Part 1:Anti-fibrotic Effect Of 6-Bromo-indirubin-3 ′ -oxime (6-BIO) Via Regulation Of Activator Protein-1 (AP-1) And Specificity Protein-1 (SP-1) Transcription Factors in Kidney Cells

Anti-fibrotic effect of 6-Bromo-indirubin-3 ′ -oxime (6-BIO) via regulation of activator protein-1 (AP-1) and specificity protein-1 (SP-1) transcription factors in kidney cells

For more information:ali.ma@wecistanche.com

Keywords: kidney, Fibrosis, SMAD, Anti-fibrotic

ABSTRACT

PAI-1 and CTGF are overexpressed in kidney diseases and cause fibrosis of the lungs, liver, and kidneys. We used a rat model of unilateral ureteral obstruction (UUO) to investigate whether 6-BIO, a glycogen synthase kinase-3β inhibitor, attenuated fibrosis by inhibiting PAI-1 and CTGF in vivo. Additionally, TGFβ-induced cellular fibrosis was observed in vitro using the human kidney proximal tubular epithelial cells (HK-2), and rat interstitial fibroblasts (NRK49F). Expression of fibrosis-related proteins and signaling molecules such as PAI-1, CTGF, TGFβ, α SMA, SMAD, and MAPK were determined in HK-2 and NRK49F cells using immunoblotting. To identify the transcription factors that regulate the expression of PAI-1 and CTGF, the promoter activities of AP-1 and SP-1 were analyzed using luciferase assays. Confocal microscopy was used to observe the co-localization of AP-1 and SP-1 to PAI-1 and CTGF. Expression of PAI-1, CTGF, TGFβ, and α -SMA increased in the UUO model as well as in TGFβ-treated HK-2 and NRK49F cells. Furthermore, UUO and TGFβ treatment induced the activation of P-SMAD2/3, SMAD4, P-ERK 1/2, P-P38, and P-JNK MAPK signaling pathways. PAI-1, CTGF, AP-1, and SP-1 promoter activity increased in response to TGFβ treatment. However, treatment with 6-BIO decreased the expression of proteins and signaling pathways associated with fibrosis in the UUO model as well as in TGFβ-treated HK-2 and NRK49F cells. Moreover, 6-BIO treatment attenuated the expression of PAI-1 and CTGF as well as the promoter activities of AP-1 and SP-1, thereby regulating the SMAD and MAPK signaling pathways, and subsequently exerting anti-fibrotic effects on kidney cells.

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1. Introduction

Kidney fibrosis is caused by various factors, including diabetes, high blood pressure, glomerulonephritis, and age-related chronic kidney disease [1–3]. Glomeruli sclerosis and tubulointerstitial fibrosis are the final pathological features of kidney diseases [4,5]. Excessive production and deposition of extracellular matrix (ECM) proteins cause structural and functional damage to the kidneys, resulting in a gradual loss of its function that culminates in end-stage kidney disease [5]. The kidneys have a complex structure that is divided into glomeruli, tubulointerstitial, and vasculature; each compartment has a different type of extracellular matrix and can be affected by fibrosis [6,7]. The ECM of the interstitial tubular cells contains various components including collagen, glycosaminoglycan, elastin, laminin, and glycoproteins. Activated proximal tubular cells can activate the tubulointerstitial [6–10]. Glycogen synthase kinase (GSK3) promotes kidney fibrosis by activating TGF-β signaling in proximal tubular and tubulointerstitial cells [11]. In this study, we aimed to determine the effects of the GSK3 inhibitor, 6-BIO, on proximal tubular and interstitial fibroblast cells by targeting plasminogen activator inhibitor-1 (PAI-1) and connective tissue growth factor (CTGF), which play a pivotal role in progressive kidney damage and tubulointerstitial fibrosis.

6-BIO is a cell-permeable derivative of an indirubin analog, 6-bro-indirubin, which is isolated from marine mollusks and has high specificity and selectivity for GSK-3 [12,13]. 6-BIO is a competitive inhibitor of ATP and exhibits similarity to the ATP binding domain of the cyclin-dependent protein kinase (CDK) that regulates anti-tumor activity [14–16]. Abnormal GSK3 activity is associated with several human diseases, including diabetes, inflammation, neurodegenerative diseases, and psychosis [17,18]. It has been demonstrated in previous studies that GSK3 inhibitors can repair damaged tubules and reduce inflammatory responses, epithelial-mesenchymal transition (EMT) associated with TGFβ1 signaling, and fibronectin accumulation in cultured glomerular intermediate cells [11,19–21]. In addition, 6-BIO may exert potential therapeutic effects in several diseases, including diabetes, neurodegenerative disorders, and cancer by regulating inflammation, oxidative stress, cell survival as well as proliferation, and cell death [22–24]. However, the potential therapeutic effects of 6-BIO on kidney fibrosis associated with the proximal tubular and tubular interstitial damage are still unknown.

PAI-1, a multifunctional glycoprotein in the kidneys, is a serine protease inhibitor, which inhibits urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) that breaks down plasminogen into plasmin [25,26]. Overexpression of PAI-1 inhibits fibrinolysis and degradation of ECM, thereby increasing ECM accumulation [27]. In addition, PAI-1 upregulates the MAPK (mitogen-activated protein kinases), SMAD, and RhoA/ROCK (Rho-associated protein kinase) signaling pathways in diabetes and chronic kidney disease [28–30]. Transcription factors, such as AP-1, SP-1, SMAD 3/4, USF2 (upstream transcription factor 2), and CTF/NF (family of site-specific DNA-binding

proteins), are also activated by MAPK and SMAD signaling [30,31]. The combined activation of these signaling pathways and transcription factors increases the expression of PAI-1 and leads to the accumulation of ECM. CTGF is an ECM-related heparin-binding protein and includes domains, such as insulin-like growth factor binding protein (IGFBP), the von Willebrand type C repeats (VWC), thrombospondin type 1 repeat (TSR), and a C-terminal domain (CT) with a cysteine knot motif. Of these four domains, the CT domain interacts with the TGFβ superfamily, fibronectin, and perlecan [32–34]. Consequently, CTGF increases MAPK, SMAD, PI3K, and YAP signaling by inducing TGFβ, and these signaling pathways activate transcription factors, such as AP-1, SP-1, NF-kB, and SMAD 2/3. Thus, upregulation of CTGF induces migration, proliferation, and inflammation of cells, thereby causing ECM accumulation [35–37].

Based on these results of earlier studies, we speculated that MAPK and SMAD signaling, as well as AP-1 and SP-1 transcription factors, maybe the common links between PAI-1 and CTGF-induced ECM accumulation in progressive kidney disease and tubulointerstitial fibrosis. Thus, we investigated whether 6-BIO could prevent chronic progressive kidney disease by regulating PAI-1 and CTGF.

2. Material and methods

2.1. Antibodies and reagents

The primary antibodies used were anti-rabbit antibodies against PAI-1 (11907), TGFβ (3711), phosphorylated SMAD 2/3 (8828), SMAD 4 (38454), phosphorylated ERK (P-ERK) (9101), phosphorylated P38 (P-P38) (4631), phosphorylated JNK (P-JNK) (9251), phosphorylated C-JUN (P-C-JUN) (3270), phosphorylated C-FOS (P-C-FOS) (5348), and histone H3 (9715), all of which were obtained from Cell Signaling Technology. CTGF (sc-365970), SP-1 (sc-17824), SMAD 6 (sc-25321) were obtained from Santa Cruz Biotechnology. Mouse β-actin antibodies (a5316) and (2’Z,3’E)-6-Bromo indirubin-3-oxime (6-BIO) (B1686) were obtained from Sigma-Aldrich.

2.2. Animal experiments

All animal experiments were performed following relevant guidelines and regulations. The experimental protocol was approved by the Animal Care Regulations (ACR) Committee of Chonnam National University Medical School (CNUH IACUC-18010, April 19, 2019). Experiments were performed using male Sprague-Dawley rats (180–200 g, Samtako, Korea) [38]. Rats were kept under controlled temperature (21 ± 2 ◦ C) in a 12 h light-dark cycle. The rats were divided into three groups: control (n = 6), unilateral ureteral obstruction (UUO, n = 6), and UUO with 6-BIO treatment (n = 6). 6-BIO (0.2 mg/kg/day) was injected intraperitoneally (i.p.) for 6 days. To develop an obstructive nephropathy rat model, the operation was performed as follows: UUO was induced by ligating the left ureter for seven days. The abdominal cavity was opened and 2–0 silk ligature was placed at the left proximal ureter. The control group received the same treatment for seven days, except for the ligature. The rats had free access to standard rat feed and tap water and were sacrificed on day 7 after the operation. The kidneys were rapidly removed and processed for immunohistochemistry and western blotting.

2.3. Histology

Kidney tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5 µm-thick sections. Hematoxylin and eosin (H&E), Periodic acid-Schiff (PAS), Sirius red, and Masson’s trichrome (MT) staining procedures were performed to assess the histological morphology. H&E, PAS, Sirius red staining were performed according to the manufacturer’s instructions. For MT staining, after deparaffinization with xylene, the sections were treated with Bouin’s solution at 56 ◦ C for 30 min and washed under running tap water until the sections were clear. The sections were subsequently stained with Weigert’s hematoxylin (A: B = 1:1), followed by staining with Biebrich Scarlet/Acid Fuchsin solution for 10 min and washing with distilled water. The sections were incubated with phosphotungstic acid/phosphomolybdic acid solution for 10 min and treated with Aniline Blue solution for 15 min. The sections were subsequently incubated with acetic acid for 1 min and dehydrated with ethanol and xylene. Collagen depositions, nuclei, and muscle fibers were stained blue, black, and red, respectively. The quantitative analysis of the stained section was performed using ImageJ software.

2.4. Western blot analysis

The cells were harvested, washed twice with ice-cold phosphate-buffered saline (PBS), resuspended in lysis buffer, and sonicated briefly. Protein extraction buffer consisted of 50 mM Tris-HCl (pH 7.2), 5 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, protease inhibitor cocktail (GenDEPOT, P3100–001), and phosphatase inhibitor cocktail

(GenDEPOT, P3200–001). After centrifugation, supernatants containing the protein extracts were collected and the protein concentrations were measured using a Pierce® BCA Protein Assay Kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Proteins were separated on 12% sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose membranes. The blots were blocked at room temperature for 1 h with 5% skim milk in PBS containing 0.1% Tween-20 (PBST). The blot was then incubated overnight with the primary antibody (1;2000) at 4 ◦ C, followed by incubation with the secondary antibody (1:2500), and finally with anti-rabbit horseradish peroxidase-conjugated antibodies, as described previously [39]. Specific protein bands were visualized using an enhanced chemiluminescence system. Quantitative analysis of

band intensity was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

2.5. siRNA knockdown

For knock-down of SP-1, SP-1 siRNA (Santa Cruz Biotechnology, Inc., sc-29487) and control siRNA (Santa Cruz, sc-37007) were transfected for 24 h into HK-2 and NRK49F cells at 60% confluence using Dhar-maFECT 1 transfection reagent (Dharmacon, T-2001–02) at a final concentration of 30 nM. Cells were incubated in serum-free medium for one day in a 37 ^◦ C incubator under a humidified 5% CO 2 atmosphere, pretreated with 6-BIO for 1 h, and stimulated with TGF-β for 24 h. The efficiency of knockdown was determined by western blot analysis.

2.6. Transient transfection of the plasmid and reporter construct

Cells were grown until 60–70% confluent, washed with DMEM-F-12 (HK-2 cells) or DMEM (NRK49F cells) media, and then incubated for 24 h in respective media without serum and antibiotics. The cells were then transfected with dominant-negative C-JUN construct, as well as PAI-1, CTGF, AP-1, and SP-1 reporter carrying pGL3 vector using FuGene HD reagent. The concentration of the reporter plasmid used in transient transfection was 2 μ g (1 μ g/ μ L). Reporter-transfected cells were pre-treated with 6-BIO for 1 h and incubated with 10 ng/mL TGFβ for 16 h.

2.7. Promotor reporter assay

The PAI-1 and CTGF reporter constructs were produced by BIO FACT (Yuseong-gu, Daejeon, Republic of Korea). The pGL3-AP-1 and pGL3-SP-1 reporter constructs were kindly provided by Professor Young Do Jung (Chonnam National University). Transcriptional regulation of PAI-1, CTGF, AP-1, and SP-1 was investigated by transient transfection of their respective pGL3 promoter-luciferase reporter constructs. HK-2 and NRK49F cells (5 × 10 5 ) were seeded and grown until 60–70% confluence was reached. Then, the promoter constructs and pGL3-empty vectors were transfected into the cells using FuGene HD, according to the manufacturer’s protocol. The pRL-null plasmid encoding Renilla luciferase was included in all the samples to monitor the transfection efficiency. At 24 h post-transfection, the levels of firefly and Renilla luciferase activities were measured sequentially from a single sample using the Dual-Glo Luciferase assay system (Promega). Firefly luciferase activity was normalized to Renilla activity and the relative amount of luciferase activity in the untreated cells.

2.8. Confocal laser microscopy

Rat kidney samples were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 3- μ m-thick sections. Kidney sections were deparaffinized and rehydrated, blocked, washed with PBS, and incubated with anti-PAI-1, anti-CTGF, anti-P-C-JUN, anti-P-C-FOS, anti-SP-1, and anti-AQP1 antibodies diluted in blocking buffer, for 24 h at

4 ^◦ C. After washing, the sections were incubated with an Alexa Fluor 488 (green fluorescence) goat anti-rabbit (Life Technologies, Carlsbad, CA, USA), Cy3 (red fluorescence) goat anti-mouse, or goat anti-rabbit (Invitrogen, Oregon) antibodies for 2 h. HK-2 and NRK49F cells (3 ×10 5 ) were seeded in 4-well chambers and incubated for 24 h at 37 ◦ C. Cells were pretreated with 5 μ M 6-BIO for 1 h and then exposed to 10 ng/mL TGFβ for 16 h. After washing with 1X PBS, cells were fixed with 4% paraformaldehyde at room temperature for 15 min and blocked for 2 h at room temperature with 1X PBS containing 1% BSA and 0.2% Triton X-100. The cells were then incubated with rabbit or mouse monoclonal antibodies against PAI-1, CTGF, P-C-JUN, P-C-FOS, and SP-1 (diluted 1:200 in blocking buffer) at 4 ℃ for 24 h. After washing, cells were incubated with respective secondary antibodies (Cy3 goat anti-rabbit and anti-mouse antibodies, Alexa Fluor 488 goat anti-rabbit and anti-mouse antibodies, diluted 1:200) for 2 h at room temperature. After washing, coverslips were mounted onto micro slides using ProLong Gold Antifade Reagent with DAPI (Life Technologies Corporation). Images were analyzed under the confocal laser-scanning microscope Leica TCS SP8 (Leica Microsystems, Mannheim, Germany).

2.9. Statistical analysis

Results were expressed as the mean ± SEM. Multiple comparisons among the groups were performed by one-way ANOVA and Tukey’s honestly significant difference (HSD) post hoc test. Results with p < 0.05 were considered statistically significant.

3. Results

3.1. 6-BIO attenuates morphological changes and the expression of fibrosis-associated markers in the UUO rat model

We evaluated the effects of 6-BIO on fibrosis in the kidneys of a UUO rat model. As shown in Fig. 1A, the UUO model exhibited markedly increased kidney tubulointerstitial damage, glomerular sclerosis, interstitial infiltration of mononuclear cells, deposition of interstitial collagen, and fibrosis compared with the controls, as determined by H&E, PAS, Sirius Red, and MT staining. In addition, the EMT-related proteins that induce kidney fibrosis as well as Collagen I and -IV that increase ECM accumulation were increased in UUO compared to the controls (Fig. S1A and C). However, these changes were attenuated by the 6-BIO treatment. Fig. 1B shows that after 7 days of obstruction, the expression of PAI-1, CTGF, α -SMA, and TGFβ proteins increased in the kidneys of UUO model rats as compared to that in controls, and this increase was attenuated by treatment with 6-BIO. The expression of fibrotic markers increased markedly in UUO compared with controls, which was counteracted by the 6-BIO treatment (Fig. 1C). Immunofluorescence staining (Fig. 1D) revealed that the expression of PAI-1 and CTGF (red fluorescence) increased in the obstructed kidneys at day-7 compared with the controls. However, the expression of PAI-1 and CTGF decreased upon 6-BIO treatment (Fig. 1D). Collectively, these findings suggest that 6-BIO attenuates tubulointerstitial damage by inhibiting PAI-1 and CTGF expression.

3.2. 6-BIO attenuates the expression of P-C-JUN, P-C-FOS, SP-1, and inhibits fibrosis-associated signaling pathways in the UUO rat model

Next, we determined whether 6-BIO affects the signaling pathways related to kidney fibrosis. First, we confirmed that 6-BIO, which is a known inhibitor of GSK3β, inhibits GSK3β phosphorylation. Fig. S1A and B show the reduction of GSK3β by 6-BIO. Fig. 2A shows the TGFβ/SMAD signaling pathway. Western blot analysis showed that the expression of phosphorylated SMAD 2/3 and SMAD 4 proteins increased in UUO kidneys, which was attenuated by the 6-BIO treatment (Fig. 2A). However, SMAD 6, which inhibits the phosphorylation of SMAD 2 and the activity of SMAD 4 [40], had opposite results. In addition, MAPK signaling plays a key role in kidney fibrosis [41,42]. We performed western blot analysis to determine whether 6-BIO affects MAPK signaling. Levels of phosphorylated ERK, JNK, and P38 increased in UUO kidneys compared to those in control kidneys (Fig. 2B). The expression of phosphorylated ERK and JNK was significantly reduced by the 6-BIO treatment, while the decrease in phosphorylated p38 level was negligible (Fig. 2B). SMAD and MAPK signaling pathways were upregulated in the UUO group, which was reversed by the 6-BIO treatment (Fig. 2C). Many transcription factors are involved in kidney fibrosis and play key roles in regulating downstream gene expression. Fig. 2D shows that after 7 days of obstruction, the expression of P-C-JUN, P-C-FOS, and SP-1 proteins increased in the kidneys of rats with UUO compared to that in controls, and this increase was attenuated by 6-BIO treatment. Similarly, the expression of AP-1 and SP-1, the important transcription factors regulating kidney fibrosis-related genes, was markedly increased in kidneys of rats with UUO but was attenuated upon treatment with 6-BIO (Fig. 2E). Fig. 2F shows the immunofluorescence staining of P-C-JUN, P-C-FOS. and SP-1. The red fluorescence of P-C-JUN, P-C-FOS, and SP-1 staining was significantly increased in the obstructed kidneys, compared to the controls, and was decreased by the 6-BIO treatment. These results confirm that AP-1 and SP-1 are important transcription factors involved in kidney fibrosis, and 6-BIO may decrease kidney fibrosis by attenuating their transcriptional activity.

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