Participation of angiotensin II in chronic kidney diseases including diabetic nephropathy (DN) has been extensively described. Similarly, several studies support a protective role for angiotensin-(1-7). However, other studies suggest that some of the cellular effects of angiotensin-(1-7) may be deleterious. The objective of this study was to determine the role of exogenous angiotensin-(1-7) on renal hypertrophy development in rats with streptozotocin-induced diabetes. A control group and three groups of rats with streptozotocin-induced diabetes: untreated diabetic rats, diabetic rats treated with captopril, and diabetic rats treated with angiotensin-(1-7), were studied. After two weeks of treatment, the kidneys were removed under anesthesia with pentobarbital. The kidneys were weighed and the renal cortex was separated for analysis of AT 1 R, TGF-β 1 , MASR, and ACE2 expression by western blot. Rats in the three groups with diabetes had hyperglycemia, increased food and water consumption, and higher urinary volume than control rats. Treatment with captopril or angiotensin-(1-7) reversed streptozotocin-induced renal hypertrophy, measured by kidney weight, protein/DNA ratio in renal cortex, glomerular area, or proximal tubular cells area, proteinuria, and creatinine clearance reduction. AT 1 R, TGF-β 1 , and MAS receptor expression in renal cortex of diabetic rats increased significantly as compared to controls (p < 0.05); treatment with captopril or angiotensin-(1-7) reversed such increments. ACE2 in the renal cortex decreased in diabetic rats, but it was increased after treatment with captopril or angiotensin-(1-7). These findings suggest that exogenous administration of angiotensin-(1-7) may be renoprotective in early stages of diabetes mellitus.
The pathophysiological mechanisms related to diabetic nephropathy (DN) development are complex [
The angiotensin-converting enzyme monocarboxipeptidase (ACE2), homologue of ACE, catalyzes the conversion of Ang II to angiotensin-(1-7) [Ang-(1-7)]. ACE2 can also cleave angiotensin I to the inactive nonapeptide angiotensin-(1-9), which in turn is cleaved by ACE to generate Ang-(1-7) [
In the present study, we used the well-established STZ-induced diabetes model. STZ destroys the pancreatic beta cells causing a state of insulin dependent diabetes. Male Wistar rats were obtained from the rat colony of the Facultad de Estudios Superiores Iztacala. Animals aged 10 weeks, with initial body weight of 250 ± 20 g were studied. Rats had free access to standard rat chow (Rodent Laboratory Chow 5001, Ralston Purina, Richmond Indiana, USA) and tap water, with 12 - 12 h light-dark cycles throughout the experiment. Diabetes was induced by a single STZ intraperitoneal (ip) injection (65 mg/kg of body weight) in 10 mM sodium citrate buffer, pH 4.5. Control (C) rats received vehicle (10 mM sodium citrate buffer, pH 4.5) alone. Forty-eight hours after STZ injection, blood glucose concentration was determined in tail vein blood samples using a reflectance meter (One Touch; LifeScan, Milpitas, CA, USA). Only animals with blood glucose levels >300 mg/dL were included in the study. Diabetic rats were randomized into three groups: 1) untreated diabetic rats (DM) receiving vehicle (saline solution), 2) diabetic rats treated with captopril, 10 mg/kg, po (DM + CAP), and 3) diabetic rats treated with Ang-(1-7), 100 µg/kg, ip [DM + Ang-(1-7)]. Once hyperglycemia with serum glucose levels ³ 300 mg/dl was confirmed, treatment with captopril or Ang-(1-7) was given daily during the 2 weeks of the experiment. Each group consisted of five animals. Two days before STZ injection and two days before the end of the experiment, the animals were placed in metabolic cages to measure food and water consumption, urinary volume, and to obtain urine samples to measure proteins and creatinine.
At the end of the study, the rats were anesthetized with sodium pentobarbital (45 mg/kg, ip). Blood samples were obtained to measure blood glucose and creatinine levels. Both kidneys were quickly removed. The left kidney was decapsulated, weighted, and dissected into cortex and medulla for total DNA and protein extraction, and immunoblot analysis. The right kidney was cannulated, and fixed with 4% formaldehyde. Animal care and procedures were performed in compliance with the Mexican Federal Regulations for Animals Investigation and Care (NOM-062 ZOO-1969, Ministry of Agriculture, Mexico), on care and use of laboratory animals, and the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). The protocol was approved by the institutional ethics review board.
Kidney weight/rat body weight ratio was used as a kidney hypertrophy index. The formaldehyde fixed right kidneys were dehydrated through ethanol graded series, embedded in paraffin, sectioned in 4 mm thick slices, mounted on glass slides and stained with hematoxylin-eosin (HE) and periodic acid-Schiff (PAS). Glomeruli and proximal tubular cells were visualized using an optic microscope and the areas were measured using a computer program (Motic Images Plus 2.0 ML, Richmond, British Columbia, Canada). For glomerular area, 50 consecutive glomeruli per rat were analyzed with 10× magnification. For proximal tubular cells area, 100 cells per field at 40× magnification were counted, and 10 fields per slide were analyzed and averaged. Finally, total DNA and protein from cortex tissue was extracted and quantified by Trizol reagent method (Invitrogen, Grand Island, New York, USA), and the protein/DNA ratio was calculated as an index of relative hypertrophy.
A 24 h urine sample was collected placing the animals in metabolic cages. Samples were immediately frozen and stored at −80˚C to measure proteins and creatinine. Protein concentration in urine was measured by the Bradford method (Bio-Rad), we used bovine serum albumin (BSA) (Sigma Chemical Co.) as standard, and creatinine was measured with Cayman reagents (Cayman Chemical, Ann Arbor, Michigan, USA). The protein/creatinine urinary excretion ratio was calculated.
Renal tissue was homogenized in 100 mM Tris (hydroxymethyl-aminomethane-tris- hydrochloride, Sigma, St Louis, MO, USA), pH 7.4, incubated with a protease-inhibitor cocktail (Mini Complete EDTA-free protease inhibitor cocktail, Roche, Germany) and centrifuged at 10,000 g for ten min to remove insoluble debris. The protein concentration of the supernatant was quantified using the Bradford method. 50 mg of protein were loaded into a 10 % SDS-PAGE mini-gel under reducing conditions and transferred to polyvinylidene fluoride (PVDF) membranes (Amersham Hybond-ECL, GE Health Care, Buckinghamshire, UK). The membranes were blocked with 5% nonfat milk in Tris buffered saline (pH 7.6) containing 0.05% Tween 20 (TBST) for two hours at room temperature. Membranes were incubated for 16 h at 4˚C with a 1:400 dilution of a rabbit polyclonal antibody to AT1R, TGF-β1, ACE2 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) and MASR respectively (Novus Biological, Littleton CO, USA). After incubation with the primary antibody, the membranes were washed with TBST buffer and incubated with a 1:1000 dilution of horseradish-peroxidase-labeled goat anti rabbit IgG secondary antibody (Zymed, Invitrogen, Grand Island, New York, USA) at room temperature for 2 hours. Visualization was performed with an enhanced chemiluminiscence (ECL) western blotting kit (Luminol, Santa Cruz Biotechnology Inc., Santa Cruz, California, USA). The obtained films were scanned and digitalized using a flatbed scanner. Band intensity was measured by computer analysis using Multi Gauge, Fuji Film Science, Lab2003 (Fuji Photo Film Co., LTD). All membranes were stripped, re-blocked and incubated with goat β-actin antibody (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) used as housekeeping protein with the same protocol.
All values are presented as mean ± SEM, and compared by ANOVA followed by Newman-Keuls test. Differences were considered statistically significant with p values £0.05.
In
Parameter | C | DM | DM + CAP | DM+Ang-(1-7) |
---|---|---|---|---|
n = 5 | n = 5 | n = 5 | n = 5 | |
Glycemia (mg/dL) | 115 ± 4.03 | 450 ± 17.6* | 492 ± 20.4* | 425 ± 12.4* |
Body weight (g) | 274.12 ± 4 | 241.2 ± 1.27* | 238.2 ± 3.7* | 253.4 ± 2.14* |
Water ingestion (mL/24 h) | 35.66 ± 2.6 | 191.8 ± 7.8* | 213.5 ± 18.7* | 201.6 ± 10.1* |
Food ingestion (g/24 h) | 23.2 ± 2.1 | 42.8 ± 3.6* | 36.8 ± 2.5* | 32.7 ± 2.8* |
Urinary volume (mL/24 h) | 7.75 ± 1.5 | 145 ± 13.6* | 137.2 ± 3.6* | 117 ± 6.6* |
Data are expressed as mean ± SEM. *p < 0.05 vs. C.
the diabetic rats increased significantly as compared to the control group (C: 85.33 ± 1.15, DM: 138.76 ± 2.083 μm2). However, treatment with captopril or Ang-(1-7) reversed this increment (DM+CAP: 103.26 ± 0.85, and DM+Ang-(1-7): 99.739 ± 0.955 μm2 (
Creatinine clearance in diabetic rats was significantly lower as compared to the control group (C: 0.36 ± 0.05, DM 0.07 ± 0.01 mL/min), treatment with captopril or Ang-(1-7) reversed this reduction (DM+CAP: 0.32 ± 0.124, DM+Ang-(1-7): 0.36 ± 0.06 mL/min) (
creatinine urinary excretion ratio in diabetic rats increased significantly as compared to the control group (C: 5.69 ± 1.43, DM: 36.47 ± 5.83 g/g), treatment with captopril or Ang-(1-7) reversed this increment (DM+CAP: 6.22 ± 2.67, DM+Ang-(1-7): 4.99 ± 1.96 g/g,
AT1R, TGF-β1, ACE2, and MASR protein expression in renal cortex homogenates were determined by western blot analysis and standardized with β-actin. AT1R protein expression in renal cortex of diabetic rats increased significantly as compared to control group; treatment with captopril or Ang-(1-7) reversed AT1R protein expression increment, but only treatment with Ang-(1-7) showed significant differences from that of diabetic rats (
protein expression in the renal cortex of control rats was not different from that of diabetic rats. Captopril increased ACE2 protein expression in the renal cortex. However, Ang-(1-7) did not modify the expression of the enzyme (
The earlier manifestations of renal injury produced by DM are structural changes consisting of tubuloepithelial and glomerular hypertrophy, followed by thickening of glomerular and tubular basement membranes and progressive accumulation of extracellular matrix proteins in the mesangium and interstitium. The structural changes start before any other measurable clinical changes are detectable (1-3). Among many factors reported to be involved in early diabetes renal hypertrophy, the RAS, through Ang II, participates in the generation of renal disturbances preceding the development of DN [
Furthermore, treatment with Ang-(1-7) reduced AT1R, TGF-β1, and MASR expression in renal cortex. Similar effects were produced by treatment with captopril. Treatment with Ang-(1-7) did not modify the expression of ACE2, and captopril produced a significant increment of the enzyme.
Numerous studies have demonstrated that Ang II induces renal hypertrophy and glomerulosclerosis by activation of its specific receptor AT1R [
Reportedly, TGF-β1 promotes the progression of renal fibrosis and acts as a major mediator of hypertrophic and prosclerotic changes in DN [
Our results suggest that treatment with Ang-(1-7) may exert a beneficial effect on the progression of DN, beyond reducing proteinuria and increasing creatinine clearance. In db/db mice Ang II induced oxidative stress and kidney inflammation through AT1R. Ang-(1-7) treatment for 28 days reduced ROS and lipid deposition through NADPH oxidase inhibition, decreasing the inflammatory response [
ACE2 is expressed in the kidney and it mediates conversion of Ang II to Ang-(1-7). The synthesis of Ang-(1-9) from Ang I and the catabolism of Ang II to produce Ang- (1-7). Classic ACE inhibitors do not inhibit this effect. Ang-(1-7) binds to the MASR and counteracts the effects of Ang II in DN [
These results are similar with those found in NRK-52E cells incubated with high glucose for 24 - 72 h. ACE2 and MASR expression were decreased while TGF-β1 expression was increased [
The results suggest that early exogenous administration of Ang-(1-7) may reduce renal hypertrophy by regulating the activity of TGF-β1 in the proximal tubule, indicating the importance of the ACE2-Ang-(1-7)-MAS receptor axis, and its potential renoprotective effect in early stages of DN.
This study was supported by the Universidad Nacional Autónoma de México, UNAM (grants PAPCA 2011, and DGAPA-PAPIIT IN210307).
The authors declare that there is no conflict of interests regarding the publication of this paper.
Amato, D., Núñez- Ortiz, A.R., del Carmen Benítez-Flores, J., Segura-Cobos, D., López-Sánchez, P. and Vázquez-Cruz, B. (2016) Role of Angiotensin-(1-7) on Renal Hypertrophy in Streptozotocin-Induced Diabetes Mellitus. Pharmacology & Pharmacy, 7, 379-395. http://dx.doi.org/10.4236/pp.2016.79046