SPF grade C57BL/6 male mice were obtained from Shanghai Laboratory Animal Company (Shanghai, China) and housed at Shanghai University of Traditional Chinese Medicine in line with their rules and regulations. The animal ethics committee of the Shanghai University of Traditional Chinese Medicine approved this study (PZSHUTCM18111601).

Male mice weighing from 18 to 22 g were anesthetized intraperitoneally with 8 mg/kg of pentobarbital before performing an abdominal incision on the left side. This was followed by either a sham operation or left ureteral ligation. Animals were assigned into 4 groups at random: (a) Sham + dimethyl sulfoxide (DMSO) (n = 6), (b) Sham + SAC (n = 6), (c) UUO + DMSO (n = 6), (d) UUO + SAC (n = 6). Intraperitoneal injections of DMSO or SAC (10 mg/kg body weight, TopScience, T3149, Shanghai, China) were administered after the surgery and for 14 consecutive days. Mice were sacrificed at day 14, with kidney tissue harvested for protein extraction and tissue embedding.

Mice were injected intraperitoneally with 5 mg/kg AAI (A9451, Sigma, St. Louis, MO, USA) in normal saline (NS) or with NS only once per week for 2 weeks (2 injections in total). One week after the second injection of AAI, the DMSO or SAC (10 mg/kg body weight, TopScience, T3149, Shanghai, China) was injected intraperitoneally and daily for two weeks (n = 6). Mice were sacrificed after the treatment, and kidneys were collected for protein extraction and tissue embedding.

Cells were grown as previously described by our group [18]. Normal rat kidney interstitial fibroblasts (NRK-49F) rat kidney interstitial fibroblast cells were obtained from the National Infrastructure Cell Line Resource, Chinese Academy of Medical Sciences (cat. 3111C0001CCC000413), which has been identified for the species by PCR and was negative for mycoplasma contamination. The cell line has been tested for the mycoplasma and been authenticated from the Chinese Academy of Medical Sciences. The cell line is orginated from American Type Culture Collection, VA, USA (Cat. No. CRL1570). The culture medium for NRK-49F cells was Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) with 10% fetal bovine serum (FBS) and 0.5% penicillin/streptomycin. NRK-49F cells were grown in 6-well plates until 60–70% confluence was reached, then starved in medium containing just 0.5% FBS for 12 h. After overnight starvation, fresh medium with 0.5% FBS was added and the cells treated with DMSO or with various concentrations of SAC (TopScience, T3149, Shanghai, China). The kidney proximal tubular epithelial cell line Human Kidney 2 (HK2, cat. SCSP-511) was purchased from the Cell Bank, Shanghai Institute of Biological Sciences (Chinese Academy of Science), which has been validated by short tandem repeat profiling and was negative for mycoplasma contamination. The cells were grown in 6-well plates to 40–50% confluence, then starved overnight with DMEM/F12 containing just 0.5% FBS. The following day, fresh DMEM/F12 with 0.5% FBS was added, together with 2.5 ng/mL TGF-$\beta$ (PeproTech, 100-21, Rocky Hill, NJ, USA) and different SAC concentrations for 48 h. The SAC concentrations used here were the same as those used in two previous publications [15, 19].

Staining with Masson’s trichrome was carried out as described earlier [18]. Briefly, paraffin-embedded renal tissue sections (4 µm) were cut and then stained using hematoxylin and ponceau, followed by incubation with phospho-molybdic acid. Kidney tissue was then stained using aniline blue and acetic acid and photographed using a Nikon 80i microscope (Nikon Eclipse 80i, Tokyo, Kanto Plain, Japan).

Proteins were obtained from the kidney tissue of mice as previously described [20]. The Bradford method was employed to measure protein concentrations. Samples were diluted with 5$\times$ SDS-PAGE loading buffer (P0015L, Beyotime Biotech, Nanjing, Jiangsu, China), separated by SDS-PAGE, then transferred to polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany) by wet blotting. Primary antibodies used for protein detection were anti-snail (1:1000 dilution, A11794, ABclonal, Woburn, MA, USA), anti-fibronectin (1:5000, ab23750, Abcam, Cambridge, UK), anti-pSmad3 (1:1000, ET1609-41, HUABIO, Woburn, MA, USA), anti-Smad7 (1:1000, AB37036, Aboci, MD, USA), anti-collagen I (1:5000, ab260043, Abcam), anti-N-cadherin (1:500, sc-59987, Santa Cruz, Dallas, TX, USA), anti-vimentin (1:1000, ET1609-36, HUABIO), anti-GAPDH (1:5000, 60004-1-lg, Proteintech, Wuhan, Hubei, China), and anti-$\alpha$-tubulin (1:1000, AF0001, Beyotime Biotech, Nanjing, Jiangsu, China). Primary antibodies were incubated overnight at 4 °C with membrane, followed by incubation with a horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG, 1:1000, A0208 Beyotime Biotech, Nanjing, Jiangsu, China; or goat anti-mouse IgG, 1:1000, A0216 Beyotime Biotech, Nanjing, Jiangsu, China). Signal detection was achieved using enhanced chemiluminescence (BeyoECL Star, P0018A, Beyotime) and protein expression quantified with Image J1.8.0 (LOCI, University of Wisconsin, Madison, WI, USA).

Immunofluorescence staining of renal tissue was carried out as described previously [21]. In short, paraffin-embedded tissue was cut into 4 µm sections, blocked with 3% BSA, incubated overnight with anti-collagen I antibody (1:500) at 4 °C, rinsed in PBS, then incubated for 60 min with secondary antibody. Cell nuclei were stained with 4′,6-Diamidino-2-phenylindole (DAPI) solution for 10 min and photographed using a Nikon 80i microscope (Tokyo, Japan). Mean fluorescence intensity for each slide was analyzed using Image J and normalized to the control.

TRIzol (15596-018, Invitrogen, Carlsbad, CA, USA) was used to obtain total RNA from NRK-49F cells. This was reverse transcribed into cDNA using a Takara PrimeScript RT kit (RR0036A, Kyoto, Japan). Primer sequences for qPCR and specific for rat were: Fibronectin (F, forward), 5′-CCGAATCACAGTAGTTGCGG-3′; Fibronectin (R, reverse), 5′-GCATAGTGTCCGGACCGATA-3′; Collagen I (F), 5′-TCAAGATGGTGGCCGTTACT-3′; Collagen I (R), 5′-CATCTTGAGGTCACGGCATG-3′; Collagen III (F), 5′-ATGAATTGGGATGCAACTAC-3′; Collagen III (R), 5′-TCTAGTGGCTCATCATCACA-3′; GAPDH (F), 5′-TAAAGGGCATCCTGGGCTACACT-3′; GAPDH (R), 5′-TTACTCCTTGGAGGCCATGTAGG-3′. qPCR was performed using FastStart Universal SYBR Green Master (Rox) (Cat. 4913850001; Merck, Darmstadt, Germany) and StepOne Plus Sequence Detection System (Applied Biosystems, Foster City, CA, United States). Relative expression levels for each gene were analyzed using 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ methods and normalized with GAPDH. Results are shown as the fold-change in mRNA expression.

Results are given as mean $\pm$ SD. Student t-test was used to compare two groups with GraphPad Prism 8.0.0 (GraphPad Software, San Diego, CA, USA). A p-value 0.05 was considered as representing statistical significance. Each experimental group had an n = 6. Representative results from at least three independent experiments are presented. *p 0.05. **p 0.01. ***p 0.001.

The effect of SAC on kidney tubulointerstitial fibrosis was first investigated in vivo. UUO is a classic mouse model for studying kidney fibrosis and was established here for this purpose. UUO mice received DMSO or SAC treatment for two weeks. Compared with sham operated mice, significant deposition of collagen was detected using Masson’s trichrome staining in UUO kidneys. However, this was attenuated by two weeks of treatment with SAC (Fig. 1A). Western blot experiments showed that expression of two ECM proteins, fibronectin and collagen I, was up-regulated in mouse kidneys after UUO surgery, but was lowered by treatment with SAC (Fig. 1B). Elevated levels of collagen I expression in the interstitial areas of UUO kidneys relative to sham kidneys was also shown by immunofluorescent staining. Again, this was significantly attenuated by SAC treatment (Fig. 1C).

Fig. 1.

SAC inhibits renal tubulointerstitial fibrosis in obstructed mouse kidneys. Sham or unilateral ureter obstruction (UUO) operation was performed on mice (n = 6), followed by two weeks of treatment with DMSO or salvianolic acid C (SAC). (A) Renal fibrosis was assessed by Masson’s trichrome staining and then quantified. Bars = 100 µm. (B) The expression of fibronectin and collagen I were analyzed by Western blotting and then quantified. (C) Representative immunofluorescent images of collagen I staining in sham or UUO mouse kidneys. These were also quantified. DAPI (nuclei) = blue, collagen I = green. Bar = 20 µm. N = 6 in each experimental group. One representative of at least three independent experiments is shown. Data represents the mean $\pm$ SD. **p 0.01. ***p 0.001.

The effects of SAC on anti-fibrosis were further studied in mice with chronic aristolochic acid nephropathy (AAN). Masson’s trichrome revealed mild staining in the interstitial areas of AAN kidneys (Fig. 2A). Treatment with SAC for two weeks significantly reduced the positive areas shown by Masson’s trichrome staining in AAN kidneys (Fig. 2A). Fibronectin and collagen I expression were increased in AAN kidneys relative to the control kidneys. SAC treatment was observed to reduce the expression of these ECM proteins (Fig. 2B). Up-regulation of collagen I in the interstitial areas was further confirmed by immunofluorescent staining observed in AAN kidneys relative to control kidneys (Fig. 2C). SAC treatment significantly reduced positive areas of collagen I staining in AAN kidneys (Fig. 2C).

Fig. 2.

SAC attenuates the development of renal tubulointerstitial fibrosis in mice with aristolochic acid nephropathy (AAN). Mice (n = 6) were injected intraperitoneally with aristolochic acid I (AAI) or normal saline (NS) once a week for two weeks. One week after the second injection of AAI or NS, mice were treated with DMSO or SAC (10 mg/kg) for two weeks. (A) Renal fibrosis was assessed by Masson’s trichrome staining and then quantified. Bar =100 µm. (B) The expression of fibronectin and collagen I were analyzed by Western blotting and then quantified. (C) Representative immunofluorescent images of collagen I staining in control or AAN mouse kidneys. These were also quantified. DAPI (nuclei) = blue, collagen I = green. Bar =20 µm. N = 6 in each experimental group. One representative of at least three independent experiments is shown. Data represents the mean $\pm$ SD. *p 0.05. **p 0.01. ***p 0.001.

Direct effects of SAC on kidney cells were further investigated in vitro by using the rat fibroblast cell line NRK-49F, as well as the human kidney proximal tubular epithelial cell line HK2. The mRNA levels of fibronectin, collagen I and collagen III in NRK-49F cells were reduced by SAC in dose-dependent fashion from 10 to 100 µM (Fig. 3A). Western blot analysis showed that SAC also dose-dependently reduced fibronectin and collagen I protein expression in these cells from 30 µM to 100 µM (Fig. 3B). The dose-dependent (10 µM to 100 µM) inhibition of fibronectin expression by SAC was further shown in HK2 cells stimulated with TGF-$\beta$ (Fig. 3C).

Fig. 3.

SAC reduces ECM protein expression in renal cells. Rat renal fibroblasts (NRK-49F) were starved for 12 h, then treated for 24 h with different concentrations (10 µM, 30 µM, 100 µM) of SAC. (A) The mRNA expression levels for fibronectin, collagen I and collagen III were analyzed by qPCR. (B) The expression of fibronectin and collagen I were analyzed by Western blotting and then quantified. (C) HK2 human renal epithelial cells were starved overnight, stimulated with TGF-$\beta$ and then treated with various concentrations of SAC. Cell lysates were extracted and the expression of fibronectin was analyzed by Western blotting and then quantified. Data represents the mean $\pm$ SD. One representative result of at least three independent experiments is shown. NS represents not significant. *p 0.05. **p 0.01. ***p 0.001.

The effect of SAC on EMT was evaluated in vivo as well as in vitro. Snail expression in UUO and AAN kidneys was increased compared to their respective controls (Fig. 4A,B). However, treatment with SAC reduced renal expression of Snail in these mouse models. Increasing doses of SAC (10 µM to 100 µM) also progressively inhibited N-cadherin and vimentin expression in TGF-$\beta$-stimulated HK2 cells (Fig. 4C).

Fig. 4.

SAC inhibits epithelial-mesenchymal transition in fibrotic kidneys and renal fibrotic cells. (A) The expression of Snail in sham or UUO kidneys (n = 6) was analyzed by Western blotting and then quantified. (B) The expression of Snail in NS or AAN kidneys (n = 6) was analyzed by Western blotting and then quantified. Data represents the mean $\pm$ SD. (C) HK2 human renal epithelial cells were starved overnight, then stimulated with TGF-$\beta$ and treated with various concentrations of SAC for 24 h. The expression of N-cadherin and of vimentin was analyzed by Western blotting and then quantified. One representative result of at least three independent experiments is shown. n.s. represents not significant. *p 0.05. **p 0.01. ***p 0.001.

We next evaluated the effects of SAC on Smad3 signaling in vivo and in vitro. Fig. 5A,B show that Smad3 phosphorylation was enhanced in fibrotic kidneys of UUO or AAN mice compared to their controls. SAC treatment decreased Smad3 phosphorylation (pSmad3) levels in UUO and AAN kidneys (Fig. 5A,B). Moreover, SAC inhibited pSmad3 in TGF-$\beta$-stimulated HK2 cells in a dose-dependent fashion from 10 µM to 100 µM. This correlated with up-regulated expression of the anti-fibrotic protein Smad7 (Fig. 5C).

Fig. 5.

SAC inactivates the Smad signaling pathway in fibrotic kidneys and renal fibrotic cells. (A) The expression of phosphorylated Smad3 (pSmad3) in sham or UUO kidneys (n = 6) was analyzed by Western blotting and then quantified. (B) The expression of pSmad3 in NS or AAN kidneys (n = 6) was analyzed by Western blotting and then quantified. (C) HK2 human renal epithelial cells were starved overnight, then stimulated with TGF-$\beta$ and treated with various concentrations of SAC for 24 h. The expression of pSmad3 and of Smad7 was analyzed by Western blotting and then quantified. Data represents mean $\pm$ SD. One representative result of at least three independent experiments is shown. n.s. represents not significant. *p 0.05. ***p 0.001.