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

Abstract Obstructive nephropathy is the end result of a variety of diseases that block drainage from the kidney(s). Transforming growth factor‐β1 (TGF‐β1)/Smad3‐driven renal fibrosis is the common pathogenesis of obstructive nephropathy. In this study, we identified petchiether A (petA), a novel small‐molecule meroterpenoid from Ganoderma, as a potential inhibitor of TGF‐β1‐induced Smad3 phosphorylation. The obstructive nephropathy was induced by unilateral ureteral obstruction (UUO) in mice. Mice received an intraperitoneal injection of petA/vehicle before and after UUO or sham operation. An in vivo study revealed that petA protected against renal inflammation and fibrosis by reducing the infiltration of macrophages, inhibiting the expression of proinflammatory cytokines (interleukin‐1β and tumour necrosis factor‐α) and reducing extracellular matrix deposition (α‐smooth muscle actin, collagen I and fibronectin) in the obstructed kidney of UUO mice; these changes were associated with suppression of Smad3 and NF‐κB p65 phosphorylation. Petchiether A inhibited Smad3 phosphorylation in vitro and down‐regulated the expression of the fibrotic marker collagen I in TGF‐β1‐treated renal epithelial cells. Further, we found that petA dose‐dependently suppressed Smad3‐responsive promoter activity, indicating that petA inhibits gene expression downstream of the TGF‐β/Smad3 signalling pathway. In conclusion, our findings suggest that petA protects against renal inflammation and fibrosis by selectively inhibiting TGF‐β/Smad3 signalling.

eration. An in vivo study revealed that petA protected against renal inflammation and fibrosis by reducing the infiltration of macrophages, inhibiting the expression of proinflammatory cytokines (interleukin-1β and tumour necrosis factor-α) and reducing extracellular matrix deposition (α-smooth muscle actin, collagen I and fibronectin) in the obstructed kidney of UUO mice; these changes were associated with suppression of Smad3 and NF-κB p65 phosphorylation. Petchiether A inhibited Smad3 phosphorylation in vitro and down-regulated the expression of the fibrotic marker collagen I in TGF-β1-treated renal epithelial cells. Further, we found that petA dose-dependently suppressed Smad3-responsive promoter activity, indicating that petA inhibits gene expression downstream of the TGF-β/Smad3 signalling pathway. In conclusion, our findings suggest that petA protects against renal inflammation and fibrosis by selectively inhibiting TGF-β/Smad3 signalling.

| INTRODUC TI ON
Renal fibrosis is the most common progressive process in the pathogenesis of chronic kidney diseases, degrading kidney function and eventually causing end-stage renal disease in patients. [1][2][3][4][5] Renal fibrosis is characterised by extracellular matrix deposition in glomerular and tubulointerstitial tissue. Increasing evidence shows that obstructive nephropathy, which is commonly caused by urolithiasis, benign prostatic hyperplasia and pelvic or ureteral tumours, leads to proximal tubular cell loss and interstitial fibrosis.
The unilateral ureteral obstruction (UUO) model is widely used to study the mechanisms of tubulointerstitial fibrosis via surgically induced obstructive renal injury. 6 In experimental animal models and patients with obstructive nephropathy, the major pathological features in local are infiltration of inflammatory cells, secretion of proinflammatory cytokines, such as interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) and the accumulation of fibrotic markers, such as collagen I, fibronectin and α-smooth muscle actin (α-SMA). The TGF-β/Smad signalling pathway may be a viable therapeutic target for treating renal fibrosis. [7][8][9] Treatments that manipulate TGF-β/ Smad signalling have shown beneficial effects in the kidneys of laboratory animals. [10][11][12][13][14][15][16] However, this treatment is not available in current clinical practice because of several associated problems, such as safety issues and immunological tolerance.
The genus Ganoderma (also called Lingzhi) is a medicinal fungal genus that includes various species, for example, G petchii, G australe and G lucidum. Ganoderma is traditionally used in China to promote health and longevity, lower the risks of cancer and heart disease, protect against liver and kidney diseases, and boost the immune system. [17][18][19][20][21] Derivatives of Ganoderma have shown a renoprotective effect in diabetic nephropathy, chronic glomerulonephritis and tubulointerstitial fibrosis. [22][23][24][25] Petchiether A (petA), a novel small-molecule meroterpenoid isolated from the fruiting body of G petchii, inhibits fibronectin in rat kidney tubular epithelial cells. 26 However, the in vivo mechanisms and functional significance of this activity remain unknown. The present study examined the effects of petA on UUO-induced obstructive kidney injury and its underlying mechanisms both in vivo and in vitro.

| 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  Figure 1D). The purity of petA was over 98%.

| 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 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.

| Morphological and immunohistochemical analysis
To examine the changes in renal morphology, we stained formalinfixed, 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 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.

| MTT assay
HK-2 cells were seeded into 96-well plates at a density of 1 × 10 5 cells/ mL in a volume of 200 μL per well and allowed to attach for 24 hours.

| 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

| 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][31][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

| 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.

| Toxicity of petA
To detect the toxicity of petA, we incubated human proximal tubu-  Figure S1).

| 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 Figure 1A). However, administration of petA (40 mg/kg), initiated either immediately after or F I G U R E 2 Petchiether A (PetA) inhibits the expression of proinflammatory cytokines and infiltration of macrophages in the obstructed kidneys of unilateral obstructive (UUO) mice. Mice receiving daily intraperitoneal injection of vehicle or petA (40 mg/kg/d) 4 d before or right after UUO operation were killed 5 d after UUO. A, interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), MCP-1 and IL-6 expression were examined by quantitative real-time PCR, as indicated. B, Immunohistochemical staining and (C) semi-quantitative analysis of F4/80 + and IL-1β expression. Data represent the mean ± SEM for 6-8 mice per group. *P < 0.05, ***P < 0.001 vs the sham group; # P < 0.05, ## P < 0.01, ### P < 0.001 vs the vehicle UUO group. Bar = 50 μmol/L. Magnification ×200. PetA Px, PetA prevention; PetA Tx, PetA treatment 4 days before the UUO procedure, markedly reduced these changes compared with those in the vehicle group ( Figure 1A (p-p65). B, Western blot and quantitative analysis of phospho-NF-κB/p65 (p-p65) protein expression. Data represent the mean ± SEM for 6-8 mice per group. **P < 0.01, ***P < 0.001 vs the sham group; # P < 0.05, ### P < 0.001 vs the vehicle UUO group. Bar = 50 μmol/L. Magnification ×200. PetA Px, PetA prevention; PetA Tx, PetA treatment F I G U R E 3 Petchiether A (PetA) inhibits the expression of fibronectin, collagen 1 and α-smooth muscle actin (α-SMA) in the obstructed kidneys at 5 d after unilateral obstructive (UUO) operation. Mice receiving daily intraperitoneal injection of vehicle or petA (40 mg/kg/d) 4 d before or right after UUO operation were killed 5 d after UUO. A, Immunohistochemical staining and quantitative analysis for the expression of α-SMA, collagen 1 and fibronectin. B, Quantitative real-time PCR analysis of α-SMA, collagen 1 and fibronectin mRNA expression. C, Western blot and semiquantitative analysis of collagen 1 and fibronectin, respectively. Data represent the mean ± SEM for 6-8 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 vs the sham group; # P < 0.05, ## P < 0.01, ### P < 0.001 vs the vehicle UUO group. Bar = 50 μmol/L. Magnification ×200. PetA Px, PetA prevention; PetA Tx, PetA treatment

| 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 upregulation 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).

| 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.

| 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

| D ISCUSS I ON
We report for the first time that petA, a novel small molecule ex- We previously demonstrated that Smad3 is a key Smad protein that mediates fibrosis in multiple organs and tissues. 3,28 Inhibition of Smad3 using specific inhibitors reduces fibrosis. [39][40][41][42] In this study, we found that petA is a potential inhibitor of TGF-β/Smad3 activation, which suggests that petA may relieve renal fibrosis as well as cirrhosis, cardiac fibrosis and lung fibrosis.
F I G U R E 5 Petchiether A (PetA) inhibits transforming growth factor-β1 (TGF-β1)/Smad3 signalling in the obstructive kidneys of unilateral obstructive (UUO) mice. Mice receiving daily intraperitoneal injection of vehicle or petA (40 mg/kg/d) 4 d before or right after UUO operation were killed 5 d after UUO. A, TGF-β1 expression was examined by immunohistochemistry and quantitative real-time PCR. Phosphorylation of Smad3 was examined by immunohistochemistry (B) and quantitative Western blot analysis (C). Data represent the mean ± SEM for 6-8 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 vs the sham group; # P < 0.05, ## P < 0.01, ### P < 0.001 vs the vehicle UUO group. Magnification ×200. PetA Px, PetA prevention; PetA Tx, PetA treatment Inflammation plays a central mechanism in the initiation and maintenance of kidney injury, and a suppressed inflammatory response reduces the extent of renal fibrosis. 43,44 Our results indicate that administering petA to prevent or treat UUO can suppress the expression of multiple proinflammatory cytokines, such as TNF-α, IL-β, MCP-1 and IL-6, and inhibit macrophage infiltration, suggesting that inhibition of the inflammatory response is also one of the mechanisms by which petA ameliorates renal fibrosis.
Furthermore, several mechanisms may contribute to the antiinflammatory effects of petA. We previously found that the activation of TGF-β/Smad3 is associated with a decreased expression of Smad7, an inhibitory Smad, in UUO, hypertensive and diabetic kidneys. 27,45,46 The decrease in Smad7 enhances NF-κB P65 phosphorylation, which consequently aggravates inflammation in the kidneys. 27,45,46 Another possible mechanism may be the overexpression of TGF-β1 in UUO kidneys ( Figure 5A). Findings from other research teams indicate that TGF-β1 activates renal tubular cells and immune system cells to produce inflammatory cytokines and further promote the inflammatory response, which, in turn, amplifies fibrosis and tubular injury. [47][48][49] The mechanisms by which petA attenuates inflammation will be explored in future studies.
In conclusion, our findings suggest that petA is a potential natural small-molecule inhibitor of TGF-β1-induced Smad3 phosphorylation. The beneficial effect of petA may occur through the inhibition of TGF-β1/Smad3 and NF-κB signalling, subsequently reducing renal fibrosis and inflammation in UUO kidneys. Our studies may provide a useful natural therapeutic agent for the prevention and treatment of renal fibrosis during the progression of fibrotic kidney disease.

ACK N OWLED G EM ENT
None.

CO N FLI C T O F I NTE R E S T
The authors have declared that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.