Human recombinant ACE2 reduces the progression of diabetic nephropathy

Abstract

Objective: Diabetic nephropathy is one of the most common causes of end-stage renal failure. Inhibition of ACE2 function accelerates diabetic kidney injury, whereas renal ACE2 is downregulated in diabetic nephropathy. We examined the ability of human recombinant ACE2 (hrACE2) to slow the progression of diabetic kidney injury.

Research design and methods: Male 12-week-old diabetic Akita mice (Ins2(WT/C96Y)) and control C57BL/6J mice (Ins2(WT/WT)) were injected daily with placebo or with rhACE2 (2 mg/kg, i.p.) for 4 weeks. Albumin excretion, gene expression, histomorphometry, NADPH oxidase activity, and peptide levels were examined. The effect of hrACE2 on high glucose and angiotensin II (ANG II)-induced changes was also examined in cultured mesangial cells.

Results: Treatment with hrACE2 increased plasma ACE2 activity, normalized blood pressure, and reduced the urinary albumin excretion in Akita Ins2(WT/C96Y) mice in association with a decreased glomerular mesangial matrix expansion and normalization of increased alpha-smooth muscle actin and collagen III expression. Human recombinant ACE2 increased ANG 1-7 levels, lowered ANG II levels, and reduced NADPH oxidase activity. mRNA levels for p47(phox) and NOX2 and protein levels for protein kinase Calpha (PKCalpha) and PKCbeta1 were also normalized by treatment with hrACE2. In vitro, hrACE2 attenuated both high glucose and ANG II-induced oxidative stress and NADPH oxidase activity.

Conclusions: Treatment with hrACE2 attenuates diabetic kidney injury in the Akita mouse in association with a reduction in blood pressure and a decrease in NADPH oxidase activity. In vitro studies show that the protective effect of hrACE2 is due to reduction in ANG II and an increase in ANG 1-7 signaling.

FIG. 1.

Human recombinant ACE2 reduces the increased urinary albumin excretion rates in diabetic Akita mice independent of hyperglycemia and with a mild blood pressure–lowering effect. A and B: Urinary albumin excretion rate (A) and urinary albumin/creatinine ratio (B) based on 24-h urine samples showing marked reduction in albuminuria after 4 weeks of daily treatment with hrACE2. n = 8 and 10 for urine albumin measurements in Ins2^WT/WT and Ins2^WT/C96Y groups, respectively. *P < 0.05 compared with all other groups and #P < 0.05 compared with placebo + Ins2^WT/C96Y group using ANOVA and multiple comparison testing. C–E: Plasma glucose and tail-cuff systolic blood pressure showing no effect of hrACE2 on the marked hyperglycemia (C) and mild elevation in systolic blood pressure in diabetic Akita mice (D) that was normalized over a 4-week period in response to daily hrACE2 administration (2 mg · kg⁻¹ · day⁻¹) (E). n = 10 for plasma glucose and n = 12 for systolic blood pressure measurements. *P < 0.05 compared with corresponding Ins2^WT/WT group (C and D) or with Ins2^WT/C96Y + placebo group (E) using Student t test.

FIG. 2.

Glomerular mesangial expansion and thickening of basement membrane were reduced by treatment with human recombinant ACE2. A–C: Representative light micrographs of periodic acid Schiff–stained kidney sections from each group of mice (magnification ×630) (A) with quantification of the glomerular volume (C) showing glomerular expansion in the diabetic Akita mice and a marked reduction in response to hrACE2. B and D: Transmission electron microscopy of the glomeruli (B) with quantification of the glomerular basement thickness (D) showing increased glomerular basement membrane thickness in the Akita Ins2^WT/C96Y mice, which was normalized by treatment with hrACE2. White arrows indicate the glomerular basement membrane. E: Mesangial matrix expansion score showing increased mesangial expansion in diabetic Akita kidneys, which was prevented by hrACE2. n = 5 for all groups. *P < 0.05 compared with all other groups and #P < 0.05 compared with placebo + Ins2^WT/WT group using ANOVA with multiple comparison testing. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

Increased glomerular expression of α-SMA and collagen III in the diabetic Akita kidneys without evidence of inflammatory changes in response to human recombinant ACE2. A–-D: Increased glomerular immunostaining for α-SMA (A) and collagen III (B) in diabetic Akita mice that was quantified based on computer image analysis scores of glomerular immunostaining of α-SMA (C) and collagen III (D). Positive controls are shown as staining in renal blood vessels for α-SMA and from a kidney after 14 days of ureteral obstruction for collagen III. n = 5 for all groups. *P < 0.01 compared with all other groups using ANOVA and multiple comparison testing. E and F: Immunohistochemical-specific staining of neutrophil and macrophage revealed no evidence of inflammation in the diabetic Akita mice without a differential impact with treatment with hrACE2. Positive controls were taken from mouse spleen and lung tissue. Scale bar, 100 μm. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

Human recombinant ACE2 alters angiotensin peptide metabolism without a differential impact on expression of the genes of the renin-angiotensin system while normalizing matrix gene expression in diabetic Akita mice. A and B: Reduction in plasma and renal cortical ANG II levels (A) and increases in plasma and renal cortical ANG 1–7 levels (B) in diabetic Akita mice after treatment with hrACE2. n = 10 for placebo group and n = 12 for hrACE2-treated group. *P < 0.05 compared with corresponding placebo group using Student t test. C–G: Decreased renal cortical ace (C) and increased ace2 (D) expression, unaltered ANG II type 1 receptor, AT1R (E), and type 2 receptor, AT2R (F), expression, and increased bradykinin type 2 receptor, B2R (G), expression in Akita mice were not affected by treatment with hrACE2. n = 8 for placebo groups; n = 10 for hrACE2 groups. †P < 0.05 compared with corresponding Ins2^WT/WT group using Student t test. H and I: Increased renal cortical expression of extracellular matrix genes, fibronectin (H) and pro–collagen III α-1 (I), in diabetic Akita mice was suppressed in response to hrACE2. n = 8 for placebo groups; n = 10 for hrACE2 groups. #P < 0.05 compared with placebo + Ins2^WT/C96Y group using ANOVA and multiple comparison testing.

FIG. 5.

Reduction of NADPH oxidase activity and PKC protein levels in response to human recombinant ACE2 in diabetic Akita mice. A–C: Elevated renal cortical NADPH activity (A) and increased expression of NOX2 (gp91^phox) (B) and p47^phox mRNA (C) in Akita mice were completely suppressed and normalized by treatment with hrACE2. n = 6 and 8 in Ins2^WT/WT and Ins2^WT/C96Y groups, respectively. *P < 0.05 compared with all other groups using ANOVA with multiple comparison testing. D–F: Western blot analysis of PKCα and PKCβ1 protein levels (D) showing a marked elevation in PKCα (E) and PKCβ1 (F) protein levels that was significantly reduced by treatment with hrACE2. β-actin was used as the loading control; n = 5 for all groups. *P < 0.05 compared with all other groups using ANOVA with multiple comparison testing.

FIG. 6.

Recombinant human ACE2 prevents high glucose (HG) and ANG II–induced oxidative stress and NADPH oxidase activation in cultured rat mesangial cells. A–D: DHF staining in cultured mesangial cells treated for 24 h with normal glucose (NG; 5.6 mmol/l) (A) or high glucose (25 mmol/l) (B) and pretreated with 250 ng/ml of ACE2 for 1 h (C) or 100 nmol/l of ANG 1–7 for 15 min (D) and then exposed to 25 mmol/l of high d-glucose for 24 h. E: NADPH oxidase activity in response to high d-glucose (25 mmol/l) or d-mannitol for 24 h. F: NADPH oxidase activity in response to 24 h of high d-glucose (25 mmol/l) and the effects of pretreatment with 100 nmol/l ANG 1–7. G–J: Dihydroethidium fluorescence in cultured mesangial cells treated with placebo (G) or stimulated by 100 nmol/l of ANG II for 18 h (H) and with pretreatment with hrACE2 (25 ng/ml) (I). Suppression of ANG II–induced NADPH oxidase activity in cultured mesangial cells by pretreatment with 25 and 250 ng/ml of hrACE2 was preventable by 1 μmol/l of the specific ACE2 inhibitor, DX600 (J). K: The effects of pretreatment with AT1 receptor blocker, losartan (10 μmol/l), and hrACE2 (250 ng/ml) on high-glucose–induced NADPH oxidase activity. L: The effects of hrACE2 (250 ng/ml) with and without the ANG 1–7 blocker, d-Ala-ANG 1–7 (10 μmol/l), on high glucose–induced NADPH oxidase activity. n = 5 for all groups. *P < 0.05 compared with all other groups, #P < 0.05 compared with the 5.6 mmol/l d-glucose group, **P < 0.05 compared with the 25 mmol/l d-glucose– and losartan-treated group, and ***P < 0.05 compared with the 25 mmol/l d-glucose– and hrACE2-treated group using ANOVA with multiple comparison testing. (A high-quality digital representation of this figure is available in the online issue.)