Diabetic nephropathy (DN), one of the most serious microvascular complications of diabetes, is a major cause of end stage renal disease.1 About fifty percent of the patients suffering from diabetes mellitus for more than 10 years develop complications from DN.2 There are increasing numbers of new cases of end stage renal disease resulting from DN in many countries. In Europe and America, 1/3 of patients with chronic renal insufficiency caused by DN need haemodialysis or kidney transplantation and the proportion of patients is also quite sizeable in China.3 Therefore, intervention for preventing and delaying the development and progression of DN is not only a medical concern but also a social issue.
Much evidence suggests that diabetes is associated with increased oxidative stress.4 One consequence of excessive intracellular glucose levels is an increased rate of oxidative phosphorylation under hyperglycemia condition, as well as the activation of the polyol pathway.5 Aldose reductase (AR), a key enzyme in the polyol pathway, catalyzes reduced nicotinamide adenine dinucleotide phosphate (NADPH) dependent reduction of glucose to sorbitol. Sorbitol is subsequently converted to fructose by sorbitol dehydrogenase with NAD+ as cofactor. Intracellular accumulation of sorbitol interferes with the uptake and metabolism of myo-inositol and decreases the activity of Na+-K+-ATPase, leading to dysfunction of renal tubular reabsorption.6 Increased polyol pathway flux has been suggested to induce overexpression of cytokines and proteins, including transforming growth factor (TGF-β) and fibronectin, through a signal transduction cascade involving protein kinase C (PKC), MAPK, the transcription factors activator protein-1 (AP-1) and cAMP response element binding protein (CREB). These events are associated with thickening of the glomerular basement membrane and progressive accumulation of extracellular matrix components in the mesangium.7-9 Thus, both oxidative stress and increased aldose reductase activity are implicated in the pathogenesis of DN.
Berberine, C20H18NO4+, one of the main constituents of Coptidis rhizoma (CR) and Cortex rhellodendri, is a type of isoquinoline alkaloid. Previous studies have demonstrated that berberine reduces hyperglycemia, serum cholesterol, triglycerides, and low density lipoprotein (LDL)-cholesterol, while it increases mRNA and protein levels of the hepatic low density lipoprotein receptor (LDLR) in the patients with hypercholesterolemia and high fat diet fed animals.10,11 Berberine also enhances the hypoglycemic action of insulin in diabetic animal models.12,13 Recent evidence has demonstrated that berberine ameliorates proteinuria in type 2 diabetic rats.14
The present study investigated the effects of berberine on oxidative stress and aldose reductase in the kidney of streptozotocin (STZ)-induced diabetic rats with DN and assesses its availability as a potential agent for treatment of DN.
Animal model and experimental protocol
Forty male Wistar rats, weighing (250±20) g, were obtained from the Center of Experimental Animals, Nanfang Medical University, Guangzhou, China (The number of the animal quality certificate is 0005201). These rats had free access to water and standard laboratory chow during the study period. All experiments were carried out in accordance with the China Animal Welfare Legislation and were approved by the Sun Yat-sen University Committee on Ethics in the Care and Use of Laboratory Animals. Rats were divided into three groups: normal (n=10), diabetic model (n=15), and berberine treatment group (n=15).
Fasted rats in the diabetic model and berberine treatment groups were given a single peritoneal injection of 60 mg/kg STZ (Sigma, USA) dissolved in sodium citrate buffer (pH 4.5) to induce diabetes. Rats in the normal group were injected with the same amount of solvent. Fasting blood glucose (FBG) was measured on the third day after STZ injection using a ONE TOUCH glucometer (Johnson & Johnson Company, USA). Rats with FBG level of above 16.7 mmol/L were considered diabetic.15,16 FBG were present, (22.42±2.50) and (22.75±3.18) mmol/L in the diabetic model and berberine treatment groups, respectively (P >0.05, n=10 per group). Ten age-matched healthy rats without STZ treatment, with a FBG level of (5.17±0.46) mmol/L, were used as the normal group. Rats in the berberine treatment group were orally administrated for 12 weeks 200 mg·kg-1·d-1 of berberine (obtained from Shanxi Scidoor Co., Ltd. China, and purified to 95.13%) dissolved in distilled water. Rats in both the normal group and the diabetic model group were given distilled water orally at the same volume. Oral administration of drug was performed between 8:30 and 9:30 in the morning everyday.
Rats were weighed before the experiment and one-day prior to sacrifice. Urine was collected from the rats housed in metabolic cages for 24 hours. All the animals were killed at the end of the 12th week. Blood was collected by puncturing the inferior vena cava at the time of sacrifice. The specimen serum was separated by centrifugation and was stored at -80°C until using for analysis. Kidney samples were rapidly excised, weighed and frozen in liquid nitrogen or fixed in 10% buffered formaldehyde solution.
Histopathological examination of kidney
Coronal slice of kidney for routine light microscopy was processed in the standard fashion and the sections were stained with hematoxylin and eosin (HE) for routine histopathological examination. The average area and volume of glomerulus were measured by HPIAS-2000 Image Analysis System V6.0. Mean values were calculated from each of 10 glomeruli per section.
Biochemical analysis of blood
FBG was determined by the glucose oxidase method with the results presented in units of mmol/L. Blood urea nitrogen (BUN), serum creatinine (Cr) were measured by methods of oxidase and phosphoglycerol oxidase dynamical enzyme with the results presented in units of mmol/L and μmol/L, respectively. Urine protein was detected by sulfosalicylic acid-sodium sulfate turbidity method with the results presented in units of g/L (Kits from Beijing Chemclin Biotech Co., Ltd. China).
Superoxide dismutase activity and malondialdehyde content in serum
Superoxide dismutase (SOD) activity in serum was detected by the method of the xanthine oxidase presented with unit of U/ml, and content of serum malondialdehyde (MDA) was detected by the thiobarbituric acid reagent method with the results presented in unit of nmol/L17 (kits from Jiancheng Bioengineering Research Institute, Nanjing, China).
Aldose reductase activity in kidney
Aldose reductase (AR) activity was assayed according to the method reported previously.18 AR activity was measured spectrophotometrically by a decrease in the absorbance of NADPH at 340 nm using DL-glyceraldehyde as a substrate. The assay mixture contained 30 mmol/L potassium phosphate buffer (pH 6.5), 5 mmol/L DL-glyceraldehyde, 0.2 mol/L ammonium sulfate, and 1.0 mmol/L NADPH. The results were presented in unit of μmol NADPH·min-1·g-1 protein (Reagents from Sigma-Aldrich Corporation, USA).
AR mRNA expression in kidney assayed by RT-PCR
Total RNA from the kidney was isolated using TRIzol reagent (Invitrogen, UAS, Lot No. 1311168) according to the manufacturer's instructions. The sequence of the sense primer for AR was 5′-ACTGCCATTGCAAAG-GCATCGTGGT-3′and that of the antisense primer was 5′-CCCCCATAGGACTGGAGTTCTAAGC-3′.19-21 The sense primer sequence for β-actin (the internal standard) was 5′-AACACCCCAGCCATGTACG-3′and that of antisense primer was 5′-ATGTCACGCACGATTTCC-C-3′ (primers sequences were synthesized by SBS Biotech, Beijing, China). RT-PCR was performed on a thermal cycler (PTC-200) using an RNA PCR kit (TaKaRa, Japan).
Total RNA was reverse transcribed into single stranded DNA with avian myeloblastosis virus (AMV) RNase Reverse Transcriptase XL and Random Primer (9 mer) programmed as follows: 30°C for 10 minutes, 42°C for 60 minutes, 99°C for 5 minutes, and 5°C for 5 minutes for 1 cycle. And the PCR amplification was performed for AR as follows: initial melt at 94°C for 3 minutes, then cycled 33 times of denaturation at 94°C for 1 minute, annealing at 60°C for 1 minute, and extension at 72°C for 2 minutes, followed by final extension at 72°C for 7 minutes. The PCR products were separated on 2% agarose gel by electrophoresis at 90 V for 40 minutes. The intensity of the PCR products was visualized by ethidium bromide staining and quantified with Gelworks LABWORK 4.0 Analysis Software.
Protein expression of AR in kidney by Western blotting
Rat kidneys from three groups were lysed in 1 ml RIPA solution containing 50 mmol/L Tris-HCl (pH 7.4), 1% NP-40, 0.25% Na-deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mmol/L Na3VO4 and 1 mmol/L NaF. Then the homogenates were centrifuged at 12 000 r/min for 30 minutes and the supernatants were stored at -80°C. The concentration of protein was determined using a BCATM Protein Assay Kit (Pierce, USA, Lot No. 23225) according to the manufacturer's instructions. Fifty micrograms of protein were separated on 12% sodium dodecyl sulfatepolyacrylamide gel by electrophoresis under reducing conditions and transferred to polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories).
The blotted membrane was blocked with 5% skimmed milk and incubated overnight at -4°C with goat polyclonal anti-AR antibody (ALR2 (P-20), 1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse monoclonal anti-β-actin antibody (1:1000; Sigma, USA), followed by incubations with horseradish peroxidase (HRP) conjugated anti-goat IgG or anti-mouse IgG (1:1000; Vector Laboratories, Burlingame, CA, USA). The blotted membrane bounded antibody was detected by enhanced chemiluminescence (ECL) method with Supersignal West Picochemiluminescent substrate (Pierce, UAS, Lot No. HD103454) and captured on X-ray film. The densitometry assay was performed using UVP's Gel Documentation System GDS8000 and Gelworks LABWORK4.0 Analysis Software.
Data from experiments were assessed by SPSS 11.5. Results were represented as mean ± standard deviation (SD). The one-way analysis of variance (ANOVA) and t test were used for statistical comparison within group and between groups. A difference was considered significant atP <0.05.
General characteristics of the animal
As shown in the Table the average kidney weight (KW) and kidney weight/body weight (KW/BW) significantly increased while BW decreased when the diabetic model group was compared with the normal group (P <0.05). In addition, glomerular area, glomerular volume, FBG, BUN, Cr, and UP24h, were significantly increased in the diabetic model group compared with the normal group (P <0.05). Berberine treatment maintained the BW, decreased the KW/BW and improved parameters mentioned above compared with the diabetic model group (P <0.05). There were no significant differences in parameters mentioned above between the normal and berberine groups (P >0.05).
Effect of berberine on SOD activity and MDA content in serum
As shown in Figure 1A, serum SOD was (292.54±20.86) U/ml, (159.46±11.96) U/ml and (283.61±16.38) U/ml in the normal, diabetic and berberine groups, respectively. MDA concentration was (4.97±0.79) nmol/ml, (13.39±1.20) nmol/ml and (6.27±0.73) nmol/ml in the normal, diabetic model and berberine groups, respectively (Figure 1B). Serum SOD activity was significantly decreased and MDA content was increased in the diabetic model group compared with the normal group (P <0.05). Berberine significantly increased the activity of SOD and blocked the increase in MDA content compared with diabetic model group (P <0.05). There were no significant differences in SOD activity and MDA content between the normal and berberine groups (P >0.05).
Effect of berberine on AR activity in the diabetic kidney
As shown in Figure 2, renal AR activity was (0.54±0.06), (1.15±0.04) and (0.62±0.03) μmol NADPH·-1·g-1 of protein in the normal, diabetic model and berberine groups, respectively. Renal AR activity in the diabetic model group was obviously increased compared with the normal group (P <0.05) and this increase was markedly inhibited by berberine (P <0.05).
Effect of berberine on AR mRNA expression in diabetic kidney
The mRNA of renal AR was detected by RT-PCR corresponding to the production of a band of 670 bp (Figure 3A). The assay showed a more intense signal of AR mRNA in the model group than in either the normal or the berberine groups. β-actin expression showed that a relatively equal amount of RNA was loaded. The average values of the relative expression of AR mRNA were 1 in normal, 2.14 in diabetic rats and 1.02 in the berberine group, respectively (Figure 3B). AR mRNA expression was upregulated in the diabetic model animals compared with the normal group (P <0.05). In the berberine group, AR mRNA expression was significantly downregulated compared with the diabetic model group (P <0.05).
Effect of berberine on the protein expression of AR in kidney
By Western blot analysis with the polyclonal anti-AR antibody the expression of AR protein was identified as a 37 kD band. The blot showed more AR protein in the diabetic model group than in the normal or berberine groups. There was no significant difference in the signal intensity of β-actin between groups (Figure 4A). The average values of relative expression were 1 in the normal, 2.20 in the diabetic model and 1.12 in the berberine group (Figure 4B). AR protein expression was significantly increased in the model group compared with that in normal group (P <0.05). In the berberine group AR protein expression was obviously downregulated compared with the diabetes model group (P <0.05).
The pathologic features of diabetic nephropathy include glomerular and tubular hypertrophy, increased basement membrane thickness, tubulointerstitial fibrosis and arteriosclerosis. These features are primarily the extent of diffuse mesangial matrix expansion and increased albuminuria that correlate best with progression to renal failure.22,23 It has been reported that in STZ-induced diabetic rats, the renal pathological changes and deteriorated functions are very similar to human diabetes.24,25 In our experiment diabetic rats induced by STZ in the model group showed a high level of blood glucose. Kidney weight/body weight, glomerular area, glomerular volume, BUN, Cr, and urine protein were significantly increased at the 12th week compared with the normal group, indicating the formation of kidney hypertrophy, glomerular injury and renal dysfunction.26-28 Berberine in the given dose significantly decrease blood glucose and reduce the kidney weight/body weight, glomerular area, glomerular volume, BUN, Cr and urine protein compared with those in model group; suggesting berberine could effectively ameliorate renal injury in STZ-induced diabetic rats.
Hyperglycemia not only generates more reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, but also attenuates antioxidative mechanisms through glycation of the scavenging enzymes including SOD and catalase. Drel et al29 has demonstrated that renal hydrogen peroxide overproduction and lipid peroxide accumulation occur at very early stages of STZ-diabetes and are associated with manifest impairment of antioxidative defense; in particular GSH and ascorbate (AA) depletion and downregulation of SOD and GSH peroxidase activities. Artenie et al30 has pointed out that a decrease in activity of SOD or in the level of GSH is associated with an increased microalbuminuria in type 1 diabetic patients. Chang et al31 has documented that both plasma and urinary levels of MDA, an important marker of lipid peroxidation, are significantly higher in patients with diabetic nephropathy than in normal controls.
Urinary MDA is significantly correlated with the degree of glomerulosclerosis and the index of mesangial expansion in DN patients. Thus, numerous studies have clearly demonstrated the close relationship between oxidative stress under hyperglycemia and renal dysfunction in diabetes. Excessive ROS interacts with the metabolites of essential biological cellular macromolecules such as lipids, proteins and DNA, resulting in renal histologic changes as well as functional abnormalities. Meanwhile, ROS mediate high glucose-induced activations of PKC, especially protein kinase C-beta (II), and the P38MAPK signal transduction cascade in the diabetic kidney. Transcription factors NF-κB and activated protein-1 (AP-1) are activated which can enhance the activity of the TGF-β1, fibronectin and laminin promoters and upregulate extracellular matrix (ECM) expression, leading to glomerular mesangial expansion and tubulointerstitial fibrosis.32,33 Therefore, hyperglycemia-induced oxidative stress plays a further role in the pathogenesis and progression of diabetic nephropathy.
Our study showed that kidney dysfunction resulting from diabetes was accompanied by a decreased activity of SOD and increased content of MDA in serum, suggesting that over-oxidative stress was implicated in impairment of renal function. The changes of SOD activity and MDA content in rats with DN were reversed by berberine treatment, indicating that antioxidative stress could be one of the mechanisms by which berberine alleviated the kidney dysfunction in STZ-induced diabetic rats.
The polyol pathway is an alternative route of glucose metabolism. An increased cellular glucose uptake shifts some glucose into this pathway. In a hyperglycemic condition, accelerated flux through the polyol pathway has been suggested to contribute to the development of diabetic complications. Aldose reductase, the first and rate-limiting enzyme in the polyol pathway, is activated by hyperglycemia resulting in overproduction of sorbitol and fructose. Accumulation of intracellular sorbitol can result in an increased intracellular osmotic pressure and depletion of myo-inositol leading to cellular swelling, decreased activity of enzymes, including ATPase, and increased cellular membrane permeability. The same changes have been seen in the kidney. The increased glomerular exudation has been suggested to be associated with abnormalities in structure and function of glomerular endothelial cells, epithelial cells and basement membrane. Renal tubule granular degeneration leads to increased albuminuria.29,34
Ghahary et al35 has demonstrated that renal AR activity and immunoreactivity are significantly increased in STZ-induced diabetic rats compared with age-matched nondiabetic controls and the increased AR activity can be in part explained by enhanced AR gene expression. Kasajima et al36 observed that the glomerular mesangial area showed diffuse positive reactions for AR in nephropathic diabetic patients and the severity of structural changes in the glomeruli correlates with the intensity of immunoreactive AR. In addition, AR contents in the renal cortex of diabetic patients were 1.5-fold greater than those from non-diabetic patients.
Increased AR activity triggers signal transduction pathways such as PKC, P38MAPK and JNK, which result in overproduction of cytokines such as TGF-β and TNF-α leading to some of the pathophysiological changes associated with diabetic nephropathy.7-9 Ishii et al37 has demonstrated that epalrestat, an aldose reductase inhibitor, abolishes the glucose-induced increases in TGF-β and PKC activity in cultured human mesangial cells in a dose-independent manner. It has been shown that epalrestat prevents the increase in urinary albumin excretion (UAE) and the reduction of anionic sites on the lamina rara externa of glomerular basement membrane in STZ-induced diabetic rats.38 Donnelly et al39 has proposed that tolrestat, an aldose reductase inhibitor, prevents glomerular hyperfiltration, renal hypertrophy, extracellular matrix accumulation and mesangial cell hypocontractility in STZ-induced diabetic rats.
The above findings have indicated that aldose reductase inhibitors can effectively prevent and delay the development and progression of diabetic nephropathy in a diabetic animal model. This suggests that AR activation is involved in the pathogenesis of diabetic complications, including diabetic nephropathy. Our present study observed that in STZ-induced diabetic rats with DN, AR activity, as well as it is mRNA and protein expression in kidney, was increased significantly; thereby suggesting the involvement of AR in the development of diabetic nephropathy. Berberine treatment significantly inhibited the increase in AR activity and downregulated both mRNA and protein expression of AR. This finding is consistent with inhibition of the polyol pathway by berberine being responsible for recovery of renal function in diabetic rats.
In summary, the present study demonstrates that STZ-induced diabetic rats developed an impairment of renal function, and that over-oxidative stress and an increased polyol pathway could be involved in this impairment. This study also showed that berberine could ameliorate renal dysfunction in DN rats through the control of blood glucose, reduction of oxidative stress and inhibition of the activation of the polyol pathway. Further studies are necessary to understand the signal pathway through which beberine exerted its protective effects on the diabetic kidney.
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