NSC 74859

Pharmacologic inhibiting STAT3 delays the progression of kidney fibrosis in hyperuricemia-induced chronic kidney disease

Jing Pan, Min Shi, Fan Guo, Liang Ma, Ping Fu
1 Kidney Research Institute, Division of Nephrology, West China Hospital of Sichuan University, Chengdu 610041, China.
2 Department of Thoracic Oncology, Cancer Center, West China Hospital of Sichuan University, Chengdu 610041, China.

Kidney fibrosis is a histological hallmark of chronic kidney disease (CKD), where hyperuricemia is a key independent risk factor. Considerable evidence indicated that STAT3 is one of the crucial signaling pathways in the progression of kidney fibrosis. Here, we investigated the pharmacological blockade of STAT3 delayed the progression of renal fibrosis in hyperuricemia-induced CKD.
Main methods:
In the study, we used the mixture of adenine and potassium oxonate to perform kidney injury and fibrosis in hyperuricemic mice, accompanied by STAT3 activation in tubular and interstitial cells.
Key findings:
Treatment with STAT3 inhibitor S3I-201 improved renal dysfunction, reduced serum uric acid level, and delayed the progression of kidney fibrosis. Furthermore, S3I-201 could suppress fibrotic signaling pathway of TGF-β/Smads, JAK/STAT and NF-κB, as well as inhibit the expression of multiple profibrogenic cytokines/chemokines in the kidneys of hyperuricemic mice.
These data suggested that STAT3 inhibition is a potent anti-fibrotic strategy in hyperuricemia-induced CKD.

1. Introduction
Hyperuricemia is a metabolic disorder that is common in patients with chronic kidney disease (CKD) and aggravated with the deterioration of kidney function [1, 2]. With the improvement of living conditions and lifestyle, the incidence of hyperuricemia is rapidly increasing worldwide [3, 4]. Increasing evidence indicated that hyperuricemia was a potential risk factor for the causation and progression of kidney disease [5, 6]. Elevated serum uric acid level could lead to renal arteriolopathy, renal inflammation, renal tubular injury, tubulointerstitial fibrosis and uric acid nephrolithiasis, ultimately leading to CKD or end-stage renal disease (ESKD) [1, 7, 8].
Signal transducer and activator of transcription 3 (STAT3) is a cytoplasmic transcription factor that modulates gene transcription in multiple tissues through relaying signals from activated plasma membrane receptors to the nucleus [9, 10]. STAT3 is transcriptionally activated by the phosphorylation of tyrosine 705 residues. Upon activation, STAT3 forms homodimers or heterodimers with other STAT proteins and enters the nucleus, where it binds to specific DNA elements to drive the target gene expression [11]. Emerging evidence suggests that STAT3 plays a critical role in the pathogenesis of chronic, fibrotic renal disease. Pang et al. observed that inhibition of STAT3 attenuated excessive deposition of extracellular matrix (ECM) and suppressed proliferation of renal interstitial fibroblasts in a murine model of the fibrotic kidney induced by UUO injury [12]. In vivo studies have shown that knockdown of STAT3 activity decreases lesion progression in diabeticglomerulopathy [13]. Enhanced expression and augmented activity of STAT3 is also reported in glomerulonephritis, HIV-associated nephropathy and polycystic kidney disease [14-16]. However, the role of STAT3 in the progression of hyperuricemia-induced CKD remained poorly understood.
S3I-201 is a STAT3 SH2 domain inhibitor identified from the National Cancer Institute (NCI) chemical libraries through structure-based virtual screening (Fig. 1A). It preferentially inhibits STAT3 DNA-binding activity and reduces STAT3 tyrosine phosphorylation [17, 18]. In this study, we established a hyperuricemic nephropathy (HN) mouse model with a mixture of adenine and potassium oxonate, a uricase inhibitor, to evaluate the therapeutic effect of S3I-201 on the progression of kidney fibrosis. Allopurinol, an established xanthine oxidase inhibitor, is the most commonly used medication in the management of hyperuricemia and was used as a positive control drug in this study [19, 20].

2. Materials and methods
2.1. Chemicals and antibodies
Adenine (A8626), potassium oxonate (156124) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Allopurinol was purchased from Shanghai xinyi Vientiane pharmaceutical Co., Ltd (Shanghai, China), and S3I-201 (S1155) was from Selleck (Shanghai, China). Antibody to p-STAT3 (ab76315), t-STAT3 (ab68153) and collagen I (ab88147) were procured from Abcam (Cambridge, MA, USA), whereas α-SMA (ET1607-53) antibody was acquired from Hangzhou HuaAn Biotechnology Co., Ltd (Hangzhou, China).

2.2. Animals and experimental protocol
All animal experiments were performed in accordance with The Guide for the Care and Use of Laboratory Animals and approved by the ethics committee for experimental research, Animal Care and Use Ethics Committee of Sichuan University in China. Eight-week-old male C57BL/6J mice weighing 25-28 g were acquired from HuaFuKang biological Technology Co Ltd (Beijing, China). The mice were housed in a controlled room of light (12:12-h light-dark cycle), temperature (23±2°C), and free access to food and water. After adapting to the environment for one week, the animals were randomly divided into two groups: Model 14DAY group were orally administered by gavage adenine (160 mg/kg) and potassium oxonate (2400 mg/kg) [21], and Control 14DAY group were given to the same volume of distilled water (Thermo Fisher Scientific, MA, USA) once daily for 14 consecutive days. Then, part of mice (n=5) in the Control 14DAY and Model 14DAY group were euthanized and the kidneys were collected for protein analysis and histologic examination. The remaining mice in the Model 14DAY group were further randomly divided into threesubgroups (n≥6/each group): Model 30DAY, Allopurinol and S3I-201 group. The mice in Model 30DAY group were given a mixture of adenine (160 mg/kg) and potassium oxonate (2400 mg/kg) by gavage once every two days. The other twogroups received 10 mg/kg allopurinol (per os, P.O, dissolved in distilled water) and 10 mg/kg S3I-201 (intraperitoneal injection, I.P, dissolved in dimethyl sulfoxide) daily together with adenine and potassium oxonate for the next 16 days, respectively. The remaining mice in Control 14DAY group were raised for another 16 days and thennamed Control 30DAY (Fig. 1B).

2.3. Assessment of serum and urine biochemistry indices
Murine blood samples were collected via cardiac puncture into the tubes without any anticoagulant, allowed to clot for 30 min at room temperature, and centrifuged at 3000 rpm for 15 min to obtain the serum. The mice were put into the metabolic cage (Techniplast, Italy) to collect 24-h urine samples before sacrifice. Urine was then centrifuged at 800×g for 10 min to remove the particulate contaminants. Serum uric acid, creatinine, and urine microalbumin were detected by an automatic biochemical analyzer (BS-240, Mindrary, Shengzhen, China).

2.4. Western blotting
The kidney tissues were homogenized in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing 1‰ proteinase inhibitors (Keygen Biotech, Nanjing, China) using a Polytron homogenizer. The homogenate was centrifuged at 13000 rpm for 15 min at 4°C and the protein concentration in supernatant was quantified by a BCA protein assay kit (Thermo Fisher Scientific, MA, USA). Then, 30µg of protein were resolved by 10-12% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto a 0.2um polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked in 5% nonfat milk for 1h at room temperature and incubated with primary antibodies overnight at 4°C. Thereafter, the membranes were incubated with appropriate secondary antibodies (HuaAn Biotechnology, Hangzhou, China) at room temperature for 2h and imaged using Bio-Rad ChemiDoc MP Imaging System (Bio-RadLaboratories Inc., Hercules, CA, USA). Secondary antibodies included Goat anti-Mouse IgG-HRP antibody (HA1006), Goat anti-Rabbit IgG-HRP antibody (HA1001).

2.5. Histology and Immunohistochemistry examination
Kidney tissues were fixed with 10% neutral buffered formalin, embedded in paraffin, and 4-μm-thicksections were stained with periodic acid-Schiff (PAS) and Masson’s trichrome (MASSON). Immunohistochemical analyses were performed using p-STAT3 (ab76315, Abcam, MA, USA), on the basis of the procedure described in our previous study [22]. The histopathological damage of kidney tissue was scored as follows: More than five random low power microscopic field (×100 or×200) scores for one mouse kidney were calculated and averaged. To measure the extent of tubular injury, <10%, 11-25%, 26-75%, >75% were scored as 1, 2, 3, or 4 according to the degree and extent of epithelial necrosis, luminal necrotic debris, and tubular dilation; Areas of positive staining for p-STAT3 and renal interstitial fibrosis were quantitatively calculated by Image J program (National Institutes of Health, Bethesda, MD, USA).

2.6. RNA-seq transcriptomic assay
We performed RNA-seq by collecting whole kidneys from Control 30DAY group, Model 30DAY group and S3I-201 group (n = 5 in each group). Total RNA from whole kidneys was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA). The purity and integrity of the RNA was estimated by spectrophotometry NanoPhotometer (Implen, Munich, Germany) and using the Bioanalyzer 2100(Agilent Technologies Inc., Santa Clara, CA, USA), respectively. Sequencing libraries were constructed using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. After size selection and quantification, libraries were sequenced using Illumina HiSeq xten/NovaSeq 6000 sequencer (2 × 150 bp read length) at Beijing Novogene Technology Co. Ltd. (Beijing, China) and the data were analyzed on the free online platform of Novogene Cloud Platform (www.novogene.com). Differentially expressed genes (DEGs) were analyzed using the DESeq2 R package. DEGs were screened using padj ≤  0.05. The ClusterProfiler software package was used to conduct gene ontology (GO) enrichment analysis of differentially expressed genes. GO terms with padj ≤ 0.05 were considered as significantly enriched.

2.7. Statistical analysis
Quantitative data are presented as the mean ± SD for each group. One-way or two-way analysis of variance was used for comparison between groups. All values were considered to indicate a statistically significant if p < 0.05. 3. Results 3.1. STAT3 activation in kidneys of hyperuricemic mice To investigate whether the activation of STAT3 during the progression of renal fibrosis, we firstly analyzed the p-STAT3 expression in kidney tissues of hyperuricemic mice at two diff erent time points (Fig. 1C). The expression of p-STAT3 protein in Control 14DAY group was low but significantly upregulated inModel 14DAY group (p < 0.0001). The 30 days after potassium oxonate/adenine challenge, the phosphorylated protein of STAT3 expression was still observed in the kidney and was more remarkable than that of day 14 (p < 0.0001)( Fig. 1D). The same changes occurred in STAT3 total protein (Fig. 1E). We next investigated the tubular injury and renal fibrosis of hyperuricemic mice. As shown in Fig. 2A, PAS staining revealed that kidney tissues developed severe tubular dilation and atrophy after 14 days of modeling, when compared with that of Control 14DAY group (p < 0.0001). The tubular injury is persistent till 30 days (Fig. 2B). Masson staining of hyperuricemic mice showed that renal interstitial fibrosis was being existed on day 14 (p < 0.0001) and further aggravated on day 30 (p < 0.0001), which was demonstrated by the quantification analysis of Masson trichrome-positive areas (Fig. 2C). Moreover, the expression of collagen I and α-SMA were also increased in kidneys of hyperuricemic mice on day 14 and more on day 30, shown by Western blot analysis (Fig. 2F-G). We also examined the change of serum creatinine and serum uric acid level of hyperuricemic mice. As compared with control group, continuous oral administration of adenine and potassium oxonate induced a markedly increase serum creatinine and serum uric acid at day 14 and 30 (Fig. 2D, E). 3.2. S3I-201 inhibited the STAT3 phosphorylation in the kidneys of HN mice To further understand the role of STAT3, we examined the effect of S3I-201 onthe expression of p-STAT3 in the kidneys of hyperuricemic mice. As shown in Fig. 3, daily administration of S3I-201 for 16 days largely suppressed the STAT3 phosphorylation in tubular and interstitial cells (p < 0.01, Fig. 3A, B). The result was further confirmed by Western blot of p-STAT3 protein (p < 0.001, Fig. 3C, D). Total STAT3 expression was not affected by the treatment compared to hyperuricemic mice (Fig. 3C, E). These data suggested that hyperuricemia-related CKD induced STAT3 activation and upregulated total STAT3 protein, and S3I-201 is an effective inhibitor of STAT3 without changing STAT3 protein level. 3.3. S3I-201 ameliorated hyperuricemia and renal dysfunction in HN mice As shown in Fig. 4A, serum uric acid concentration was increased by oral administration of adenine and potassium oxonate for 30 days when compared with that of control mice (192.2±39.9 μmol/L of Control 30DAY vs. 225.8±10.4 μmol/L of Model 30DAY, p < 0.05). Treatment with S3I-201 for 16 days could markedly reduce serum uric acid level of hyperuricemia-induced CKD to be lower than that of Model 30DAY group (186.2±7.5 μmol/L, p < 0.01). Allopurinol at 10 mg/kg, as a positive control, had better effect on serum uric acid reduction and even lower than that of Control 30DAY group. We also detected serum creatinine and urine microalbumin to evaluate renal function of mice. As shown in Fig. 4B, serum creatinine of HN mice were significantly elevated compared with that of Control 30DAY group (17.9±1.6 μmol/L of Control 30DAY vs. 42.3±5.0 μmol/L of HN mice, p<0.0001), which was effectively attenuated by S3I-201 at 10 mg/kg to 33.7±5.0 μmol/L (p < 0.01). Allopurinol for 16 consecutive days had no effect on serum creatinine reduction, even higher than that of Model 30DAY group (48.9±5.1 μmol/L, p< 0.05). In addition, potassium oxonate/adenine induced a remarkable increase in urine microalbumin ofmice compared with that of Control 30DAY group (181.2±31.7 mg/L of Control 30DAY vs. 257.9±19.7 mg/L of HN mice, p < 0.01). S3I-201 decreased the levels of urine microalbumin in hyperuricemic mice, but there was no statistical difference (215.7±16.0 mg/L) (Fig. 4C). 3.4. S3I-201 ameliorated histopathological damage and suppressed tubulointerstitial fibrosis of HN mice PAS staining was performed to observe histological changes in the kidneys (Fig. 5A). Compared with Control 30DAY mice, the kidneys of hyperuricemic mice developed severe glomerulosclerosis and tubulointerstitial damage with tubular atrophy and dilatation (p <0.0001). S3I-201 administration preserved kidney architecture and attenuated tubular damage (p<0.01), which was indicated by the quantification of tubular score (Fig. 5B). To test the effect of S3I-201 on delaying hyperuricemia-induced renal fibrosis, collagen I and α-SMA protein were evaluated in response to 30 days of modeling. As indicated in Fig. 6A, α-SMA and collagen I was barely detected in the kidneys of Control 30DAY mice. Hyperuricemia resulted in a marked increase in their corresponding expression (p<0.0001). Administration of S3I-201 dramatically decreased the expression of α-SMA and collagen I (p<0.01), which was observed at 16 days of treatment (Fig. 6B, C). Similarly, an increase in Masson’s trichrome positive areas was observed within the tubulointerstitium in the kidneys of hyperuricemic mice (p<0.0001). Quantification analysis of Masson-stained kidneysshowed that S3I-201 treatment significantly reduced such areas (p<0.01)( Fig. 6D, E). However, allopurinol had no inhibitory effect on renal fibrosis induced by hyperuricemia, even if it could effectively reduce serum uric acid level (Fig. 4A). 3.5. Identification of the potential STAT3 target genes and signaling pathways involved in hyperuricemia-induced renal fibrosis Compared with the Control 30DAY group, 4950 transcripts were significantly increased in the kidney of Model 14Day group, and 6294 transcripts were significantly increased in the Model 30Day group. Among them, a total of 4380 transcripts were up-regulated in both 14days and 30days Model group. Treatment with S3I-201 downregulated 621 of them (Fig. 7A). Similarly, among the 4077 downregulated genes under hyperuricemia condition, 407 were upregulated by S3I-201 (Fig. 7B). In Figure 7C, the significantly enriched gene ontology (GO) functions of a total number of 1028 potential STAT3 regulatory genes included extracellular matrix structural constituent, extracellular matrix binding, platelet-derived growth factor binding, fibronectin binding and MHC protein complex. Because STAT3 is primarily a transcriptional activator that drives the transcription of genes in response to diverse extracellular signaling agents, we decided to focus our analysis on the 621 genes that were elevated in the Model group and decreased in the S3I-201 group. The GO term analysis of 621 genes in Fig. 7D showed that in the cellular components category, the top three enrichments were external side of plasma membrane, extracellular matrix, and extracellular matrix component; In the molecular function category were cell adhesion molecule binding, cytokine binding, and fibronectin binding. Inflammation is the initial and key step of uric acid-induced renal fibrosis, so we further explored the role of STAT3-mediated proinflammatory cytokines/chemokines in kidneys by RNA-seq transcriptomic profiling (Fig. 7E). Our results revealed that the expression of TNF-α, IL-1β, IL-6, IL-18, ICAM-1, CRP and CCL5 were significantly upregulated in renal tissues of both 14days and 30days Model mice, and treatment with S2I-201 downregulated their corresponding expression. We also identify the potential regulatory signaling pathway of STAT3 that may mediate kidney fibrosis. As shown in Fig. 7C, the heatmap showed that JAK/STAT, TGF-β/Smads and NF-κB signaling pathways were the potential mechanisms of S3I-201 delaying the progression of renal fibrosis in hyperuricemic mice. 4. Discussion STAT3 belongs to the signal transducer and activator of transcription family and has key roles in physiological functions including cell growth, differentiation, immunity and inflammatory response [11]. STAT3 has been reported to be activated in different compartments of damaged kidney, including podocyte, mesangial cell, tubular cell and interstitial fibroblast, and was involved in the process of renal fibrosis and inflammation [23-25]. However, little is known about the functional role of STAT3 in hyperuricemia-induced CKD. In this study, we identified the crucial role of STAT3 in the pathogenesis of hyperuricemia-induced renal fibrosis. Firstly, we detected the expression of STAT3 in renal tissue of hyperuricemic mice at different time points. It was observed that hyperuricemia induced STAT3 phosphorylation at tyrosine 705 in kidneys as early as day 14 and reaching a higher level at day 30, indicating that STAT3 activation gradually increased during the progression of renal fibrosis. Furthermore, treatment with S3I-201 for 16 days slowed down renal fibrosis, attenuated renal injury and suppressed multiple proinflammatory cytokine production in kidneys of hyperuricemic mice. Blockage of STAT3 by inhibitor S2I-201 also suppressed the activation of JAK/STAT, TGF-β/Smads and NF-κB signaling pathways in kidneys of hyperuricemic mice, supporting the idea that pathways involving STAT3 are critical for hyperuricemia-induced CKD. TGF-β1/Smads signaling plays a major role in renal interstitial fibrosis [26, 27]. TGF-β1 plays its biological role by binding to TGF-β receptor and subsequent activation of downstream Smad molecules [28]. STAT3, as a transcription factor, has been reported to induce the expression of a multitude of genes, including TGF-β1 [12]. Our results showed that the expression of TGF-β1 was upregulated in kidneys of hyperuricemic mice, and S3I-201 treatment suppressed its corresponding expression. Meanwhile, the expression of other molecules related to the TGF-β1/Smads signaling pathway, such as TβRII, Smad3, and Smad4, also significantly differed in hyperuricemic mice compared with that of Control 30DAY group. These results indicated that S3I-201-elicited inhibition of renal fibrosis is likely mediated by antagonizing TGF-β1/Smads signaling. Accumulation of inflammatory cells and proinflammatory cytokine/chemokine production were closely associated with the progression of hyperuricemia-induced renal fibrosis [29, 30]. In the current study, we observed that administration of S3I-201 could markedly reduce the expression of IL-1β, IL-6, IL-18, TNF-α and CCL5 in the kidneys of hyperuricemic mice. Moreover, S3I-201 also down-regulated renal C-reactive protein (CRP) expression. CRP is commonly used to assess inflammation and also plays a role in hyperuricemia-induced CKD. It has been reported that anti-C-reactive protein (CRP) treatment of human umbilical vein endothelial cells (HUVECs) could reverse the reduction of nitric oxide production induced by uric acid (UA), suggesting that CRP expression may be involved in UA-induced vascular remodeling [31]. Activation of NF-κB signaling pathway iscritically involved in the inflammatory response [32], and the activation and interaction between STAT3 and NF-κB have been observed [33]. We found that STAT3 inactivation inhibited NF-κB signaling, however, the exact mechanism has not yet been explored in our experiments. These results suggest that inhibition of inflammatory response may be another mechanism by which S3I-201 attenuates renal fibrosis. Allopurinol is a first-line drug for the treatment of hyperuricemia, which effectively improved the symptoms of hyperuricemia [34]. However, allopurinol is cleared through the kidney, so patients with CKD may have an increased risk of allopurinol toxicity [35]. Many studies have investigated the effects of allopurinol on renal function [36, 37], but more large-scale studies are needed to verify its efficacy and safety. In addition, there are few data for the effect of allopurinol on the progression of ESRD. It was reported that allopurinol treatment had no effect on estimated glomerular filtration rate (eGFR) in patients with stage 3 or 4 CKD [38]. In our study, allopurinol, as a positive control, significantly reduced serum uric acid of hyperuricemic mice, but had no effect on inhibiting STAT3 activation, improving renal function and could not delay the progression of renal fibrosis. Therefore, it is imperative to find therapeutic strategies for hyperuricaemia-associated CKD. Many small-molecule compounds that directly inhibited the activity and function of STAT3 have been used in preclinical and clinical studies [39]. S3I-201 (NSC 74859) selectively inhibited STAT3 dimerization and STAT3 DNA-binding, and has been reported to prevent kidney fibrosis and nephropathy in STZ-induced diabeticmice [40]. S3I-201 also could attenuate angiotensin II-induced renal fibrosis and dysfunction [41]. Furthermore, S3I-201 could also ameliorate the fibrosis of peritoneum, liver and heart in different animal models [42-44]. These observations showed that S3I-201 may have a beneficial effect on hyperuricemia-induced renal insufficiency, and its therapeutic potential and associated anti-fibrosis mechanism have been investigated in our current experiment. 5. Conclusion In summary, our results firstly reported that STAT3 activation was an important molecular basis of hyperuricemia-induced renal fibrosis. 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