A total of 24 adult male Wistar rats (65–70 days old, 190–220 g) were purchased from the Experimental Animal Center, Faculty of Pharmacy, King Saud University (Riyadh, Saudi Arabia). Animals were housed in the animal facility of the College of Science, Taif University (Taif, Saudi Arabia) at 22°C and 55% humidity with a 12 h light/dark cycle. The rats were fed a standard pellet diet and provided with water ad libitum. The present study was approved by the ethical committee of Taif University.

As curcumin is water insoluble, it was added to the food of the animals at a concentration of 1.5 g/kg of food. The food pellet was crushed to a powder and mixed thoroughly with curcumin powder (Nature's Purest, West Los Angeles, CA, USA). A small volume of water was added to create a paste and the pellets were reformed. The pellets were left to air dry for 4 days. The concentration of curcumin was selected based on the average body weight of the rats (~200 g) and the estimated average daily feed consumption (~25–30 g/day). The NC (Merck Millipore, Darmstadt, Germany) concentration was also selected according to the average body weight and the average daily water consumption (~25 ml/day).

Following 1-week acclimatization, the rats were divided into 2 groups as follows: 18 rats were fasted for 8 hrs, after which they were injected intraperitonealy with a single dose of streptozotocin (STZ; 60 mg/kg body weight; Sigma-Aldrich, St. Louis, MO, USA) freshly dissolved in citrate buffer (pH 4.5); and a negative control group (6 rats) were injected with an equivalent volume of vehicle (citrate buffer, pH 4.5). Following injection, the rats were provided with free access to food and water, and were given a 5% glucose solution to drink overnight to counteract hypoglycemic shock. DM in the rats was identified by moderate polydipsia and marked polyuria. After 3 days, the fasting blood glucose levels were measured in tail blood samples (Accu-Chek Aviva Blood Glucose Meter; Roche Diagnostics, Indianapolis, IN, USA). The rats with glucose levels >200 mg/100 ml were considered to be diabetic and selected for further experimentation. The diabetic rats were divided into three groups of six rats each as follows: i) The DM group; ii) the DM + NC group; and iii) the DM + NC + curcumin group. NC was added to the drinking water of the DM + NC and the DM + NC + curcumin group rats at a concentration of 12 µg/ml to attain a treatment dose of 1.5 mg/kg body weight. The food of the DM + NC + curcumin group rats was additionally supplemented with curcumin at a concentration of 1.5 g/kg body weight to obtain a final concentration of 1.5 mg/kg body weight. Treatments were continued for the 8 weeks following induction of DM. Random blood glucose levels were measured from tail blood samples every week to ensure the rats remained diabetic. At the end of the experiment, the rats were fasted for 8 h and anesthetized with diethyl ether (≥99.0%; Sigma-Aldrich), while blood samples were taken from the medial canthus of the eyes into heparinized tubes. The rats were sacrificed by head dislocation and tissue samples were taken for RNA extraction and stored at −80°C prior to use. Samples for histological examination were stored in 10% formalin (Sigma-Aldrich) until processing.

Urea and plasma creatinine assays were performed using the UREA liquicolor (cat. no. 10505) and CREATININE liquicolor (cat. no. 10051) Complete Test kits purchased from HUMAN Gesellschaft für Biochemica und Diagnostica mbH (Wiesbaden, Germany). Cholesterol, triacylglycerol (TG) and high density lipoprotein (HDL) levels were measured using kits (cat. nos. 024-100, 059L-050 and 041-050, respectively) purchased from United Diagnostics Industry (Dammam, Saudi Arabia). Commercial kits were purchased from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China) to measure the levels of plasma malondialdehyde (MDA; A003-1), and the activities of γ-glutamyltranspeptidase (GGT), superoxide dismutase (SOD; A001-1) and glutathione peroxidase (GPx; A005). The plasma phospholipid levels were determined using the Phospholipid Assay kit (MAK122; Sigma-Aldrich), according to the manufacturer's protocol. The concentrations of the measured biochemical constituents were calculated according to the manufacturer's instructions.

For preparation of total RNA, 50 mg of kidney tissue sample was homogenized in 1 ml QIAzol (Qiagen, Inc., Valencia, CA, USA) and 0.3 ml chloroform (Nanjing Jiancheng Bioengineering Research Institute) was added to the homogenate. The mixtures were then shaken for 30 sec followed by centrifugation at 4°C and 12,000 × g for 20 min. The supernatant layers were transferred to a new set of tubes, and an equal volume of isopropanol (Sigma-Aldrich) was added to the samples. The samples were shaken for 15 sec and centrifuged at 4°C and 12,000 x g for 15 min. The RNA pellets were washed with 70% ethanol, briefly dried and dissolved in diethylpyrocarbonate (DEPC) water. The prepared RNA integrity was determined by 1.5% agarose gel electrophoresis. The RNA concentration and purity were determined spectrophotometrically at 260 nm using the GelDoc-It Imaging System (UVP, Inc., Upland, CA, USA). The ratio of the 260/280 nm optical density of all RNA samples was 1.7–1.9.

DNase treatment of the extracted RNA was performed prior to cDNA synthesis using the RQ1 RNase-Free DNase kit (M6101; Promega Corporation, Madison, WI, USA). For synthesis of cDNA, a mixture of 2 µg total RNA and 0.5 ng Oligo (dT)15 primers (Macrogen, Inc., Seoul, Korea) in a total volume of 11 µl sterilized DEPC water was incubated in the Thermo Hybaid PXE 0.2 Thermal Cycler (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 70°C for 10 min for denaturing. Subsequently, 4 µl 5X RT-buffer, 2 µl 10 mM dNTPs and 100 units RevertAid Premium reverse transcriptase (Fermentas; Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA) were added to the reaction mixture and DEPC water was used to bring the total volume to 20 µl. The mixture was then incubated in the thermal cycler at 30°C for 10 min, 42°C for 1 h and 90°C for 10 min. The resulting cDNA was then preserved at −20°C until use.

The mRNA expression levels of gene markers of kidney damage, kidney protection and integrity, and inflammation and fibrosis were analyzed by semi-quantitative PCR using the corresponding gene-specific primers (Table I). The genes analyzed were vimentin, desmin, synaptopodin (SPD), connexin 43 (Cx43), sterol regulatory element-binding protein-1c (SREBP-1c), transforming growth factor β1 (TGF-β1) and erythropoietin (EPO). As a reference gene, the expression of GAPDH mRNA was determined. The primers were designed using the Oligo Primer Analysis software, version 4 (www.oligo.net) and nucleotide sequences published in Genbank (www.ncbi.nlm.nih.gov/genbank; Table I). The primers were synthesized by Macrogen, Inc. PCR was conducted in a final reaction volume of 25 µl consisting of 1 µl cDNA, 1 µl of each 10 pM forward and reverse primers, 12.5 µl PCR master mix (Promega Corporation) and nuclease-free deionized water. PCR was conducted using the thermal cycler with a cycle sequence of denaturing at 94°C for 4 min for 1 cycle, followed by 25–32 cycles of denaturation at 94°C for 1 min, annealing at the specific temperature corresponding to each primer (see Table I) and extension at 72°C for 1 min, with an additional final extension step at 72°C for 5 min. A pilot study was performed to optimize the specific number of cycles for each gene according to its expression level. The PCR products were electrophoresed on a 1% agarose A (Bio Basic Canada, Inc., Markham, ON, Canada) gel in Tris-EDTA buffer at 100 V for 30 min, and stained with ethidium bromide. The PCR products were visualized under UV light and images were captured using the GelDoc-It Imaging System. The intensities of the bands were densitometrically quantified using NIH Image software (National Institutes of Health, Bethesda, MD, USA; rsb.info.nih.gov/nih-image).

Small specimens (1.5×0.5 cm) from the pancreas and kidneys were fixed in 10% neutral-buffered formalin (Sigma-Aldrich) for 24 h, then washed under a running tap and preserved in 70% ethanol. The samples were dehydrated in ascending grades of ethanol, cleared in xylene (Sigma-Aldrich), embedded in Paraplast Plus (Sigma-Aldrich) and sectioned to 5 µm thickness. Tissue sections were mounted onto positively charged, coated slides (Thermo Fisher Scientific, Inc.). Sections were stained with hematoxylin and eosin (Sigma-Aldrich) for studying the general histological analyses (13).

For immunohistochemical detection of insulin granules, paraffin-embedded sections of the pancreas were deparaffinized in xylene, rehydrated in descending grades of alcohol and then immersed in 0.3% hydrogen peroxide (Sigma-Aldrich) for 30 min to inactivate endogenous peroxidase activity. The samples were heated in 10 mM citrate buffer (Sigma-Aldrich) at 121°C for 30 min for antigen retrieval, blocked in 5% normal serum (Sigma-Aldrich) for 20 min, and incubated with a rabbit polyclonal anti-insulin [dilution, 1:100 in phosphate-buffered saline (PBS); sc-9168; Santa Cruz Biotechnology Inc., Dallas, TX, USA] antibody overnight at 4°C. Subsequently, three PBS washes were performed and the sections were incubated with a goat anti-rabbit biotin-conjugated secondary antibody (dilution, 1:2,000 in PBS; sc-2040; Santa Cruz Biotechnology, Inc.) for 20 min at room temperature. After further incubation with horseradish peroxidase-labeled streptavidin (Vector Laboratories, Inc., Burlingame, CA, USA), the antibody binding was visualized with diaminobenzidine (Sigma-Aldrich) and sections were counterstained with hematoxylin. Negative controls, in which the primary or secondary antibodies, or the avidin-biotin complex reagent was omitted, produced no positive staining. Positive controls were used according to the instructions provided by the manufacturer's of the primary antibodies.

Photomicrographs were captured using a Leica DM LB light microscope (Leica Microsystems, Inc., Buffalo Grove, IL, USA) and a Leica EC3 digital camera (Leica Microsystems, Inc.).

Statistical analysis was performed using analysis of variance and Scheffe's protected least-significant difference test on SPSS software, version 13.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference. Results are expressed as the mean ± standard error.

In the present study, plasma levels of the anti-oxidant enzymes, SOD and GPx, were significantly decreased in diabetic rats compared with controls (Table II), which may be caused by oxidative stress generated by the development of DM. Further reduction of these anti-oxidant enzyme levels was observed in diabetic rats treated with NC, however, the levels were increased to a level similar to that of the control by curcumin supplementation (Table II). The plasma concentrations of the oxidative markers, MDA and γ-glutamyltranspeptidase (GGT), and the lipid profile, including cholesterol, TG and phospholipids, were increased in diabetic rats compared with control rats. This increase was enhanced by NC treatment. Curcumin supplementation reversed these effects of DM and NC. The diabetic nephropathy markers, urea and creatinine, were increased in rats of the diabetic group compared with levels in the control group. These were further enhanced in the DM + NC group, while almost normalized with curcumin supplementation.

Diabetic rats demonstrated upregulation of vimentin and desmin mRNA expression levels compared with control rats (P<0.05; Fig. 1). Treatment of diabetic rats with NC decreased the expression levels of these genes compared with untreated diabetic rats (P<0.05), however, the expression remained increased compared with the control group (P<0.05). Following curcumin treatment, the DM and NC-induced upregulation of vimentin and desmin mRNA expression levels were comparable to the control group (Fig. 1).

Diabetic rats demonstrated no changes to SPD mRNA expression level. However, diabetic rats treated with NC exhibited significantly reduced SPD mRNA expression levels compared with diabetic or control rats (P<0.05; Fig. 2A). Diabetic rats treated with NC and curcumin exhibited increased levels of SPD mRNA expression compared with the diabetic rats with NC treatment (P<0.05; Fig. 2A).

DM significantly decreased the Cx43 mRNA expression compared with the control levels (P<0.05; Fig. 2B). Administration of NC to DM rats resulted in a further reduction of the Cx43 mRNA expression level. However, curcumin supplementation abrogated the combined inhibitory effect of DM and NC on the Cx43 mRNA, restoring its expression, as compared with the DM + NC group (P<0.05; Fig. 2B).

Diabetic rats exhibited a downregulation of EPO mRNA expression levels compared with the control rats (P<0.05; Fig. 2C). NC treatment of diabetic rats caused further suppression of EPO mRNA expression compared with the diabetic group (P<0.05). Curcumin supplementation prevented the diabetic and NC-induced suppression of EPO (P<0.05) and further increased EPO expression levels compared with control levels (P<0.05; Fig. 2C).

Diabetic rats exhibited upregulated SREBP-1c mRNA expression levels compared with control rats (P<0.05; Fig. 3A). NC treatment of diabetic rats did not demonstrate any significant change to the SREBP-1c mRNA expression compared with diabetic rats. However, the DM-induced SREBP-1c mRNA expression was abolished by curcumin supplementation, as compared with the DM + NC group (P<0.05; Fig. 3A).

Diabetic rats exhibited a significant increase of TGF-β1 mRNA expression levels compared with the control group (P<0.05; Fig. 3B). NC treatment of diabetic rats decreased TGF-β1 mRNA expression levels compared with diabetic rats, however, the levels remained significantly higher compared with the control rats (P<0.05). The DM-induced increases in the TGF-β1 mRNA expression level was significantly inhibited by curcumin supplementation, as compared with the DM + NC group (P<0.05), and was normalized to control levels (Fig. 3B).

Diabetic rats exhibited upregulated iNOS mRNA expression levels compared with control rats. NC treatment of diabetic rats further increased iNOS mRNA expression levels, as compared with diabetic rats. However, the increased level of iNOS mRNA was inhibited by curcumin supplementation, compared with the DM and NC group (P<0.05; Fig. 3C).

The effect of curcumin on the histological structure of the pancreas was investigated. Pancreatic sections from control rats exhibited islets of Langerhans cells, and contained lightly stained acidophilic cells arranged in branching and anastomosing cords (Fig. 4A). Sections immunostained for the presence of insulin displayed positive β-cells within the islets (Fig. 4A). Sections from diabetic rats displayed damaged islets, represented by cytoplasmic vacuolations, pyknotic nuclei and a decrease in insulin antibody reaction (Fig. 4B). β-Cells from diabetic rats that received NC exhibited more severe islet alterations (Fig. 4C). Sections from diabetic rats that received NC and curcumin exhibited decreased β-cell damage and increased numbers of positive insulin-reacted cells (Fig. 4D).

Kidney sections of control rats displayed the cortex to be occupied by renal corpuscles and surrounding proximal and distal convoluted tubules (Fig. 5A, left panel), and the medulla tissue exhibited straight proximal and distal tubules, collecting ducts and thin tubules distinguished from medullary blood capillaries (Fig. 5A, right panel). Kidney tissues from DM rats displayed tissue damage, including glomerular hypertrophy, hypercellularity, mesangial expansion, pronounced glomerular sclerosis, severe tubulo-interstitial changes represented by lipid accumulation in the cortical tubules, tubular dilatation, interstitial mononuclear cell infiltration and fibroplasia (Fig. 5B). NC treatment further enhanced tissue damage, which was represented by increases in glomerular hypertrophy, sclerosis, mesangial expansion and severe tubulo-interstitial changes (Fig. 5C). However, curcumin treatment reduced the incidence of glomerular sclerosis, lipid accumulation and tubular dilatation compared with that of diabetic rats receiving NC without curcumin treatment (Fig. 5D).