Catalpol attenuates EMT by inhibiting Wnt/β-catenin and TGF-β/Smads signaling to alleviate kidney fibrosis

Purpose: To determine the anti-fibrosis effect and underlying mechanism of action of catalpol (CAT) in chronic kidney disease (CKD)


INTRODUCTION
The proportion of chronic kidney disease (CKD) is increasing worldwide, with 10.8 % of Chinese adults having some degree of CKD [1].Renal fibrosis, a hallmark and common feature of all types of CKD, contributes to the deterioration of renal function ultimately resulting in end-stage renal failure (ESRD) [2].Accordingly, inhibiting the development of renal fibrosis is crucial for CKD patients and this has been taken as an effective way to delay the progression of CKD and reduce the high treatment costs paid for alternative therapies, such as renal dialysis and kidney transplantation.At present, existing drugs are unable to reverse the course of renal fibrosis; hence, it is necessary to find accessible and effective therapeutic methods to prevent renal fibrosis generation and CKD progression.
Catalpol, the first iridoid glycoside monomer isolated from Rehmannia glutinosa (Gaertn.)DC. (RG), is also the primary iridoid compound of the aqueous extracts of dried RG [3].CAT has a variety of pharmacological actions, such as renal protection against adriamycin-induced nephropathy [4], diabetic nephropathy [5] and acute kidney failure [6].Strikingly, CAT attenuated pulmonary fibrosis as well as liver fibrosis [7,8].Preliminary studies revealed that dried RG has a higher CAT content and exhibits more antifibrotic action than steamed RG.In addition, it has been shown that the anti-fibrotic potential of CAT is involved in the regulation of Wnt/β-catenin signaling in vitro [9].In this context, this study was designed to further elucidate the effect of CAT on renal fibrosis and determine its potential mechanism of action in order to provide a reference for the clinical application of CAT.

Cell culture and treatment
Human kidney proximal tubule epithelial (HK-2) cells were cultured in K-SFM supplemented with the K-SFM kit and maintained at 37 °C in a humidified atmosphere containing 5 % CO2.Cells stimulated with TGF-β1 (10 ng/mL) were co-cultured with CAT, LiCl or DKK1 for 24 h.

Animal protocol
Forty (40) male Sprague-Dawley rats (8 weeks old, 200 ± 20 g) were maintained at the animal care facility of the Chongqing Engineering Technical Research Center for the Characteristic Diagnosis and Treatment of Traditional Chinese Medicine (TCM) [10].The rats were provided standard rodent chow and water ad libitum.The study was approved by the Animal Care and Use Committee of the Chongqing Hospital of TCM (approval no.2018-DWKY-WQ) and followed international guidelines for animal studies [11].
Rat models were prepared using the unilateral ureteral obstruction (UUO) method, which was performed as described previously [12].Shamoperated rats received identical surgical procedures except that the left ureter was simply manipulated without ligation.UUO model rats were further randomly divided into 3 groups (n = 10 per group) based on the treatments: vehicle only; Low dose CAT group (100 mg/kg) and High dose CAT group (200 mg/kg).Catalpol was dissolved in purified water and administered by oral gavage from post-UUO on days 1 to 2 or 14 days.The sham group was administered vehicle only.Five rats in each group were sacrificed on day 2 after treatment and the other 5 rats were sacrificed on day 14.Kidney tissues were collected for pathological examination and Western blot analysis.

Histological analysis
Kidney samples were collected from the rats under anesthesia.The samples were fixed in 4 % paraformaldehyde, embedded in paraffin and sectioned at 5μm thickness.Hematoxylin and eosin staining (H&E staining) and Masson's trichrome staining were performed according to the standard procedure while images were acquired with confocal microscopy (Leica, Germany).

Immunohistochemical studies
After deparaffinization, the sections were incubated in 3 % H2O2 deionized water and microwaved to retrieve the antigens.Next, the paraffin sections were incubated with primary antibodies overnight at 4 °C after they were blocked with goat serum.Thereafter, they were treated with secondary antibody of streptavidin-HRP conjugation followed by diaminobenzidine (DAB) staining, hematoxylin counterstaining and gummy neutral balsam mounting.Finally, images were acquired with confocal microscopy (Leica, Germany).

Statistical analysis
All data are reported as mean ± standard deviation (SD).Statistical analyses of the data were performed using GraphPad Prism software.
Comparisons between groups were made using one-way ANOVA and statistical significance was set at p < 0.05.

CAT attenuates EMT in TGF-β1-stimulated HK-2 cells
Epithelial-mesenchymal transition (EMT) is a process characterized by both loss of epithelial traits and gain of mesenchymal characteristics in epithelial cells.This transition promotes excessive generation of fibroblast and myofibroblast and is accompanied by an increased expression of vimentin and α-SMA as well as a decreased expression of E-cadherin [13].Adding TGF-β1 stimulation to HK-2 cells is a classical model for studies related to EMT.In this study, therefore, the intensities of α-SMA and Ecadherin immunofluorescence were determined to estimate the inhibitory action of CAT on EMT induced by TGF-β1 in HK-2 cells.Compared with HK-2 cells without TGF-β1 stimulation, TGF-β1stimulated HK-2 cells had increased expression of α-SMA (p < 0.05), and decreased expression of E-cadherin (p < 0.05; Figure 1).However, compared with TGF-β1-induced HK-2 cells, CAT significantly depressed the expression of α-SMA (p < 0.05) and upregulated the expression of Ecadherin (p < 0.05) in a dose-dependent manner (Figure 1), suggesting that CAT represses EMT progression stimulated by the TGF-β1.

CAT inhibits hyperactivation of Wnt/β-catenin pathway
When HK-2 cells were stimulated with TGF-β1 and LiCl (the activator of Wnt/β-catenin signaling), the expression levels of α-SMA were found to increase significantly.In contrast, Ecadherin was found to significantly decrease (p < 0.05) when compared with the pre-treatment levels.However, CAT significantly reversed this observation in HK-2 cells (p < 0.05; Figure 2   The relative intensity of immunofluorescence evaluated using ImageJ (k-n).*P < 0.05 vs. control group, # P < 0.05 vs. TGF-β1 and LiCl-treated group A similar inhibition pattern/trend/intensity was observed between CAT treatment (50 and 100 µg/mL) and DDK1.Treatment of stimulated HK-2 cells with DDK1 presented similar inhibition as treatment with 50 and 100 µg/mL CAT (Figure 2 f -j).The above data showed that CAT had bioactivity in preventing EMT in TGF-β1-treated HK-2 cells, and the mechanism is related to neutralizing the activation of Wnt/β-catenin and TGF-β1 signaling pathways.

CAT alleviates kidney fibrosis
To confirm the antifibrotic activity of CAT on renal fibrosis in vivo, a UUO model which is a typical interstitial fibrosis model, was utilized in this study [12].The results of H&E staining demonstrated that UUO resulted in pathological changes, such as tubule simplification, swelling and vacuolization, inflammatory cell infiltration and tubular cell death by apoptosis and necrosis.The presence of mild tubular abnormalities was observed on day 2 and severe tubular injury with interstitial matrix accumulation was observed on day 14 after surgery (Figure 3 a).Furthermore, compared with the 2-day UUO rats, the 14-day UUO rats had increased tubular interstitial fibrosis with severe collagen deposition, as assessed by Masson staining (Figure 3 b).In addition, compared with the UUO group, CAT alleviated tubular injury, including tubular expansion, vacuolization and dilation, tubular interstitial fibrosis and collagen deposition in the 2-day and 14-day CAT-treated UUO groups (Figure 3 a and b).Moreover, the ameliorative effect on interstitial fibrosis and collagen deposition occurred strongly in a dose-related manner in the 2-day CAT-treated UUO group (Figure 3 b).These data indicate that CAT has a notable antifibrotic effect, especially at the early stages of kidney injury, and delays the development and progression of renal fibrosis.

CAT inhibits UUO-induced EMT
EMT is a key promoter of fibrosis development and progression [14].To study the effect of CAT on EMT in UUO rat kidneys, α-SMA and vimentin expression were determined by immunohistochemistry and Western blot.The results indicated that compared with the sham group, the UUO group had positive expression for α-SMA and vimentin, whereas treatment with CAT decreased this positive expression after surgery at 2 days and 2 weeks, in a dosedependent manner (Figure 4).These results indicate that CAT prevents UUO-induced EMT progression during chronic injury.

CAT inhibits the hyperactivation of TGFβ/Smads and Wnt/β-catenin signaling pathways
Based on the in vitro results, the expression levels of Wnt3a, p-GSK3β, β-catenin, TGF-β1 and Smad2/3 were examined by Western blot to verify further the inhibitory effects of CAT on the TGF-β/Smad and Wnt/β-catenin signaling pathways in UUO rats.Compared with the sham group, the UUO rats had upregulated expression levels of Wnt3a, p-GSK3β, β-catenin, TGF-β1 and Smad2/3 at day 2 and 14 post-injury (Figure 5 a -b), which revealed hyperactivation of TGFβ/Smad and Wnt/β-catenin signaling pathways in UUO rats.In contrast to the hyperactivation induced by UUO, CAT significantly downregulated the increased expression of Wnt3a, p-GSK3β, β-catenin, TGF-β1 and Smad2/3 in UUO rats in a dose-dependent manner at day 2 and 14 (Figure 5 c -g).
Snail1, an essential transcription factor for the EMT process, is a downstream target gene of the TGF-β/Smad and Wnt/β-catenin signaling pathways [15].Compared with the sham group, Snail1 was significantly overexpressed in the UUO group.Still, CAT administration reversed the Snail1 overexpression induced by UUO in a concentration-dependent manner at days 2 and 14, which is similar to the trend seen in the expression patterns of Wnt3a, p-GSK3β, βcatenin, TGF-β1 and Smad2/3 (Figure 5 h).Therefore, CAT blocked the aberrant activation of the TGF-β/Smad and Wnt/β-catenin signaling pathways simultaneously and then downregulated the expression of Snail1, hence preventing the development of EMT, and eventually alleviated renal fibrosis.

DISCUSSION
Renal injury leads to the production and secretion of a series of profibrotic factors such as TGF-β, Wnt and hedgehog ligands, inducing epithelial cells to transform into fibroblasts and myofibroblasts, leading to ECM accumulation, and ultimately to renal fibrosis and the progression of CKD [16].Numerous studies have demonstrated that the TGF-β and Wnt signaling pathways work synergistically in mesenchymalderived cells of different organs to accelerate fibroblast activation, proliferation and ECM accumulation [17].The TGF-β and Wnt/β-catenin signaling pathways interconnect and converge at β-catenin activation, leading to EMT transcriptional program activation.TGF-β plays a crucial role in progressive renal fibrosis.This study revealed that TGF-β1 promoted the expression of α-SMA and decreased the expression of E-cadherin in vitro.The Wnt/βcatenin signaling pathway is activated and GSK-3β is phosphorylated.Meanwhile, active βcatenin induced the activation of target gene Snail1.Notably, Snail1 is a transcription factor expressed during renal embryogenesis and is maintained in a silent state in renal epithelial cell precursors during adulthood.The reactivation of Snail1 promotes renal fibrosis [15].In this experiment, the results show that after UUO in vivo, the expression of α-SMA and vimentin and collagen deposition increased in rat kidney tissue, which indicates renal tissue fibrosis.Simultaneously, the study showed that the expression levels of key protein factors increased in the TGF-β and Wnt/β-catenin signaling pathways.Therefore, inhibiting the common target genes in these two signaling pathways would be an underlying therapeutic strategy for improving renal fibrosis.In this study, CAT effectively inhibited the EMT hyperactivated by TGF-β1 in a dose-dependent manner, alleviated tubular injury, interstitial fibrosis and collagen accumulation, and significantly downregulated the overexpression levels of α-SMA and vimentin in UUO rats at days 2 and 14 after the obstruction in a dosedependent manner.In UUO rats, CAT inhibited the increased expression levels of Wnt3a, βcatenin, p-GSK-3β and Snail1 induced by ureteral obstruction in a dose-dependent manner at 2 and 14 days, meaning that CAT prevented EMT progression and alleviated renal fibrosis by repressing the hyperactivation of Wnt/β-catenin signaling pathway induced by obstructive injury.Meanwhile, CAT downregulated the overexpression of TGF-β1 and Smad2/3 induced by obstructive injury, suggesting that CAT has excellent antifibrotic effects in UUO rats, which is also associated with deactivating the TGFβ/Smads signaling pathway.

CONCLUSION
This study reveals that catalpol has anti-fibrotic pharmacological action, effectively prevents EMT and relieves renal fibrosis.The underlying mechanisms are related to repressing the hyperactivation of the TGF-β/Smad and Wnt/βcatenin signaling pathways.Therefore, catalpol is a promising candidate for ameliorating renal fibrosis.

Figure 4 :
Figure 4: CAT delayed the progression of UUOinduced EMT.Immunohistochemistry detection of the myofibroblast and mesenchymal cell markers α-SMA (a) and vimentin (b) in the medulla from UUO rats with or without CAT treatment for 2 and 14 days (5x, 40x).Vimentin and α-SMA protein expression was determined by Western blot, with GAPDH expression as a control (c-f).UUO + LC: UUO rats treated with CAT at a low dosage (100 mg/kg), UUO + HC: UUO rats treated with CAT at a high dosage (200 mg/kg).The arrow indicates positive α-SMA and vimentin staining.The data are expressed as the means ± SD from 3 independent experiments.**P < 0.05, *p < 0.01, compared with the sham group.# P < 0.05, ## p < 0.01, compared with the UUO group.△ P < 0.05, compared with the CAT group (100 mg/kg) Activated TGF-β1 binds to cell membraneembedded signal receptors and initiates Smad signaling pathways, which induce the transcription of target gene Snail1 [18,19].