Hyperlipidemia and hyperglycemia result in oxidative stress and play a major role in the development of diabetic nephropathy (DN). We explored the effects of proanthocyanidin (PA) on the induction and progression of DN in apolipoprotein E-deficient mice. Diabetes Mellitus was induced in ten-week-old male apoE−/−mice using streptozotocin (STZ). Mice were fed with a high-fat diet in presence or absence of PA. PA treatment significantly reduced the high cholesterol levels, restored renal functions, and reduced albuminuria in the PA-treated diabetic mice compared with the diabetic untreated mice. In addition, the glomerular mesangial expansion in the diabetic mice was attenuated as a result of PA supplementation. Moreover, PA treatment restored the elevated levels of MDA and CML and the reduced activity of SOD and GSH in the diabetic mice. Furthermore, PA feeding reduced the activation and translocation of NF-
Diabetes mellitus (DM) is considered as the leading cause of end-stage renal disease. The relative risk for cardiovascular diseases is 10 folds higher for type-1 diabetic patients with nephropathy compared with those without diabetic nephropathy (DN) [
Hyperglycemia is a major cause for the increased glycation of proteins and lipids that, in turn, enhances the generation of reactive oxygen species (ROS) [
In diabetic patients with suboptimal glycemic control, hyperlipidemia may develop. In addition, even in subjects with optimal glycemic control, the abnormal lipid profile that is potentially atherogenic may exist. In diabetes mellitus, hyperlipidemia is considered an independent and major determinant of progression of renal disease [
Proanthocyanidin (PA) are polyphenolic compounds, which are derivatives of flavan-3-ol flavonoids. They are mainly composed of dimers, oligomers, and polymers of (+)-catechin, (−) epicatchin, and their gallic acid esters. Their degree of polymerization is generally distributed between 2 and 15. The wide presence of PAs in plants makes them an important part of the human diet [
PAs have been reported to show various beneficial properties. In addition to their free radical scavenging and antioxidant activity [
The present study examined the effect of dietary PA supplementation on diabetes-mellitus-induced kidney damage in apoE−/mice, an animal model previously demonstrated to have a series of pathological conditions including dyslipidemia and atherosclerosis. Hyperlipidemia per se is associated with the development of early renal lesions in apoE−/mice [
All of the animal experiments were carried out under protocols approved by the Institutional Animal House of the University of King Abdulaziz at Jeddah, Saudi Arabia. Male apoE−/mice on C57BL/6J genetic background (14 weeks old) were divided into four groups, both control and diabetic groups, without or with PA supplementation. Diabetes mellitus was induced in the mice by intraperitoneal injections of STZ at a dose of 40 mg/kg bodyweight in citrate buffer, pH 4.5 (Sigma, St. Louis, MO, USA) for 5 days [
Kidneys from each mouse were divided into several parts. The first part was used to prepare the kidney homogenate as discussed later. This homogenate was used for assaying the oxidant/antioxidant parameters as well as CML and IL-6 in the mice kidney. The second part was used for isolation of total RNA and DNA from the kidney cortex. The third part was used for preparation of the nuclear and cytoplasmic extracts for EMSA and Western blot analyses. The fourth part was used for histopathological examination.
The diagnostic kits for determinations of levels of glucose, creatinine (Cr), blood urea nitrogen (BUN), sodium, potassium, cholesterol, and triglycerides were purchased from BioSystem (Barcelona, Spain). Serum cytokines and antioxidant markers as well as AGEs were assayed using commercially available kits. All analyses were performed in accordance with the manuals provided by the manufacturer.
Part of the kidney was cut into small pieces and washed by cold phosphate-buffered saline (PBS). Furthermore, it was ground in a homogenization buffer {0.05 M Tris-HCl pH 7.9, 25% glycerol, 0.1 mM EDTA, and 0.32 M (NH4)2SO4} containing a protease inhibitor tablet (Roche, Germany). The solution was sonicated in an ice bath for 15 seconds followed by centrifugation at 12000 rpm, 4°C for 5 minutes. The supernatant was aliquoted, stored at −80°C, and assayed for protein concentration using BCA kit (Pierce, Rockford, IL, USA) using albumin diluted in a lysis buffer as standard [
The activities of total SOD as well as the concentrations of MDA and GSH in the kidney homogenate were determined using commercially available kits from BioVision Research Products (Linda Vista Avenue, Pasadena, CA, USA) according to the methods described by Nishikimi et al. [
IL-6 concentration in the serum and in the kidney homogenate was determined by an ELISA kit. The ELISA for determination of IL-6 was performed using a commercially available kit from R&D (Mannheim, Germany) according to the instructions of the manufacturer.
Urine samples were collected by placing the mice in individual metabolic cages for 24 h before diabetes mellitus had been induced and the day before the end of experiment. The urine albumin concentration was determined with an ELISA kit (Nephrat II, Exocell, Philadelphia, PA, USA), and the concentration of Cr in pooled urine samples was determined by the commercial assay kit. All analyses were performed in accordance with the manuals provided by the manufacturers.
Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels were determined using an ELISA kit from Genox Corporation (Baltimore, MD, USA) according to the method of Matsubasa et al. [
The supernatant of the kidney homogenate was tested for CML using the anti-CML mouse autoantibody ELISA kit which employs the semiquantitative enzyme immunoassay technique. The ELISA kit for CML was provided by Roche Diagnostics (Mannheim, Germany). The absorbance of the resulting yellow product was measured at 450 nm [
Renal tissues were collected after animal sacrifice, fixed in 10% formalin, processed routinely, and embedded in paraffin. 5
Total RNA of mice in different groups was isolated from kidney cortex by using TRIzol reagent (Invitrogen, Darmstadt, Germany) according to the manufacturer’s protocol. Gene expressions were determined using real-time quantitative reverse transcription polymerase chain reaction.
Cytoplasmic and nuclear fractions of the mice kidneys were separated as described [
Western blot was performed as described. Proteins were separated on a polyacrylamide gel and transferred to a nitrocellulose membrane (Amersham Pharmacia, Braunschweig, Germany). Membranes were blocked with nonfat milk, incubated with primary antibodies directed against NF-
All of the biochemical assays were carried out in duplicates. Statistical analysis was performed with JMP software (SAS Institute, Cary, NC, USA). Diabetes mellitus effect, PA effect, and their interaction were analyzed using the two-way ANOVA, and Tukey-Kramer HSD test was applied for post hoc pairwise comparisons. Time effect was analyzed by MANOVA for repeated measurement.
Results obtained showed thatthe diabetic untreated mice suffer from loss of bodyweight, but the diabetic mice treated with PA were able to keep their bodyweights (
Effects of STZ treatment and dietary PA feeding on the food intake, bodyweight, albuminuria, biochemical parameters, serum AGE, TNF
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Initial bodyweight, g |
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Final bodyweight, g |
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Glucose, mg/dL |
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HbA1c, % |
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Albumin, g/L |
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Total protein, g/L |
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GSH, mmol/L |
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Catalase, U/g Hb |
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Glutathione reductase, U/gHb |
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Glutathione peroxidase, U/gHb |
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Data shown represent mean ± SE of mice, 12 weeks after the treatment with STZ or with the buffer (control) in presence or absence of PA. a,bSignificance difference between groups.
Although both bodyweight loss and hyperglycemia were reduced in mice within the pre-STZ group, the obtained data from mice of the three dietary groups were not markedly affected by the time of PA supplementation.
Diabetes mellitus results in a significant increase of oxidative stress markers, as reflected by the reduction in serum and renal cortex GSH and increase of MDA and AGEs. Dietary PA administration ameliorated all of these changes (Tables
Oxidant/antioxidant parameters as well as concentration of carboxymethyl lysine (CML) in rat retinas.
Control | Control + PA | Diabetic | Diabetic + PA | |
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MDA, nmol/mg protein |
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GST, nmol substrat·mg protein−1·min−1 |
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GSH-Px, nmol substrat·mg protein−1·min−1 |
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Catalase, IU·mg protein−1 |
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SOD, nmol substrat·mg protein−1·min−1 |
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GSH, nmol/mg protein |
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CML, pg/mg protein |
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Oxidative stress markers in the kidney homogenate of apoE−/mice. Mice treated either with or without STZ in presence or absence of PA. PA treatment was continued for 12 weeks after the onset of diabetes. Data shown represent mean ± SEM. a,bSignificance difference between groups.
As a result of oxidative stress and formation of AGE in the diabetic mice, we examined the activation of NF-
NF-
Previous data [
Activation of NF-
Nephrin mRNA expression (a), TGF
All of the diabetic mice suffered from polyuria. PA treatment improved to some extent this disorder, although the effect was not statistically significant (Table
In the diabetic apoE−/mice, the kidneys appeared to be unaltered. Examinations with light microscope revealed mesangium hypercellularity, no dular or diffuse MME, and rare foci of mesangiolysis in diabetic mice without PA treatment. On the other hand, diabetic mice treated with PA had significantly reduced MME compared with the diabetic untreated mice (Figure
Mesangial expansion and ultrastructural changes in diabetic apoE−/mice after 12 weeks after induction of diabetes mellitus. (a) Nondiabetic mice without PA. (b) Diabetic mice without PA. (c) Diabetic mice with PA. PAS staining, original magnification
Our current findings are in line with the previous observations that PA treatment attenuated both hyperglycemia in diabetes mellitus and DN [
The effects of dietary PA supplementation on hyperglycemia and hyperglycemia-induced nephropathy are not fully understood [
The obtained data indicated the increase of oxidative stress in the diabetic mice. The oxidative stress markers like the elevated urinary 8-isoprostane, which is considered a reliable parameter of
The role of dietary PA supplementation in the attenuation of AGEs formation
It has been reported that AGEs trigger the activation of NF-
PA feeding can improve the general health of the diabetic animals, including prevention of severe weight loss, suggesting that glucose metabolism is responsible for this reduction. PA is a naturally occurring antioxidant and could play an important role in the activity of several mitochondrial enzymes that are involved in the oxidation of glucose and ATP production. Therefore, the beneficial effects of PA on diabetes mellitus and DN could be attributed to the combined anti-inflammatory/antioxidant effects, the metabolic regulations that include increasing of the oxidation of glucose, and the attenuation of NF-
The present study demonstrated that dietary supplementation of PA significantly improved hyperglycemia and renal function in apolipoprotein E-deficient mice. In addition, it attenuated NF-
This paper was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, Saudi Arabia, under Grant no. 394-130-D1432. The authors, therefore, acknowledge with thanks the DSR technical and financial support.