Loss of angiotensin-converting enzyme-2 (Ace2) accelerates diabetic kidney injury

Abstract

Diabetic nephropathy is one of the most common causes of end-stage renal failure, but the factors responsible for the development of diabetic nephropathy have not been fully elucidated. We examined the effect of deletion of the angiotensin-convert-ing enzyme 2 (Ace2) gene on diabetic kidney injury. Ace2(-/-) mice were crossed with Akita mice (Ins2(WT/C96Y)), a model of type 1 diabetes mellitus, and four groups of mice were studied at 3 months of age: Ace2(+/y)Ins2(WT/WT), Ace2(-/y)Ins2(WT/WT), Ace2(+/y) Ins2(WT/C96Y), and Ace2(-/y)Ins2(WT/C96Y). Ace2(-/y) Ins2(WT/C96Y) mice exhibited a twofold increase in the urinary albumin excretion rate compared with Ace2(+/y)Ins2(WT/C96Y) mice despite similar blood glucose levels. Ace2(-/y)Ins2(WT/C96Y) mice were the only group to exhibit increased mesangial matrix scores and glomerular basement membrane thicknesses compared with Ace2(+/y)Ins2(WT/WT) mice, accompanied by increased fibronectin and alpha-smooth muscle actin immunostaining in the glomeruli of Ace2(-/y) Ins2(WT/C96Y) mice. There were no differences in blood pressure or heart function to account for the exacerbation of kidney injury. Although kidney levels of angiotensin (Ang) II were not increased in the diabetic mice, treatment with an Ang II receptor blocker reduced urinary albumin excretion rate in Ace2(-/y)Ins2(WT/C96Y) mice, suggesting that acceleration of kidney injury in these mice is Ang II-mediated. We conclude that ACE2 plays a protective role in the diabetic kidney, and ACE2 is an important determinant of diabetic nephropathy.

Figure 1

Breeding strategy. Female Ace2^−/−Ins2^WT/WT mice were bred with male Ace2^+/yIns2^WT/C96Y mice.

Figure 2

Body weight and blood glucose measurements. a: Body weights were measured monthly and the values were similar in the four groups of mice. b: Blood glucose was also measured monthly in each group of mice using an Ascensia Breeze glucometer. Both diabetic groups developed early and sustained hyperglycemia compared with the nondiabetic groups. Data are presented as mean ± SEM. *P < 0.05 compared with Ace2^+/yIns2^WT/WT mice, ^†P < 0.05 compared with Ace2^−/yIns2^WT/WT mice, and **P < 0.05 compared with Ace2^+/yIns2^WT/C96Y mice.

Figure 3

Development of increased urinary AERs in the diabetic mice. The urinary AER was measured with an enzyme-linked immunosorbent assay in 24-hour urine samples collected at 3 months. The AER rate increased in both groups of diabetic mice but the AER was twofold greater in the Ace2^−/yIns2^WT/C96Y mice compared with the Ace2^+/yIns2^WT/C96Y mice at 3 months of age. Data are presented as mean ± SEM. *P < 0.05 compared with Ace2^+/yIns2^WT/WT mice, ^†P < 0.05 compared with Ace2^−/yIns2^WT/WT mice, and **P < 0.05 compared with Ace2^+/yIns2^WT/C96Y mice.

Figure 4

Analysis of the mesangial matrix scores in glomeruli. a: Representative light micrographs of PAS-stained kidney sections from each group of mice. b: Mesangial matrix scores were generated on a quadrant scale (0 to 4) based on the amount of PAS-positive material in 34 glomerular profiles per animal. The Ace2^−/yIns2^WT/C96Y mice were the only diabetic group to develop a significant increase in the mesangial matrix score at 3 months of age compared with the control Ace2^+/yIns2^WT/WT mice. Data are presented as mean ± SEM on n = 6 per group. *P < 0.05 compared with Ace2^+/yIns2^WT/WT mice, and ^†P < 0.05 compared with Ace2^−/yIns2^WT/WT mice. Original magnifications, ×600.

Figure 5

Histological analysis of fibronectin immunostaining in glomeruli. a: Representative light micrographs of fibronectin immunostaining in kidney sections from each group of mice. b: Fibronectin immunostaining in glomeruli was analyzed by computer image analysis. Fibronectin expression increased in both groups of diabetic mice, but there was a marked increase in fibronectin expression in the glomeruli of the Ace2^−/yIns2^WT/C96Y mice compared with the Ace2^+/yIns2^WT/C96Y mice at 3 months of age by measuring brown staining pixel density. Data are presented as mean ± SEM. *P < 0.05 compared with Ace2^+/yIns2^WT/WT mice, ^†P < 0.05 compared with Ace2^−/yIns2^WT/WT mice, and **P < 0.05 compared with the Ace2^+/yIns2^WT/C96Y mice. Original magnifications, ×600.

Figure 6

Histological analysis of α-SMA immunostaining in glomeruli. a: Representative light micrographs of α-SMA immunostaining in kidney sections from each group of mice. b: Semiquantitative scoring of α-SMA immuno-staining in glomeruli. α-SMA immunostaining was increased sixfold in the Ace2^−/yIns2^WT/C96Y mice compared with the Ace2^+/yIns2^WT/C96Y mice at 3 months of age. Data are presented as mean ± SEM. *P < 0.05 compared with Ace2^+/yIns2^WT/WT mice, ^†P < 0.05 compared with Ace2^−/yIns2^WT/WT mice, and **P < 0.05 compared with the Ace2^+/yIns2^WT/C96Y mice. Original magnifications, ×600.

Figure 7

Effect of diabetes mellitus on ACE, ACE2, and the bradykinin B2 receptor expression in the kidney. mRNA levels were measured in the renal cortical tissue by real-time RT-PCR and related to 18S mRNA levels. a: Hyperglycemia was associated with a decrease in ACE expression in the kidney cortex of both groups of diabetic mice. b: ACE2 expression increased in the Ace2^+/yIns2^WT/C96Y mice compared with the Ace2^+/yIns2^WT/WT mice and, as expected, was not detected in the mice with deletion of the AceII gene. c: Bradykinin B2 receptor expression increased in both groups of diabetic mice. Data are presented as mean ± SEM. n.d., not detectable. *P < 0.05 compared with Ace2^+/yIns2^WT/WT mice, and ^†P < 0.05 compared with both Ace2^+/yIns2^WT/WT and Ace2^−/yIns2^WT/WT mice. AU, arbitrary unit.

Figure 8

Western blot analysis of ACE2 expression in the kidney. a: Western blots for ACE2 and β-actin protein levels in the renal cortical tissue in the four groups of mice. b: Densitometry measures of ACE2 protein levels were related to β-actin. Hyperglycemia was associated with an increase in ACE2 expression in the kidney cortex of the Ace2^+/yIns2^WT/C96Y mice compared with the Ace2^+/yIns2^WT/WT mice. No ACE2 protein expression was detected in the Ace2^−/yIns2^WT/WT mice and the Ace2^−/yIns2^WT/C96Y mice. Data are presented as mean ± SEM. *P < 0.05 compared with Ace2^+/yIns2^WT/WT mice.

Figure 9

Effect of angiotensin II type 1 receptor blockade on the urinary AER. Four groups of mice were studied: Ace2^+/yIns2^WT/C96Y mice and Ace2^−/yIns2^WT/C96Y, either untreated or treated with the angiotensin II type 1 receptor blocker irbesartan (IRB) from weaning until 3 months of age (n = 6 in each group). The urinary AER was measured with an enzyme-linked immunosorbent assay in 24-hour urine samples. The AER was greater in untreated Ace2^−/yIns2^WT/C96Y mice compared with untreated Ace2^+/yIns2^WT/C96Y mice but this difference was prevented by treatment with IRB. Data are presented as mean ± SEM. **P < 0.05 compared with Ace2^+/yIns2^WT/C96Y mice, ^§P < 0.05 compared with IRB Ace2^+/yIns2^WT/C96Y mice, and ^‡P < 0.05 compared with Ace2^−/yIns2^WT/C96Y mice.