Petchiether A attenuates obstructive nephropathy by suppressing TGF-β/Smad3 and NF-κB signalling

2.1 Drug isolation and identification

The fruiting bodies of G petchii and G australe were purchased from a market selling Chinese medical materials in Zhonghao-Luoshi-Wan, Kunming, Yunnan Province, China. The material was identified by Prof. Zhu-Liang Yang at the Kunming Institute of Botany, Chinese Academy of Sciences.

The procedure of PetA isolation was previously described.26 The powders of fruiting bodies of G petchii were extracted by reflux with 70% ethyl alcohol (EtOH). The extraction was suspended in water, followed by the participation with ethyl acetate (EtOAc). Eight parts (Fr1-Fr8) were separated from the EtOAc extract using a MCI gel CHP 20P column (75-150 μm) washing with gradient aqueous methyl alcohol (MeOH) from 10%-100%. The Fr5 was further separated into seven portions (Fr5.1-Fr5.7) using a MCI gel CHP 20P column eluting with gradient aqueous MeOH (20%-100%). Among the fragments, pet A was separated from fr5.6 using Sephadex LH-20 (MeOH) followed by an RP-18 column (MeOH/H₂O, 30:70-100:0), and preparative TLC (CHCl₃/Me₂CO, 8:1). Because of the low content of petA in G petchii, the compound was also enriched from G australe (176 mg from 90 kg fungus) using a similar isolation procedure and structurally identified using multiple spectroscopic methods and further confirmed by the Mosher's method (Figure 1D). The purity of petA was over 98%.

2.2 Animal model

Ten- to 12-week-old male C57BL/6J mice (bodyweight 20-25 g) were used for this study. Unilateral ureteral obstruction was performed using an established protocol as described previously.27, 28 To evaluate the effect of petA on renal fibrosis, we randomised the mice into four groups (n = 5-8 per group): (a) the sham-operated group; (b) the UUO group, in which mice received intraperitoneal (ip) injections of vehicle for four consecutive days; (c) the prevention group, in which mice received 4 consecutive days of petA (ip) before UUO; and (d) the treatment group, in which mice received 4 days of petA (ip) after UUO. Petchiether A was dissolved in dimethyl sulfoxide (DMSO). All mice were killed on day 5 after left ureter ligation. Kidney tissues were collected for histology, immunohistochemistry, Western blotting and real-time reverse transcript-PCR analysis as previously described.29 All studies were approved by the Animal Experimentation Ethics Committee of the University of Hong Kong, and the experimental methods were performed in accordance with the approved guidelines.

2.3 Morphological and immunohistochemical analysis

To examine the changes in renal morphology, we stained formalin-fixed, paraffin-embedded sections (3 µm) with haematoxylin and eosin or a Masson's trichrome staining kit (ScyTek Laboratories, West Logan, UT) according to the manufacturer's instructions and as previously described.29, 30 Immunohistochemistry was performed in paraffin sections using a microwave-based antigen retrieval method.31 The primary antibodies used in this study included antibodies against TNF-α, IL-1β, TGF-β1, fibronectin (Santa Cruz Biotechnology, Santa Cruz, CA), F4/80 (AbD Serotec, Kidlington, UK), phospho-Smad3 (Rockland Immuno-chemicals, Gilbertsville, PA), α-SMA (Sigma, St. Louis, MO), collagen I (Southern Biotech, Birmingham, AL) and phospho-nuclear factor κ light chain enhancer of activated B cells (NF-κB)/p65 (Abcam, Cambridge, MA). Positive signals were analysed with the quantitative Image Analysis System (Image-Pro Plus 7.0; Media Cybernetics, Bethesda, MD) as described previously.32, 33

2.4 Cell culture

Human kidney (proximal tubular epithelial) cell line 2 (HK-2) cells, a normal adult human renal tubular epithelial cell line, were cultured in serum-free DMEM/Ham's F12 medium (Invitrogen Life Technologies, Gaithersburg, MD). To investigate the effect of petA on TGF-β1-induced phosphorylation of Smad3, we pre-treated HK-2 cells with the indicated doses of petA for 12 hours before adding TGF-β1 (2.5 ng/mL) for another 12 hours. To determine the preventive and therapeutic effects of petA on TGF-β1/Smad3 signalling-induced collagen I expression, we incubated HK-2 cells with petA (25 μmol/L) for 12 hours before or after 12 hours of TGF-β1 (2.5 ng/mL). Each experiment was repeated independently at least three times.

2.5 MTT assay

HK-2 cells were seeded into 96-well plates at a density of 1 × 10⁵ cells/mL in a volume of 200 μL per well and allowed to attach for 24 hours. The cells were starved with serum-free medium for 24 hours and then incubated with the indicated amounts of petA (1, 2.5, 5, 10, 25, 50, 100 μmol/L) for 12 hours. Cell viability was determined by adding 20 μL of the reagent 3-[4,5-dimethylthiazol-2-yl-]-2,5-diphenyltetrazolium bromide (MTT) at a concentration of 5 mg/mL and incubating the cells for 4 hours at 37°C. The MTT-containing medium was removed, and 150 μL of DMSO was added to dissolve the formazan crystals. The absorbance value was measured at 540 nm using a microplate reader (Bio-Tek Instruments, Inc, Ontario, Canada).

2.6 Smad3-responsive promoter assay

HK-2 cells were transiently transfected with the Smad3-responsive promoter p(CAGA)12-Luc (kindly provided by Professor Hong-Jian Zhu, University of Melbourne) as described previously.28 The PGL3 Basic plasmid was co-transfected into the cells as a control. After transfection, cells were treated with petA (5, 25, 50 μmol/L) for 12 hours, followed by the addition of TGF-β1 (2.5 ng/mL) for another 12 hours. The luciferase activity of p(CAGA)12 was analysed using a Promega Luciferase Assay kit (Promega Corporation, Wisconsin, USA) according to the manufacturer's instructions and measured using a PerkinElmer 2030 Multilabel Luminescence Microplate Reader (PerkinElmer Life and Analytical Sciences, Finland). Three independent experiments were performed.

2.7 Western blot analysis

Protein was extracted from the kidney tissues and cultured HK-2 cells using radio-immunoprecipitation assay lysis buffer, and Western blot analysis was performed as described previously.28 Five percent bovine serum albumin (BSA) was used to block non-specific binding before the membranes were incubated with primary antibody overnight at 4°C. The antibodies used in this study included primary antibodies against collagen I (Southern Biotech), fibronectin (Santa Cruz Biotechnology), phospho-NF-κB/p65, NF-κB/p65, phospho-Smad3 and Smad3 (Cell Signalling Technology Inc, Danvers, MA) and secondary antibodies labelled with LI-COR IRDye 800 (Rockland Immuno-chemicals). Signal detection was performed using the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE) and quantified by Imagej software (National Institutes of Health). The expression level of each protein was normalised to the expression level of β-actin and is expressed as the mean ± SEM

2.8 RNA extraction, quantitative real-time PCR

Total RNA was isolated from the renal tissues and cultured HK-2 cells using TRIzol reagent from Invitrogen according to the manufacturer's instructions, and real-time PCR was performed using Bio-Rad IQ SYBR Green Supermix with Option 2 (Bio-Rad, Hercules, CA) as previously described.27 The primers used in this study for mouse IL-1β, TNF-a, TGF-β1, a-SMA, collagen I and fibronectin mRNAs have been described previously.27, 28, 30-32, 34 The housekeeping gene β-actin was used as an internal control. The expression level of each mRNA of interest was normalised to that of β-actin and expressed as the mean ± SEM

2.9 Statistical analysis

All the data obtained from this study are expressed as the mean ± SEM from at least three independent experiments or groups of five to eight mice each. Statistical analyses were performed with one-way ANOVA followed by the Newman-Keuls post hoc test. The tests were performed in GraphPad Prism 5 (GraphPad Software, La Jolla, CA). A P-value <0.05 was considered significant.

3.1 Toxicity of petA

To detect the toxicity of petA, we incubated human proximal tubular cells (HK-2) with the indicated doses of petA. Cell viability was detected by MTT (3-[4,5-dimethylthiazol-2-yl-]-2,5-diphenyltetrazolium bromide) experiments. Petchiether A did not affect the viability or proliferation of the cells at any of the tested doses (1, 2.5, 5, 10, 25, 50, 100 μmol/L; Figure S1).

3.2 PetA attenuates kidney injury after UUO

We evaluated the effect of petA on renal fibrosis in a typical model of renal interstitial fibrosis caused by UUO. In the preliminary study, based on our previous study of G lucidum,35 petA was administered immediately after UUO operation by ip injection at dosages of 20 and 40 mg/kg/d (n = 8 in the 20 mg/kg group, n = 5 in the 40 mg/kg group). Mice were killed on day 5 after UUO. Morphological and Western blot analyses showed that 40 mg/kg petA significantly decreased histological injury and Smad3 phosphorylation and collagen I expression (Figure S2). Therefore, 40 mg/kg of petA was used in the following prevention and treatment studies.

The haematoxylin and eosin and Masson's trichrome staining analyses indicated the obstructed kidney showed severe tubulointerstitial damage, such as tubular dilatation, atrophy, infiltration of inflammatory cells and accumulation of collagen deposition compared to the sham-operated kidney (Figure 1A). However, administration of petA (40 mg/kg), initiated either immediately after or 4 days before the UUO procedure, markedly reduced these changes compared with those in the vehicle group (Figure 1A); this result was further confirmed by semi-quantification of the tubulointerstitial damage observed in haematoxylin and eosin- and Masson's trichrome-stained sections of the kidney tissue (Figure 1B,C). Taken together, these results indicate that the use of petA for the prevention and treatment of renal fibrosis in mice protects against renal histological damage and fibrosis.

3.3 PetA ameliorates renal inflammation and fibrosis in the kidney after UUO

We then examined the effect of petA on renal inflammation and fibrosis in UUO mice. Immunohistochemistry and real-time PCR analysis revealed that, compared with the sham-operated mice, UUO vehicle mice developed moderate renal inflammation including a marked up-regulation of proinflammatory cytokines/chemokines (TNF-α, IL-1β, MCP-1 and IL-6) and renal infiltration of F4/80⁺ macrophages (Figure 2). However, all these inflammatory features were significantly decreased in the obstructed kidney after 5 days of UUO in the petA (40 mg/kg) prevention and petA (40 mg/kg) treatment group mice (Figure 2). Further studies also revealed that moderate renal fibrosis, as indicated by the expression of α-SMA, collagen I and fibronectin mRNA and the accumulation of the corresponding matrix proteins, occurred in UUO vehicle mice but was substantially attenuated in mice that received petA (40 mg/kg) 4 days before or immediately after UUO operation (Figure 3).

3.4 PetA ameliorates renal inflammation and fibrosis in UUO mice and is associated with the suppression of the NF-κB and TGF-β1/Smad3 signalling pathways

We next investigated the underlying signalling mechanisms by which petA protects against obstructive kidney injury. First, we examined the NF-κB inflammation signalling pathway. Immunohistochemistry and Western blot analysis revealed significantly elevated concentrations and nuclear translocation of phosphorylated p65 subunit in the obstructed kidneys of the vehicle UUO group, and these measures were markedly decreased in the obstructive kidneys of the petA (40 mg/kg) prevention and petA (40 mg/kg) treatment group mice (Figure 4), suggesting that petA may protect against renal inflammation during UUO via NF-κB signalling.

Furthermore, we also found a significant up-regulation of renal TGF-β1 at the mRNA and protein levels (Figure 5A) in UUO vehicle mice, and this up-regulation was associated with enhanced Smad3 signalling, as detected by increased concentrations and nuclear localisation of phosphorylated Smad3 in glomerular and tubulointerstitial cells (Figure 5B,C). However, these increases were abolished by petA (40 mg/kg) as prevention or treatment in UUO mice (Figure 5). These findings suggest that petA may protect against renal fibrosis in UUO via the TGF-β/Smad3 signalling pathway.

3.5 PetA attenuates fibrosis by inhibiting phosphorylation of Smad3

To examine whether petA protected against fibrosis by suppressing TGF-β1-induced Smad3 phosphorylation, HK-2 tubular epithelial cells were treated with TGF-β1 with/without petA. Western blot results demonstrated that petA significantly inhibited TGF-β1-induced Smad3 phosphorylation in a dose-dependent manner (Figure 6A), and real-time PCR analysis revealed a dramatic decrease in endogenous TGF-β1 and collagen 1 expression (Figure 6B,C). However, the baseline level of endogenous TGF-β1 in PetA treated HK-2 cells had no significant change. Further, HK-2 cells were pre-treated with or without petA (25 μmol/L) for 12 hours either before (prevention group) or immediately after (treatment group) stimulation with TGF-β1 (2.5 ng/mL). Western blot analysis showed that the addition of petA (25 μmol/L) inhibited the phosphorylation of Smad3 and the up-regulated expression of the fibrotic marker collagen I by TGF-β1 (Figure 6D-F). The Smad3-responsive promoter assay results demonstrated that petA dose-dependently suppressed TGF-β1-induced Smad3-responsive promoter activity, indicating that petA significantly inhibited gene expression downstream of the TGF-β/Smad3 signalling pathway (Figure 6G).