also increased, and insulin decreased it partly but significantly after several weeks of administration. The increase in urinary ACE2 activity in db/db mice reflected an increase in enzymatically active protein with two bands identified of molecular size at 110 and 75 kDa and was associated with an increase in kidney cortex ACE2 protein at 110 kDa but not at 75 kDa. ACE2 activity was increased in isolated tubular preparations but not in glomeruli from db/db mice. Administration of soluble recombinant ACE2 to db/m and db/db mice resulted in a marked increase in serum ACE2 activity, but no gain in ACE2 activity was detectable in the urine, further demonstrating that urinary ACE2 is of kidney origin. Increased urinary ACE2 was associated with more efficient degradation of exogenous ANG II (10(-9) M) in urine from db/db compared with that from db/m mice. Urinary ACE2 could be a potential biomarker of increased metabolism of ANG II in diabetic kidney disease.

Keywords: angiotensin-converting enzyme 2; biomarker; diabetes; nephropathy; urinary.

Fig. 1.

Urinary angiotensin-converting enzyme 2 (ACE2) activity (A) and albumin/creatinine ratio (B) measured several consecutive times at 1- to 4-wk intervals apart in db/m (triangles) and db/db mice (squares). Significant differences between the two groups were found by repeated-measures general linear model: ***P < 0.001. From the measurements in A and B, 2–3 adjacent measurements were averaged each time to better show the differences in urinary ACE2 activity/creatinine ratio (C) and albumin/creatinine ratio (D) between db/m and db/db mice. RFU, relative fluorescence units. As the animals become older and albuminuria increases, urinary ACE2 activity increases further: *P < 0.05, **P < 0.01, ***P < 0.001 vs. db/m.

Fig. 2.

Urinary angiotensin-converting enzyme 2 (ACE2) activity in 18-wk-old female db/m (n = 5) and db/db mice (n = 5) measured in urines collected over 12 h in metabolic cages. Urinary ACE2 activity in db/db mice was about 3-fold higher using both 12-h ACE2 excretion rates (A) and using ACE2 activity/creatinine ratio (B). A significant correlation between 12-h ACE2 excretion rates (A) and ACE2 activity/creatinine ratio (B) in db/m (n = 5, circle data points) and db/db mice (square data points) measured in urines collected over 12 h in metabolic cages (C): **P < 0.01, ***P < 0.001 vs. db/db. A significant positive correlation between ACE2 activity/12 h and urine volume (D) and between ACE2 activity/creatinine ratio and urine volume (E) were also found. Significant positive correlations were also observed between ACE2 activity and albumin/creatinine ratio measured in spot urines collected from db/m (n = 20) and db/db mice (n = 20) over a period of 20 wk (18–38 wk of age) (F).

Fig. 3.

A: urinary ACE2 activity measured at 1- to 3-wk intervals several consecutive times in mice on a normal salt intake, on high-salt (8% NaCl), and on low-salt (0.1% NaCl) diet. The differences between db/db and db/m mice were significant by repeated measures using GLM analysis throughout the three experimental periods (P < 0.001). The four consecutive ACE2 activity measurements for each experimental period in A were averaged as shown in B. A significant increase in urinary ACE2 activity during high-salt diet intake in both db/m and db/db mice was observed compared with respective groups on a normal-salt diet. Compared with normal-salt diet, low-salt diet was associated with significantly lower ACE2 activity in db/db mice. In db/m mice there was no significant difference between low- and normal-salt diet. *High salt vs. normal salt (control) db/m (P < 0.05). ***High salt db/db vs. normal salt (control) db/db and low salt db/db (P < 0.001). #Low salt db/db vs. normal salt (control) db/db (P < 0.05).

Fig. 4.

Urinary ACE2 activity/creatinine ratio (A) and albumin/creatinine ratio (C) measured at 1- to 3-wk intervals 3–4 consecutive times in mice under control conditions, then receiving an ACE inhibitor, captopril, and an AT1 receptor blocker, telmisartan. A 1-wk washout of drug administration was allowed between periods. Significant differences between db/db and db/m were observed by repeated-measures analysis using GLM during the control period and were sustained during captopril or telmisartan administration. *P < 0.05, **P < 0.01, ***P < 0.001, db/db vs. db/m. B: four consecutive ACE2 activity/creatinine ratio measurements for each experimental protocol in A were averaged. No significant differences in urinary ACE2 activity/creatinine ratio between control conditions and either captopril or telmisartan administration were found in either db/m or db/db mice. D: three to four consecutive albumin/creatinine ratio measurements for each experimental protocol in B were averaged. Significant differences in urinary albumin/creatinine ratio between control conditions and either captopril or telmisartan administration were found in db/db mice (#P < 0.05 by ANOVA followed by LSD post hoc test) but in db/m mice the differences were not significant.

Fig. 5.

The effect of an acute recombinant (r)ACE2 infusion on serum (top panels) and urinary ACE2 activity (bottom panels) in db/m (left panels) and db/db mice (right panels). Sera and urines were collected before (–) and 3 h after rACE2 ip bolus (1 mg/kg BW). Administration of rACE2 resulted in a marked increase in serum ACE2 activity in both db/m and db/db mice, but not in urinary ACE2 activity in either db/m or db/db mice.

Fig. 6.

ACE2 activity in kidney tubular preparations (A) and glomeruli isolated using Dynabeads (B) in 32-wk-old female db/m (white bars) and db/db mice (gray bars). Tubular ACE2 activity in db/db mice was significantly higher than in db/m controls (A): ***P < 0.001 vs. db/m. In glomerular isolates, ACE2 activity was not significantly different between the two groups (B).

Fig. 7.

A: urinary ACE2 activity measured multiple times at 1- to 3-wk intervals apart in vehicle controls (triangles) and streptozotocin (STZ)-treated diabetic mice (squares). After STZ administration there was a significant increase of urinary ACE2 activity in STZ-treated mice throughout the entire period of follow-up (P < 0.001 by repeated-measures GLM analysis).

Fig. 8.

Blood glucose (A), urinary ACE2 activity/creatinine ratio (B), and urinary albumin/creatinine ratio (C) measured in control (n = 5, white filling), STZ-mice (n = 9, black filling), and STZ mice that received insulin (INS) pellets for 12 wk (n = 9, gray filling). Insulin almost completely normalized blood glucose levels in STZ mice early on (A). Bars in B and C represent means of 3 consecutive spot urine collections 1–2 wk apart for each experimental group. STZ-treated mice have higher levels of ACE2 activity and albumin/creatinine ratio than vehicle controls. ACE2 activity was not significantly reduced by insulin in STZ-treated mice at 5–7 wk of its administration but it fell significantly after 8–12 wk of its administration (P < 0.01). Albumin/creatinine ratio was reduced by insulin administration but not significantly. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. control. #P < 0.05 and ##P < 0.01 vs. STZ.

Fig. 9.

Degradation of exogenous ANG II by urine from different groups. A: urines from C57BL/6 wild-type mice (WT, n = 3) degraded ANG II more efficiently than those obtained from ACE2 deficient mice on the same genetic background (ACE2KO, n = 3). **P < 0.01 WT vs. ACE2KO as determined by repeated-measures GLM analysis. B: urines from db/db mice (n = 11) degraded ANG II more efficiently than those collected from db/m (n = 9) *P < 0.05, db/db vs. db/m as determined by repeated-measures GLM analysis. C: degradation of ANG II in the urines from the db/db (n = 11) and db/m mice (n = 9) was not significantly different when a specific ACE2 inhibitor, MLN-4760 (10⁻⁶ M), was added to the urine.

Fig. 10.

ACE2 protein in urines from db/m and db/db mice. A representative Western blot of concentrated urines from 20- to 24-wk-old female db/m and db/db mice showing two main ACE2 immunoreactive bands at around 75 and 100–110 kDa. Graph below depicts densitometric analysis of Western blots showing that both 75 and 100–110 kDa ACE2-immunoreactive proteins were significantly increased in urines from db/db mice (n = 16) compared with db/m (n = 14). *P < 0.05.

Fig. 11.

A: Western blot of concentrated urines from db/m and db/db mice (each group n = 5) showing ACE2 immunoreactive bands at around 100–110 kDa and also at 75 kDa variably expressed (see also Fig. 10). B: kidney cortex membrane fraction (upper image) and whole lysates (middle image) collected from the db/m and db/db mice shown in A were probed in Western blot with ACE2 specific antibody showing a single immunoreactive band at ∼100–110 kDa.

Fig. 12.

ACE2 immunoreactive proteins in pooled urines from wild-type (WT) and ACE2 knockout mice (KO). Urines were concentrated (∼50×) on ultrafiltration device before testing. Concentrated mouse urines were separated on SDS-PAGE and probed in Western blot using a specific anti-ACE2 antibody. Two distinct bands (∼75 kDa and ∼100–110 kDa) were seen in WT (lane 2) but not in ACE2KO urines (lane 3). For comparison, soluble recombinant mouse ACE2 (rACE2) was run in parallel (lane 1) and showed a main band at ∼100–110 kDa as well as an additional band at a higher molecular size, which is likely its homodimer.