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Human Recombinant ACE2 Reduces the Progression of Diabetic Nephropathy

Oudit GY, Liu GC, Zhong J, Basu R, Chow FL, Zhou J, Loibner H, Janzek E, Schuster M, Penninger JM, Herzenberg AM, Kassiri Z, Scholey JW - Diabetes (2009)

Bottom Line: The effect of hrACE2 on high glucose and angiotensin II (ANG II)-induced changes was also examined in cultured mesangial cells.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 studies show that the protective effect of hrACE2 is due to reduction in ANG II and an increase in ANG 1-7 signaling.

Affiliation: Division of Cardiology, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada. gavin.oudit@ualberta.ca

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.

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 Ins2WT/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 + Ins2WT/WT group using ANOVA with multiple comparison testing. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 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 Ins2WT/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 + Ins2WT/WT group using ANOVA with multiple comparison testing. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: Given the marked protective effect of hrACE2 on the urinary albumin excretion in the diabetic mice, we sought to relate this functional change to kidney histomorphology. As expected, kidney hypertrophy (Table 1) was associated with an increase in glomerular volume in the Ins2WT/C96Y + placebo mice compared with the control Ins2WT/WT mice, and glomerular volume was reduced by hrACE2 treatment (Fig. 2A and C). In accordance with the light microscopic changes, increased glomerular basement membrane (GBM) thickness in the Akita Ins2WT/C96Y mice was also significantly reduced in response to hrACE2 treatment (Fig. 2B and D). Diabetic nephropathy is characterized by an accumulation of extracellular matrix proteins in the glomerular mesangium. A semiquantitative and blinded assessment of the mesangial matrix expansion showed a significant increase in the diabetic Akita mice that was reduced by treatment with hrACE2 (Fig. 2E).

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Human Recombinant ACE2 Reduces the Progression of Diabetic Nephropathy

Oudit GY, Liu GC, Zhong J, Basu R, Chow FL, Zhou J, Loibner H, Janzek E, Schuster M, Penninger JM, Herzenberg AM, Kassiri Z, Scholey JW - Diabetes (2009)

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 Ins2WT/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 + Ins2WT/WT group using ANOVA with multiple comparison testing. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 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 Ins2WT/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 + Ins2WT/WT group using ANOVA with multiple comparison testing. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: Given the marked protective effect of hrACE2 on the urinary albumin excretion in the diabetic mice, we sought to relate this functional change to kidney histomorphology. As expected, kidney hypertrophy (Table 1) was associated with an increase in glomerular volume in the Ins2WT/C96Y + placebo mice compared with the control Ins2WT/WT mice, and glomerular volume was reduced by hrACE2 treatment (Fig. 2A and C). In accordance with the light microscopic changes, increased glomerular basement membrane (GBM) thickness in the Akita Ins2WT/C96Y mice was also significantly reduced in response to hrACE2 treatment (Fig. 2B and D). Diabetic nephropathy is characterized by an accumulation of extracellular matrix proteins in the glomerular mesangium. A semiquantitative and blinded assessment of the mesangial matrix expansion showed a significant increase in the diabetic Akita mice that was reduced by treatment with hrACE2 (Fig. 2E).

Bottom Line: The effect of hrACE2 on high glucose and angiotensin II (ANG II)-induced changes was also examined in cultured mesangial cells.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 studies show that the protective effect of hrACE2 is due to reduction in ANG II and an increase in ANG 1-7 signaling.

Affiliation: Division of Cardiology, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada. gavin.oudit@ualberta.ca

ABSTRACT

Background:

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.

View Similar Images In: Results  - Collection
View Article: Pubmed Central -  PubMed
Show All Figures - Show MeSH
getmorefigures.php?pmc=2809962&rFormat=json&query=null&req=5