Alantolactone Attenuates Renal Fibrosis via Inhibition of Transforming Growth Factor β/Smad3 Signaling Pathway

Animals and UUO model

Male C57BL/6 8-week-old mice, weighing 25±2 g, were purchased from the Koatech Technology Corporation (Pyeongtaek, Korea). All experiments were approved by the Animal Care and Use Committee of Daegu Gyeongbuk Institute of Science and Technology (DGIST-IACUC-19052105-01). Mice were bred under standard room temperature (22°C±2°C) and humidity (60%±10%) conditions with a 12-hour light/dark cycle. Animals were randomly divided into the following three groups: control (CON, n=7), UUO (n=7), UUO with 5 mg/kg AL treatment (UUO/AL, n=7). AL (Carbosynth, Compton, UK) was dissolved in a filtered solution (1.25% dimethyl sulfoxide [DMSO], 2% polyethylene glycol [PEG] 400, 0.5% Tween 20 in saline), which was administered orally by gavage in equivalent volumes to CON and UUO mice once per day for 15 days, including 5 days of pretreatment. The UUO surgery was performed as previously described [22]: briefly, anesthetized mice underwent laparotomy to ligate the left with sterilized 5–0 silk for 10 days. Fibrosis-induced kidneys were fixed in a 10% formalin solution (Sigma-Aldrich, Burlington, MA, USA) or stored in liquid nitrogen for use as experimental samples.

Blood analysis

The serum of mice, separated using centrifugation of blood, was used to measure the levels of serum creatinine (Scr), blood urea nitrogen (BUN), alanine aminotransferase and aspartate aminotransferase using an automatic biochemical analyzer (Cobas Integra 800, Roche, Basel, Switzerland).

Cell culture

Human proximal tubule cell line (HK-2) and rat fibroblast cell line (NRK-49F) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). HK-2 cells were grown in keratinocyte serum-free medium (Gibco, Waltham, MA, USA), supplemented with 50 µg/mL bovine pituitary extract and 5 ng/mL human recombinant epidermal growth factor. NRK-49F cells were cultured in Dulbecco’s modified Eagle medium/nutrient mixture F-12 medium (Gibco) with 5% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (Welgene, Gyeongsan, Korea). Cells were cultured in an atmosphere of 5% CO₂ at 37°C in a humidity chamber and treated with 5 and 2 ng/mL of TGF-β1 (R&D Systems, Minneapolis, MN, USA), respectively, for the indicated time periods.

Western blot analysis

Total protein from cells and kidney tissues were obtained by homogenization in radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing cOmplete, Mini Protease Inhibitor Cocktail (Roche), and Halt Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Samples with an equal protein concentration were electrophoresed on Mini-PROTEAN Gels (Bio-Rad, Hercules, CA, USA), and then electrotransferred onto polyvinylidene fluoride membranes (Bio-Rad). After blocking with 5% bovine serum albumin (Sigma-Aldrich) for 1 hour, the membranes were immunoblotted overnight at 4°C with one of the following primary antibodies: anti-collagen type I (1:500) and anti-fibronectin (1:1,000) from Abcam (Cambridge, UK); anti-vimentin (1:1,000), anti-α-smooth muscle actin (α-SMA; 1:1,000), anti-plasminogen activator inhibitor-1 (PAI-1; 1:500) from BD Biosciences (Franklin Lakes, NJ, USA); and anti-phospho-Smad3 (1:500), anti-phospho-signal transducer and activator of transcription 3 (STAT3; 1: 1,000), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:1,000), and anti-β-actin (1:1,000) from Santa Cruz Biotechnology (Dallas, TX, USA). The membranes were subsequently treated horseradish peroxidase (HRP)-linked secondary antibody (1:3,500). Protein expression was measured by visualizing the proteins bands using ChemiDoc XRS+ (Bio-Rad) and performing densitometric analysis using the Image J program 1.53e (National Institutes of Health, Bethesda, MD, USA).

mRNA expression analysis using real time-quantitative polymerase chain reaction

An RNA Extraction Kits (Takara, Tokyo, Japan) was used to extract total RNA. cDNA was synthesized using a Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA) and, mRNA levels were detected using an SYBR Green polymerase chain reaction (PCR) Master Mix kit (Applied Biosystems) on an ABI 7500 quantitative polymerase chain reaction (qPCR) system. The qPCR conditions and primer sequences were designed using the Primer Express Software ver. 3.0 (Thermo Fisher Scientific). The sequences of all the primers are provided in Table 1.

Table 1.

Sequences of real time-quantitative polymerase chain reaction primers for collagen type I, fibronectin, α-SMA, and PAI-1

Histological analysis and immunohistochemical staining

Sirius red and Masson’s trichrome (Sigma-Aldrich) and immunohistochemical staining (Thermo Fisher Scientific) were performed on paraffin sections (4 μm thick) according to the manufacturer’s instructions. Kidney sections were deparaffinized using xylene and rehydrated using graded ethanol series. Tubulointerstitial collagen deposition was assessed using Sirius red or Masson’s trichrome staining. For Sirius red staining, the slides were immersed for 18 hours in saturated picric acid with 0.1% Sirius red F3BA, and 0.01N hydrochloric acid for 2 minutes. For Masson’s trichrome staining, slides were treated with Bouin’s solution and incubated sequentially with hematoxylin for 10 minutes, Biebrich scarlet-acid fuchsin for 5 minutes, phosphotungstic acid/phosphomolybdic acid for 10 minutes, aniline blue for 15 minutes, and 1% acetic acid for 5 minutes. Immunohistochemical staining was performed with anti-collagen type I (1:200), anti-fibronectin (1:200, Abcam), anti-α-SMA (1:300), anti-p-Smad3 (1:200, Santa Cruz Biotechnology), and DAB staining kit (Roche). The kidney sections were dehydrated with a graded series of ethanol and xylene and mounted on glass slides for microscopic investigation. After staining, sections from each kidney of five different animals were selected and the images (×200) were taken using Leica Microscope (Leica Microsystem, Wetzlar, Germany) and Leica Application Suite V3.8 software (Leica Microsystems). Quantification of the aniline blue (collagen, blue), Sirius red (collagen fiber, red), and immunostaining (brown color) positive areas was performed using computer-based morphometric analysis.

Immunofluorescence staining

HK-2 and NRK-49F cells, seeded on 8-well glass slides (Millipore, Burlington, MA, USA) were treated with AL or TGF-β1. For fixation, the glass slides were incubated with 2% paraformaldehyde for 2 hours at room temperature (20°C to 25°C). After washing the cells with phosphate-buffered saline (PBS), they were permeabilized for 2 minutes using 0.3% Triton X-100 (Sigma-Aldrich) in PBS. Cells were blocked with CAS-Block Histochemical Reagent (Thermo Fisher Scientific) for 1 hour, and then incubated overnight with anti-p-Smad3 antibody (1:100, Abcam) at room temperature. The slides were washed three times for 5 minutes each with PBS and then incubated with Alexa Fluor 488 anti-rabbit antibody for 1 hour in the dark. After staining the nuclei with 4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI, Invitrogen) for 5 minutes, the cells were washed three times with distilled water prior to mounting with a fluorescent mounting medium (Sigma-Aldrich). Immunofluorescence was detected using a laser scanning confocal microscope (Zeiss, Oberkochen, Baden-Württemberg, Germany) and analyzed using the ZEN 2.6 (blue edition) software.

Statistical analysis

Data are expressed as the mean±standard error of the mean of three independent experiments. Statistical analysis was performed using Student’s t-test. Statistical significance was set at P<0.05.

Toxicity of alantolactone

To examine the cell toxicity of AL, NRK-49F and HK-2 cells were incubated with the indicated concentrations of AL and their viability was measured using the cell counting kit-8 (CCK-8) assay. AL did not affect the cell viability at 0.25 to 4 µM but at 10 µM the viability of both the renal cell lines was less than 90% (Fig. 1A and B). Therefore, we used 1, 2, and 4 µM of AL for the subsequent in vitro experiments. In vivo toxicity of AL, was assessed in mice orally administered 5 or 10 mg/kg of AL daily for 15 days; these mice did not exhibit mortality, any unusual behavior, and liver toxicity (Supplementary Fig. 1).

Fig. 1.

Effects of alantolactone (AL) on the cell viability of renal cells and on renal function in mice with unilateral ureteral obstruction. (A, B) Serum-starved rat fibroblast cell line (NRK-49F) and human proximal tubule cell line (HK-2) cells were incubated with the indicated concentration of AL for 24 hours and the viability was assessed using the cell counting kit-8 (CCK-8) assay. Quantification of data from three independent experiments was done, and values are shown as the mean±standard error of the mean. (C, D) Analysis of blood urea nitrogen (BUN) and serum creatine (Scr) levels. ^aP<0.05 vs. 0 μM; ^bP<0.05 and ^cP<0.01 vs. control (CON); ^dP<0.05 and ^eP<0.01 vs. unilateral ureteral obstruction (UUO).

Alantolactone attenuate UUO-induced renal dysfunction and renal fibrosis

As shown in Fig. 1C and D, the levels of Scr and BUN were increased in the UUO group. Administration of AL in UUO mice led to lower Scr and BUN levels compared with those in the UUO mice that were not treated.

A comparison of results for the UUO and UUO/AL groups (Fig. 2A-C) showed that UUO kidneys exhibited significantly increased tubular atrophy and tubulointerstitial fibrosis. Conversely, UUO-induced renal fibrosis was markedly decreased in the kidneys of AL-treated mice. Masson’s trichrome and Sirius red staining showed that interstitial collagen deposition in UUO kidneys increased significantly, whereas less intense staining was observed in the kidneys of AL-treated mice. Immunohistochemical staining showed that the expression of collagen type I, fibronectin, and α-SMA was increased in UUO kidneys. In contrast, the expression of all the fibrotic factors was decreased in AL-treated UUO kidneys (Fig. 2A and D-F). Similar results were also observed in immunoblot analysis of kidney tissue lysates. The expression of these fibrotic factors proteins was significantly increased in UUO kidneys and decreased in AL-treated UUO kidneys (Fig. 2G-J).

Fig. 2.

Alantolactone (AL) alleviates unilateral ureteral obstruction (UUO)-induced renal fibrosis. Mice received AL (5 mg/kg) by oral gavage for 5 days before UUO and for 10 additional days thereafter. (A) Images showing the staining of kidney sections with Masson’s trichrome and Sirius red and immunostaining for collagen type I, fibronectin, and α-smooth muscle actin (α-SMA) at 20× magnification. (B, C, D, E, F) Quantification of fibrotic scores or positively stained areas using computer-based morphometric analysis. Data in all the bar graphs were normalized against the control (n=1) and are expressed as a fold increase relative to the control. (G, H, I, J) Representative band (two or three cases) obtained in the Western blot analysis of UUO kidney lysates for collagen type I, fibronectin, and α-SMA. Expression levels of these proteins in different groups were quantified using densitometry and normalized with the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and against the control group (n=1). Data in all the bar graphs are expressed as a fold increase relative to the control. ^aP<0.01 vs. control (CON); ^bP<0.05 and ^cP<0.01 vs. UUO (n=7 for each group); ^dP<0.05 vs. control (CON); ^eP<0.05 and ^fP<0.01 vs. UUO.

Alantolactone inhibits the expression of TGF-β-induced renal fibrotic factors in renal fibroblasts and renal proximal tubule cells

Next, the effects of AL on TGF-β-stimulated profibrotic target gene inhibition and on protein expression in NRK-49F and HK-2 cells were evaluated. Real-time qPCR analysis showed that AL dose-dependently inhibited the expression of TGF-β-stimulated collagen type I, fibronectin, PAI-1, and α-SMA mRNAs in NRK-49F cells (Fig. 3A-D). In addition, AL effectively inhibited the expression of TGF-β-stimulated profibrotic factor proteins in NRK-49F cells (Fig. 3E). AL also inhibited TGF-β-stimulated expreesion of collagen type I and PAI-1 mRNA and protein in HK-2 cells (Fig. 3F-H).

Fig. 3.

Alantolactone inhibits the expression of transforming growth factor β (TGF-β)-induced renal fibrotic factors in renal fibroblasts and renal proximal tubule cells. Serum-starved rat fibroblast cell line (NRK-49F) and human proximal tubule cell line (HK-2) cells were incubated with 2 ng/mL (NRK-49F) or 5 ng/mL (HK-2) TGF-β for 24 hours in the presence of alantolactone (0 to 4 μM). (A, B, C, D) Real-time quantitative polymerase chain reaction (qPCR) analysis of the expression of collagen type I, fibronectin, plasminogen activator inhibitor-1 (PAI-1), and α-smooth muscle actin (α-SMA) in TGF-β-stimulated NRK-49F cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal control. (E) Quantification analysis of collagen type I, fibronectin, PAI-1, and α-SMA bands obtained using Western blot analysis of lysates of TGF-β-stimulated NRK-49F cells. (F, G) Real-time qPCR analysis of the expression of collagen type I and PAI-1 in TGF-β-stimulated HK-2 cells. GAPDH mRNA was used as an internal control. (H) Quantification of collagen type I and PAI-1 bands obtained using Western blot analysis of lysates of TGF-β-stimulated HK-2 cells. Data represent mean±standard error of the mean values from three independent experiments. ^aP<0.05, ^bP<0.01 and ^cP<0.001 vs. control (TGF-β [–]); ^dP<0.05, ^eP<0.01, and ^fP<0.001 vs. TGF-β.

Alantolactone ameliorates renal fibrosis by inhibition of Smad3 phosphorylation in UUO kidneys and renal cells

TGF-β regulates renal fibrotic genes and matrix proteins through Smad signaling, and subsequently contributes to renal fibroblast activation and tubulointerstitial fibrosis. To elucidate the inhibitory mechanism of renal fibrosis, we investigated whether AL regulated Smad3, which is activated in UUO kidneys and TGF-β-treated renal cells. In the UUO kidneys, phospho-Smad3 levels increased compared with those in CON kidneys and were significantly decreased in AL-treated UUO kidneys (Fig. 4A). In addition, the protein expression in UUO kidneys was significantly inhibited by AL administration (Fig. 4B). As shown in Fig. 5A and B, AL inhibited TGF-β secretion in cultured renal fibroblasts and tubular epithelium-like cells. Immunoblot analysis showed that AL inhibited TGF-β-stimulated Smad3 phosphorylation as well as Smad3 translocation (Fig. 5E) to nucleus in both the renal cells (Fig. 5C and D).

Fig. 4.

Alantolactone (AL) inhibits Smad3 phosphorylation in unilateral ureteral obstruction (UUO) kidneys and renal cells. Mice received (5 or 10 mg/kg per day) by oral gavage for 5 days before UUO and for 10 additional days thereafter. (A) Images showing immunostaining of kidney sections for phospho-Smad3 (p-Smad3) at 20× magnification and quantification of positively stained areas using computer-based morphometric analysis. All data were normalized against the control (n=1) and are expressed as a fold increase relative to the control. (B) Representative bands (two or three cases) obtained in the Western blot analysis of UUO kidney lysates for p-Smad3 (upper). Expression levels of p-Smad3 in different groups were quantified using densitometry and normalized with the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and against the control (n=1). Data in all bar graphs are expressed as afold increase relative to the control (lower). ^aP<0.01 vs. control (CON); ^bP<0.01 vs. UUO (n=7 for each group); ^cP<0.01 vs. UUO.

Fig. 5.

Alantolactone (AL) inhibits the transforming growth factor β (TGF-β)/Smad3 signaling pathway in renal cells. Serumstarved rat fibroblast cell line (NRK-49F) and human proximal tubule cell line (HK-2) cells were incubated with 2 ng/mL (NRK-49F) or 5 ng/mL (HK-2) TGF-β for 24 hours in the presence of AL (0 to 4 μM). (A, B) Measurement of TGF-β secretion in TGF-β-stimulated NRK-49F and HK-2 cells with or without 2 μM AL treatment. (C, D) Western blot analysis of protein expression of p-Smad3 in TGF-β-stimulated NRK-49F and HK-2 cells. Quantification of Western blot data is from three independent experiments; data are shown as the mean±standard error of the mean. (E) Immunofluorescence staining of p-Smad3 (red in NRK-49F; green in HK-2) and 4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI) nuclear counterstaining (blue) in TGF-β-stimulated NRK-49F and HK-2 cells with or without 2 μM AL treatment (20× magnification). ^aP<0.01 and ^bP<0.001 vs. control (CON); ^cP<0.01 vs. TGF-β; ^dP<0.01 vs. control (TGF-β [–]); ^eP<0.05 and ^fP<0.001 vs. TGF-β.