Drugs and reagents
RP (the major component was purarine, then daidzin and daidzein; genistin and genistein were the least abundant) [12] and FC (essential components are flavonoids and organic acidic compounds) were provided by Shanghai Second Military Medical University. They were extracted separately using the following process: two times reflux extraction of one kilogram of each medicinal herb with 60% alcohol, the amounts of solvent of 5 folds, successively, and the extraction time of 90 min each [7]. The extract was reduced-pressure evaporated till the volume was 590 ml. RP and FC solutions were mixed at a volume ratio of 1:1 and used as the RPFC solution in this study.
Animals and diabetic model
Male 8-week-old Sprague-Dawley (SD) rats, weighing between 250 and 300 g, were purchased from the Laboratory Animal Center of the Academy of Zhejiang Medical Sciences (Zhejiang, Certificate No. 0012371). All rats were housed at a SPF-grade laboratory animal room in the Animal Laboratory Center of Ningbo University with 20–25 °C room temperature, 50–60% humidity, a 12/12 h light/dark cycle. The Standard diet was provided by Animal Laboratory Center of Ningbo University and the high fat diet was purchased from Pu Luteng Bio-Technique Co. Ltd. The high fat diet contained 16.9% fat and 10.2% casein in the standard diet.
Rats were fed with the high fat diet (30 g per day for each rat) for 8 weeks to induce insulin resistance. The experimental diabetic model was produced by intraperitoneal injection of STZ (diluted to 1% in a 10 mmol/L citrate buffer at pH 4.5) at 25 mg/kg body weight after the rats were fasted for 12 h [13]. Rats in the control group were injected with an equal volume of the citrate buffer. Blood glucose was measured 72 h after STZ injection, and rats with blood glucose over 16.7 mmol/L were considered to be diabetic [14]. Rats were continued on the high fat diet except the rats of control group, in order to keep their blood glucose levels high.
Experimental design
The experimental procedure lasted 15 weeks, during which rats were kept on the standard or high fat diet according to their group assignments. RPFC were administered by intragastric gavage during the 7th–9th weeks, and rats not receiving RPFC were given normal saline. The day after the last administration of RPFC, STZ was injected, and rats not receiving STZ were injected with the vehicle (citrate buffer). 20 rats were randomly divided into five groups with four rats in each group: 1) Normal group, fed with the standard diet. 2) High fat diet (HF) group, fed with the high fat diet. 3) Diabetes mellitus (DM) group, fed with the high fat diet, gavaged with normal saline, and injected with STZ. 4) High fat diet plus RPFC prevention group (HP), fed with the high fat diet, gavaged with RPFC, and injected with the citrate buffer. 5) Diabetes mellitus plus RPFC prevention group (DP), fed with the high fat diet, gavaged with RPFC, and injected with STZ (Fig. 1). As increased time of high fat diet feeding, obesity and resistance to insulin would be more apparent [15]. The beta-islet cells susceptibility of STZ increase in rats feed with the high fat diet. So we add HF group to observe whether high-fat rats have metabolism disorders or renal injury, and add HP group to confirm whether RPFC has impacts on the above changes.
Measurement of body weight, blood glucose, and urinary protein
Blood glucose was measured once a week and body weight was measured once a month. Blood samples were collected from the tail vein to measure the blood glucose level. The fasting blood glucose (FBG) was measured after a 12 h fasting, and postprandial blood glucose (PBG) was measured at 2 h after a meal. Blood glucose level was measured with One Touch Ultra test strips and blood glucose meter (Johnson& Johnson Medical Ltd., Shanghai, China).
Clinically, microalbuminuria was defined as urine albumin excretion rate (UAER) in the range of 20–200 μg/min or urine albumin at 30–300 mg/g creatinine [16]. Rats were considered to have nephropathy if they displayed microalbuminuria. Rats were housed individually in metabolic cages for 24 h to collect urinary samples analyzed with a MODULAR P800 Automation Biochemist Analyzer (Roche, Basel, SWIT).
Oral glucose tolerance test (OGTT), and measurement of insulin and other biochemical parameters
The oral glucose tolerance test was performed on overnight-fasted rats at the end of the study. Rats were fasted for 12 h and then given a glucose solution (2 g/kg body weight), and blood samples were collected before and at 30, 60, and 120 min after the glucose solution was given [17]. Insulin and other biochemical parameters were measured at the end of study. Blood samples were collected from the femoral artery into EDTA-anticoagulant tubes, collected the serum for the detection of serum insulin, total protein (TP), high-density lipoprotein cholesterol (HDL-C), low-density lipoproteins (LDL-C), total cholesterol (TC), triglycerides (TG), ureanitrogen (BUN), creatinine (CREA) and uric acid (UA). The levels of serum insulin were measured with a Rat Insulin ELISA kit (Qiaodu, Shanghai, China). Other biochemical indicators were measured with a MODULAR P800 Automation Biochemist Analyzer (Roche, Basel, SWIT). At last, rats were sacrificed and kidney tissues were saved standby.
Histological examination of the kidney
Renal tissues were fixed for 48 h, dehydrated through a graded series of ethanol, embedded in paraffin wax, and cut into 3 μm sections. The sections were subjected to hematoxylin and eosin (H&E), periodic acid schiff (PAS), and Masson trichrome staining and observed under a microscope.
Real time reverse transcriptase-polymerase chain reaction (RT-qPCR)
Total RNA was extracted from renal tissues using TRIzol reagent (Invitrogen, USA). 1 microgram of total RNA was for reverse transcription. Polymerase chain reactions were performed in a Lightcycler 480 II Authorized Thermal Cycler (Roche, Basel, SWIT) using the following protocol: denaturation at 95 °C for 5 min, followed by 45 cycles of 95 °C 10 s, annealing 20 s, and 72 °C 30 s [18]. The primers used were as follows: α-SMA, F: 5′-CATTGCTGACAGGATGCAGAA-3′, and R: 5′-GAAGCATTTGCGGTGGACAA-3′; collagen IV, F: 5′-GTTGGTCTACCGGGACTCAA-3′, and R: 5′-GTTGGTCTACCGGGACTCAA-3′; β-actin, F: 5′-CTGAACCCTAAGGCCAACCG-3′, and R: 5′-GACCAGAGGCATACAGGGACAA-3′. Relative mRNA levels were determined with the 2 − △△Ct method using the gene β-actin as the internal reference.
Western blot analysis
Renal tissues of 40 mg were lysed for protein extraction. Samples (50 μg) were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene fluoride membranes. The membranes after blocked were incubated with primary antibodies to Phosphoinositide 3-kinase (PI3K) (p85) (Abcam, Cambridge, MA, 1:1000), protein kinase B (AKT) (Signalway Antibody LLC, Maryland, USA, 1:1000), and α-SMA (Abcam, Cambridge, MA, 1:200) at 4 °C overnight. Then incubated with a HRP-labeled secondary antibody (Beyotime, China). The immunoreactions were detected with a gel imaging and analysis system (Tanon, Shanghai, China). The density values of bands were quantified using the software Image J (NIH, Maryland, USA).
Immunohistochemical staining
Renal slides (3 μm) were used to perform immunohistochemical staining of α-SMA and collagen IV on renal tissue sections [19] with the following primary antibodies: monoclonal mouse anti-rat α-SMA (Abcam, Cambridge, MA, 1:50) and polyclonal rabbit anti-rat collagen IV (Abcam, Cambridge, MA, 1:200). Quantitative analysis of the brown positive staining in glomerulus and tubules was performed using Image-Pro Plus 6.0 (Media Cybernetics, USA).
Statistical analysis
All the data are presented as mean ± standard error of the mean (S.E.M.). The differences among groups were analyzed by randomized block design analysis of variance by using SPSS (version 13.0) software. P values below 0.05 were considered statistically significant.
