All animal experiments received approval from the Animal Ethics Committees of the Shandong Provincial Hospital and Shandong University Postgraduate College (Shandong, China) and were performed strictly in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

A total of 76 rats (56 male and 20 female) purchased from the Laboratory Animal Center of Shandong University (Shandong, China) and weighing 150–200 g underwent a two-step 5/6 nephrectomy under sterile conditions. Six rats in the sham surgery group (Group A) received the same surgical procedures, but their kidneys were retained. The rats were allowed to drink water and eat freely following surgery. Once the 5/6 nephrectomy rat models with renal failure had been established, the model rats were further randomly divided into three groups: i) Group B/surgery control group, no treatment (n=10); ii) Group C/blank control group, treatment with FBs transfected with empty vector [FB (LXSN) cells] (n=10); and iii) Group D/treatment group, treatment with FB (LDCNSN) cells (n=10) (12).

Four time-points, the 10th, 20th, 40th and 60th days, were selected for the dynamic observation of lipid levels in the plasma and erythrocyte membrane, and three-time points, the 10th, 30th and 60th days, were selected for the lipid levels in the aortic arches. Eight and 12 rats, with equal number of males and females, were selected for the experimental and control groups, respectively, at each time-point. The experimental and control groups underwent the first and second surgeries simultaneously. For the experimental group, two-thirds of the left kidney was resected in the first procedure and the whole right kidney was resected in the second procedure 10 days later. The control group only underwent the surgical incision. Identical test conditions were maintained in the two groups. Two hours after the 12 rats in the control group revived from the anesthesia of the second surgery, blood was sampled from the heart to measure the levels of T-Ch and TG of the plasma and the level of T-PL in the erythrocyte membrane, and the aortic arches were separated as the day 0 control samples. The heart blood of the experimental and control groups was additionally collected following the second surgery to prepare the plasma and erythrocyte membrane, and the aortic arches and kidney (two rats for pathological monitoring) were obtained at different time-points. A total of 10 rats were left in the experimental group at each time-point due to the possibility of death during the experiment and the failure of the surgery and for pathomorphological observation during the formation of CRF (13).

Tissues, such as the fat on the surface of the vascular outer wall, were removed from the separated aortic arches and any blood was blotted using filter paper. The samples were then cut into 5- to 6-mm pieces and weighed with a One Over 10,000 Analytical Balance (Mettler-Toledo, Shanghai, China). The average weight of the experimental group samples was 261.106±15 mg and that of the control group was 260.156±16 mg. Each sample was then placed into the homogenizer and ground for 10 min. Lipids were extracted by the Rose-Gottlieb extraction (14,15) method and dried with nitrogen. T-Ch and TG levels were measured with the same method utilized to measure the levels in the plasma. The T-PL level was determined using the coefficient between inorganic phosphorus and T-PL. The level of inorganic phosphorous was measured using the molybdenum blue colorimetric method following acid digestion. Acid digestion was performed by adding 140 ml concentrated sulfuric acid to 250 ml distilled water, agitating the mixture, and then adding 32.5 ml 70% perchloric acid and distilled water to make a total volume of 500 ml. This mixture was then further agitated. The results are expressed in μmol per gram of aortic tissue (16).

Measurements of TG, T-Ch and T-PL levels were made using the glycerol-3-phosphate oxidase-phenol-aminophenazone (PAP), cholesterol oxidase-PAP and ammonium molybdate reduction methods, respectively. Measurements of urea and creatinine (Cr) were made using the PAP rate method. Five samples were added to each batch of samples as the intra- and inter-assay quality controls (17). The relevant kits were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China). The procedures were performed strictly in accordance with the manufacturer’s instructions.

T-Ch and T-PL were extracted from the erythrocyte membrane by the Rose-Gottlieb method, and measured by the plasma method. The results are expressed in mmol/l hematocrit. Hematoxylin and eosin (HE) staining was used for renal pathological monitoring and observed using a light microscope. The instruments used included a Monarch22000 automatic biochemical analyzer (Arris, IL company, USA) and a Beckman-700 biochemical analyzer (Beckman Coulter, Miami, FL, USA).

FB (LDCNSN) and FB (LXSN) cells were cultured in Dulbecco’s modified Eagle’s medium containing 300 ng/ml G418 and 10% fetal calf serum. The cells were then digested and collected using EDTA-trypsin, and washed three times with high-pressure sterilized normal saline. The cell concentration was adjusted to 1×10⁷/ml. The FB (LXSN) and FB (LDCNSN) cells were administered to the renal medulla in the control and treatment groups, respectively, via multi-point injection during the 5/6 nephrectomy. The injection points were distributed uniformly across five to six sites, with 1×10⁶ cells/kidney in total. The rats had free access to water and food following surgery.

Prior to treatment and in the first and fourth weeks after treatment, the body weights (BW) of the rats in each group were measured, femoral arterial blood was taken and serum TG, T-Ch, Cr and urea nitrogen (BUN) levels were detected using an automatic biochemical analyzer. The renal tissue samples were fixed in 10% formalin solution and liquid nitrogen respectively.

The renal tissues were embedded in paraffin, and sliced into 3- to 4-μm sections. Following conventional deparaffinization, the slices that underwent HE, Periodic Acid Schiff and Masson staining were observed under light microscopy. The tubulointerstitial lesions were then subjected to semi-quantitative grading, as follows: 0, normal; I, lesion scope ≤25%; II, lesion scope 26–50%; and III, lesion scope >50%.

The EnVision™ immunohistochemistry system (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) was used to detect the changes in DCN and TGF-β1 expression in the renal tissues. Following conventional deparaffinization, microwave-induced antigen retrieval was performed for 10 min and a drop of 3% H₂O₂ was added to each slice. Incubation was then carried out for 20 min at room temperature. The primary antibody, rabbit anti-rat monoclonal anti-TGFβ1 (cat. no. RS-0105R; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.), was added at a 1:200 dilution followed by rabbit anti-rat monoclonal anti-DCN (cat. no RS-0017R; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) at a 1:200 dilution, prior to incubation for 2 h at room temperature. Following incubation, the slice was rinsed with phosphate-buffered saline (PBS) three times for 5 min, avidin was added and the slice was further incubated for 20 min at room temperature. The slice was then rinsed again with PBS three times for 3 min, and horseradish peroxidase-labeled goat anti-rabbit secondary antibody (cat. no PV-0202R; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) was added prior to incubation for 30 min at room temperature. Following incubation, the slice was rinsed with PBS three times for 5 min, 3,3′-diaminobenzidine was added and the slice was examined under a light microscope for 5 min. The slices were restained with hematoxylin and 0.1% hydrochloric acid and washed with water, following which they turned blue. They were subsequently dehydrated in gradient ethanol, vitricated by dimethylbenzene, mounted with neutral balsam (mounting medium) and examined subsequent to air drying. The results from the immunohistochemistry of TGF-βl and DCN were subjected to semi-quantitative analysis performed by three personnels simultaneously. The distribution of staining in the tubulointerstitial area was graded into levels, as follows: 0, no staining; 1, occasional staining; 2, focal staining; and 3, diffuse staining.

All statistical analysis was performed by using SPSS 17.0 software (SPSS, Chicago, IL, USA). The Student’s t-test was applied for intergroup comparisons and linear correlation analysis. P<0.05 was considered to indicate a statistically significant difference.

The concentrations of urea and Cr peaked on the 10th day (27.93±2.46 mmol/l and 308.50±19.65 μmol/l, respectively). The concentrations began to decline on the 20th day and were minimal on the 40th day (18.30±4.14 mmol/l and 193.45±22.32 μmol/l, respectively); however, at both time-points the concentrations were significantly increased compared with those in the control group (8.67±3.00 mmol/l and 96.47±21.33 μmol/l, respectively) (P<0.01). The urea and Cr concentrations started to increase again on the 60th day (Fig. 1).

The levels of the three lipids started to increase on the 10th day and presented a rising trend thereafter. The TG level was increased significantly compared with that of the control group at the same time-point on the 30th day (P<0.05) and on the 60th day (P<0.01). The T-Ch and T-PL levels started to increase on the 10th day and were significantly higher than those of the control group on the 60th day (P<0.01 and P<0.05, respectively).

The TG and T-Ch levels in the plasma of the experimental group rats started to increase from the 10th day and continuously rose; the levels were significantly higher than those of the control group on the 10th, 30th, 40th and 60th days (P<0.05; Table I). The T-PL levels increased significantly on the 10th day, then decreased on the 20th and 30th days, and slightly increased again on the 60th day. The T-Ch level in the erythrocyte membranes began to increase on the 10th day and was significantly higher compared to that of the control group on the 40th and 60th days (P<0.05). The T-PL level in the erythrocyte membranes was slightly lower than that of the control group on the 20th (P<0.05), and significantly decreased on the 40th and 60th days (P<0.01). The results are shown in Table II. The TG and T-Ch levels in the plasma were significantly positively correlated with those in aortic tissue (r=0.99 and r=0.97), while the T-PL level in the plasma was significantly negatively correlated with that in aortic tissue (r=−0.92).

No significant differences were observed in the BWs of rats among the groups prior to the experiment. In the fourth week, the weights of rats in all the groups increased to different degrees, but the differences among the groups were not statistically significant (data not shown).

The blood lipid levels in the rats were significantly increased after four weeks compared with those at week 0 (Table III), and the BUN and serum Cr levels in Group D were significantly lower than those in Group C on the 4th week (P<0.05). Although a slight increase was observed in group D compared with group A for the values in week 0, the difference was not statistically significant (Table IV).

TGF-βl expression was predominantly found in the fibrotic renal interstitium, renal tubular epithelial cells and glomerulus. DCN was mainly distributed in interstitial fibrotic sites, the sclerotic glomerulus and vascular adventitia. Four weeks after the treatment with FB (LDCNSN) cells, the DCN expression in the renal tissues was increased significantly. The expression of TGF-β1 in Group D was not significantly different from that in Groups B and C (Table V).