Hematoxylin-eosin (H&E) and Masson-trichrome staining kits were obtained from Beyotime Biotech (Shanghai, China). Hydroxyproline, total urinary protein, serum creatinine and blood urea nitrogen (BUN) assay kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Trizol reagent was obtained from Invitrogen Co. (Carlsbad, CA, USA). Primary antibodies for TMEM45A, fibronectin (FN) and connective tissue growth factor (CTGF) were obtained from Abcam Biotech (Cambridge, MA, USA). Primary antibodies for Jagged-1and Notch1 were obtained from Santa Cruz Biotech (CA, USA). Primary antibody of GAPDH was obtained from CST Inc. (Cell Signaling Technology, Beverly, MA, USA). ELISA kits to measure tumour necrosis factor-alpha (TNF-alpha), IL-6, Collagen-I (COL-I) and Collagen-III (COL-III) concentrations were purchased from CST. Recombinant human TGF-beta 1 was purchased from PeproTech (Rocky Hill, NJ, USA). DAPT (Notch pathway inhibitor (21)) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum was obtained from Gibco Company (Cleveland, USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Hyclone (Logan, Utah, USA).

NRK-49F cells were obtained from the SIBCB (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China). NRK-49F cells were incubated in DMEM with 10% FBS and penicillin (100 U/ml) (37 °C with 5% CO₂) to exponential phase. Cells were then seeded into 6 well culture-plates (5×10⁵ per well) and used for later experiments when the cell density reached over 50%.

Six-week-old male Wistar rats (170 ± 20 g) were provided by the Shanghai Animal Lab Center (Shanghai, China). All animals were kept in plastic cages with free access to standard commercial rodent diet and water, in a room with controlled temperature (24 – 26 °C) and on a 12-h light/12-h dark cycle.

We used the experimental model of 5/6 nephrectomy or the remnant kidney model for generation of fibrosis in the kidney. The reduction of renal mass was achieved by surgical excision of both poles, with removal of the contralateral kidney (20). After one week of the adaptive feeding, a total of 24 rats were randomly divided into 2 groups (n = 12): control group and 5/6 (model) group. For the first step, rats were anaesthetized and two poles of the left kidney were cut out. One week later, the right kidney was removed. In addition, sham operations were carried out in the control group.

Rats were sacrificed at 4 and 8 week after operation (6 rats each group at each time point) and samples were collected. Serum creatinine, total urinary protein and BUN concentration were detected to evaluating the renal function. Relative mRNA expression and protein levels of TMEM45A and Notch1 were also examined.

To evaluate histological changes and collagen-like matrix deposition, renal tissue sections (4 ± 1 μm thickness) were stained with H&E staining kit and Masson trichrome staining kit according to manufacturers’ instructions. Briefly, renal tissues were fixed in 10% neutral buffered formalin followed by the standard methods of dehydration, clearing in xylene, and paraffin embedding. After de-paraffinizing, renal head tissue sections were stained by H&E and Masson trichrome. Finally, histopathological changes were examined by optical microscope (Olympus, Japan).

Relative contents of serum creatinine, total urinary protein and BUN were determined using colorimetric method according to the manufacturer’s protocol.

Cell samples were centrifuged at 12,000 × g for 5 min at 4 °C to obtain the cells. The total protein of each sample was determined by BCA method. Equal volumes of hydrolytic reagent was added and boiled for 20 min. After cooling to room temperature, samples were adjusted to pH 6.6 - 6.8 with phosphoric acid (or NaOH) and treated with activated carbons to decolorize for 30 min. Samples were then centrifuged at 12,000 × g for 15 min. The absorbance of supernatant fraction (1 mL for each sample) was determined at 550 nm using an automatic biochemistry analyzer (HITACHI 7170S, Japan). Finally, using standard curve, the concentration of hydroxyproline was calculated. IL-6, TNF-alpha, Col-I and Col-III contentsin NRK-49F cells were determined using ELISA assay kits according to the manufacturer’s protocol.

Relative transcript levels of genes were investigated by quantitative real time (RT)-PCR. Total RNA was isolated using Trizol reagents, and 2 μg of total RNA was reverse transcribed into cDNA by SuperScript II kit (Life technologies, Carlsbad, California, USA). PCR was performed on qRT PCR machine (ABI-7500, USA) using SYBR Green reagents (Thermo, USA).

Primers used in Real-time Fluorogenic PCR Assays were designed by Primer 5.0 and synthesized by JRDun Biotech (Shanghai, China) (Table 1). Relative mRNA expression was normalized to internal control GAPDH by the delta-delta Ct method.

Protein lysates (35 μg) from the renal tissues and cultured cells was extracted. After the standent operation of loading to SDS-PAGE and transferred onto nitrocellulose membranes, which were subsequently blocked in 5% non-fat milk for 1 h at room temperature followed by incubation with primary antibodies overnight at 4 °C. After washing, membranes were incubation with HRP-conjugated secondary antibodies. Relative protein ratios were determined and normalized to GAPDH.

Data obtained from this study were expressed as mean ± SD. Statistical data analyses were carried out by Student's t-test and ANOVA on SPSS 20.0 software (SPSS Inc., USA).

According to a previous report, glomerulosclerosis occurred at 8 weeks after operation and the untreated control group showed progression with more severe sclerosis 12 weeks after 5/6 Nx (22). In the present study, histopathological changes of rat renal tissues were examined at 8 weeks after operation by H&E staining and Masson’s trichrome staining. As shown in Figure 1a, renal cells were arranged normally and there were no signs of histopathological abnormalities in normal rats, whereas significant fibrosis and degeneration were detected in 5/6 Nx rats (n=6, 100%). Moreover, creatinine and BUN contents in serum, as well as total urinary protein content, were significantly higher in the 5/6 Nx rats than in control rats at both 4 and 8 weeks after operation. Furthermore, these contents were increased in a time-dependent manner (Figure 1b). Relative mRNA expression and protein levels of TMEM45A and Notch1 in 5/6 Nx rats were also up-regulated in a time-dependent manner (Figure 1c).

Figure 1

Effects of 5/6 Nx on rats. (a): H&E staining and Masson trichrome staining in control rats and 5/6 Nx rats at 8 weeks after operation. Scale bars: 5 μm. (b): Effects of 5/6 Nx on serum creatinine, BUN and total urinary protein contents in rats at 4 and 8 weeks after operation. (c): Effects of 5/6 Nx on mRNA expressions and protein levels of TMEM45A and Notch1 in renal tissues at 4 and 8 weeks after operation. Statistical data analyses were carried out by Student's t-test, ^**p < 0.01, compared with control group (n=6).

Effects of different concentrations of TGF-beta 1 on mRNA and protein expression of TMEM45A and Notch1 in NRK-49F cells were determined at the end of experiment. The results indicate that both mRNA expression and protein levels of TMEM45A and Notch1 in NRK-49F cells were dose-dependently increased by TGF-beta 1 (Figure 1a-b). The optimum concentration for treatment with TGF-beta 1 was 5 ng/mL, which was used in subsequent experiments. If the treatment concentration was higher than 5 ng/mL, an excessive dose (4 times the optimum concentration) of exogenous TGF-β1 might lead to cytokine abnormalities and trigger a feedback system to suppress the expression of TMEM45A and Notch1.

The effect of TMEM45A overexpression on hydroxyproline concentration in NRK-49F cells were measured by UV spectrophotometry. The protein levels of CTGF, FN, Notch-l and Jagged-l were examined by Western blot. The results showed that hydroxyproline concentration in NRK-49F was increased by TMEM45A OE and this effect was weakened by DAPT (Figure 3a). Moreover, the protein expression of CTGF, FN, Notch-1 and Jagged-1 displayed similar trend as that of hydroxyproline (Figure 3b).

The effect of TMEM45A siRNA on hydroxyproline, Col-I, Col-III, TNF-alpha and IL-6 contents as well as was of CTGF, FN, Notch-1 and Jagged-1 protein levels were determined at the end of experiment. As shown in Figure 4, the concentrations of hydroxyproline, Col-I, Col-III, TNF-alpha and IL-6 in NRK-49F cells were elevated by TGF-beta 1 treatment, and these effects were weakened upon TMEM45A knockdown by siRNA (Figure 4a). Relative protein levels showed the same trend (Figure 4b).