Supplementary Data
Methods
Preparation methods for drugs
Res was stored in the dark and dissolved in 0.5% CMC-Na at 30 mg/L immediately before administration. Rsg was dissolved in 0.5% CMC-Na at 0.075 mg/mL and could be stored at 4°C for at least 1 week. CHH was dissolved in 0.5% CMC-Na at 1.125 g/mL and could be stored at 4°C for at least 1 week. Each was administered at a dose of 4 ml/kg by gavage on 6 days of the week for 8 weeks, and the same volume of 0.5% CMC-Na was administered to the vehicle group.
Monitoring and sample collection
The general condition of the diabetic rats was monitored daily by measuring their body weight, and food and water intake, and their FBG was measured every 2 weeks. After 4 and 8 weeks of treatment, 24-hour urine collection was performed using metabolic cages. The volume of urine produced was recorded and it was frozen at −80°C . After 8 weeks of treatment and 8 h of fasting, the rats were terminally anesthetized with 20% urethane and blood was collected from the abdominal aorta for biochemical measurements. The kidneys were removed and weighed, then sectioned and stained with hematoxylin and eosin (HE), prior to histopathologic characterization under a light microscope. The kidneys were fixed in 10% formalin, embedded in paraffin before using it.
The calculation formula
The renal index was calculated as the mean of the ratio of kidney weight/ body weight for each kidney.
HOMA-IR was calculated as FINS (mIU/L) FBG (nmol/L) /22.5. Twenty-four-hour endogenous creatinine clearance (24 h-Ccr) was calculated as urine creatinine (μmol/L) 24-h urine volume (L)/serum creatinine (μmol/L).
Results
Effect on body weight, food intake and water intake of rats in each group
Prior to the administration of STZ, all the rats appeared healthy and were gaining weight. After STZ injection, the rats manifested signs of diabetes mellitus, including hyperphagia, polydipsia, polyuria, growth retardation, depression, and lethargy. The body weights of the rats are shown in Supplementary Figure 1. After 4 weeks of treatment, the body weights of the rats in the treatment groups were significantly higher than those of the vehicle-treated and non-diabetic groups. However, after 8 weeks of treatment, the body weights of the vehicle-treated were significantly lower than those of the non-diabetic group. The food and water intake of the treatment groups were lower than those of the vehicle-treated group between the 4- and 6-week time points. After 8 weeks of treatment, the water intake of the rats in all the treatment groups had decreased significantly.
Supplementary Figure 1: Effects of each treatment on food and water intake, and the body weight of the rats. Res: Resveratrol group; Rsg: Rosiglitazone group; CHH: Compound herba Houttuyniae group.
Effect on FBG and blood biochemical indexes of rats in each group
As shown in Supplementary Table 1 and Figure 2, between 2 and 8 weeks the FBGs of each treatment group were lower than that of the vehicle-treated group (P<0.05). The Res group had significantly lower serum Cr, ALT, and BUN concentrations, and a significantly higher glutathione concentration (all P<0.05), than the vehicle-treated group. This implies a higher level of antioxidant activity in the Res group. The CHH group demonstrated much lower serum ALT, AST, BUN, UA, Cr, and GSP concentrations than the vehicle-treated group (P<0.05). Finally, the Rsg group showed much lower ALT, Cr, BUN, and UA concentrations than the vehicle-treated group (P<0.05).
Supplementary Table 1: Effects of each treatment on the blood biochemistry of the rats
Groups ALT (IU/L)
AST (IU/L)
BUN (mmol/L)
Scr (µmol/L)
SUA (µmol/L)
GSP
（µmol/L)
GSH
（µmol/L) Normal 38.60±5.41 109.25±17.50 4.64±0.15 32.33±4.04 49.5±16.05 0.48±0.05 111.38±32.17
Model 118.60±12.46* 153.25±19.00* 12.98±1.87* 39.33±5.13*† 86.67±10.21* 0.75±0.05* 82.26±16.39* Res 54.00±17.70† 140.33±21.12 5.22±0.22† 23.33±1.03*† 73.83±14.16 0.68±0.07* 135.93±2.59† Rsg 55.88±11.78† 131.14±25.57 8.06±1.41*† 26.71±2.63*† 60.17±20.44† 0.71±0.25* 89.34±13.35 CH 46.75±11.85† 119.00±29.96† 7.72±0.87*† 33.63±5.07* 60.00±16.69† 0.55±0.08† 88.72±10.24 Data are mean±SD; n=5. *P < 0.05 vs. the non-diabetic group; †P < 0.05 vs. the vehicle-treated group;
ALT: alanine transaminase; AST:aspartate transaminase; BUN:blood urea nitrogen; Cr: creatinine; UA: uric acid;
GSP: glycated serum protein; GSH:glutathione.
FBG ( mmol·L-1) pre-model post-model 2 weeks 4 weeks 6 weeks 8 weeks
0 1 0 2 0
3 0 N o r m a l
m o d e l R e s R s g C H
Supplementary Figure 2: The effect of drug treatment group on fasting blood glucose in diabetic rats.
Res: Resveratrol group; Rsg: Rosiglitazone group; CHH: Compound herba Houttuyniae group.
Effect on the glomerular filtration rate (GFR) of rats in each group
Content change of total protein (TP) and urinary albumin (ALB) of 24-h urine
After 4 weeks of treatment, the concentrations of TP and ALB in the 24-h urine samples in the vehicle-treated, Res, and Rsg groups were significantly different to those of the non-diabetic group (P<0.05), but there was no significant difference between the CHH and non-diabetic groups (P>0.05).
Compared with the vehicle-treated group, only the CHH group showed significantly lower urine concentrations of TP and ALB.
Changes of creatinine clearance rate (Ccr) and renal index of rats in each group
The 24h-Ccr and renal index were significantly different in all the treatment groups (P<0.05), except when the CHH and non-diabetic groups were compared, suggesting that there was early renal pathology in the rats. The 24-h Ccr and renal index of the CHH group were significantly lower than those of the vehicle-treated group (P<0.05), but there were no differences between the CHH and non-diabetic groups (P>0.05). In addition, the Res group also had a lower renal index than the vehicle-treated group (P<0.05).
Changes of the contents of serum GLP-1 and FINS concentrations of rats in each group
Compared with the non-diabetic group, the serum concentrations of GLP-1 in the other groups were significantly lower (P<0.05), but the FINS concentrations were similar in all the groups (P>0.05).Compared with the vehicle-treated group, serum GLP-1 was higher in the Res and CHH groups. All of the treatments increased serum FINS (P<0.05). In addition, HOMA-IR was significantly lower in all the treatment groups (P<0.05).
Effect of drugs on histological changes
Supplementary Figure 3 shows the renal histology in each of the groups. The shape and structure of the glomeruli were normal, there were no signs of invasion of the renal interstitium by pro-inflammatory cells, and mesangial proliferation could not be observed by light microscopy in the non-diabetic group.
However, enlarged glomeruli, mesangial proliferation, and inflammatory cell infiltration into the renal interstitium were all present in the vehicle-treated group. These early pathologic changes were ameliorated by all of the treatments.
Supplementary Figure 3: The effects of each treatment on the renal histology of the rats (Hematoxylin and eosin staining, original magnification ×400). A: Non-diabetic group; B:
Vehicle-treated group; C: Resveratrol group; D: Rosiglitazone group; E: Compound herba Houttuyniae group.
