Inhibition of STAT3 activation mediated by toll‐like receptor 4 attenuates angiotensin II‐induced renal fibrosis and dysfunction

Abstract

Background and Purpose

Hypertension adversely affects the kidney and is the second leading cause of kidney failure. Overproduction of angiotensin II greatly contributes to the progression of hypertensive kidney disease. Angiotensin II has recently been shown to activate STAT3 in cardiovascular cells. However, the underlying mechanisms of STAT3 activation by angiotensin II and downstream functional consequences in the kidneys are not fully understood.

Experimental Approach

C57BL/6 mice were treated with angiotensin II by subcutaneous infusion for 1 month to develop nephropathy. Mice were treated with either adeno‐associated virus expressing STAT3 shRNA or STAT3 inhibitor, S3I‐201. Human archival kidney samples from five patients with hypertension and five individuals without hypertension were also examined. In vitro, STAT3 was blocked using siRNA or STAT3 inhibitor S3I‐201 in the renal proximal tubular cell line, NRK52E, after exposure to angiotensin II.

Key Results

Angiotensin II activated STAT3 in kidney epithelial cells through engaging toll‐like receptor 4 (TLR4) and JAK2, which was independent of IL‐6/gp130 and angiotensin AT₁ receptors. Angiotensin II‐mediated STAT3 activation increased fibrotic proteins and resulted in renal dysfunction. Both STAT3 inhibition by the low MW compound S3I‐201 and TLR4 deficiency normalized renal fibrosis and dysfunction caused by Ang II in mice, without affecting hypertension.

Conclusions and Implications

Our study reveals a novel mechanism of STAT3 activation, induced by angiotensin II, in kidney tissues and highlights a translational significance of a STAT3 inhibitor as potential therapeutic agent for hypertensive kidney disease.

Abbreviations

AAV

adeno‐associated virus

Ang II

angiotensin II

MD2

myeloid differentiation protein 2

p‐STAT3

phosphorylated STAT3

RAS

renin‐angiotensin system

What is already known

• Angiotensin II is a major contributor to hypertensive kidney disease.

• STAT3 is involved in renal injuries induced by ischaemia/reperfusion and diabetes.

What this study adds

• Inhibition of STAT3 normalized angiotensin II‐induced renal fibrosis and dysfunction in mice.

• Angiotensin II activates STAT3 in kidney epithelial cells through engaging TLR4 and JAK2.

What is the clinical significance

• STAT3 inhibition is a novel potential therapeutic strategy for hypertensive kidney disease.

1. INTRODUCTION

Hypertension affects approximately a quarter of the world population and causes an estimated 7 million deaths each year (Fagard, 2012). Prevalence of hypertension is expected to rise to 30% by 2025 (Kearney et al., 2005). Hypertension adversely affects the kidneys and is one of the most common causes of kidney failure. Hypertensive kidney disease is characterized by tubulointerstitial fibrosis, inflammatory infiltration, loss of renal parenchyma and tubular atrophy, and capillary and podocyte loss (Brenner, 2002; Liu, 2006).

The renin‐angiotensin system (RAS) is important in blood pressure control and the pathogenesis of hypertension‐associated organ damage (Navar, Prieto, Satou, & Kobori, 2011). In this system, angiotensin II (Ang II) is the main effector molecule. Ang II primarily mediates its actions by activating angiotensin AT₁ and AT₂ receptors. Treatment of patients with chronic kidney disease and persistent proteinuria with the aldosterone antagonist, spironolactone, reduced proteinuria after 4 weeks (Sekizawa et al., 2011). Clinical evidence also shows reno‐protective effects of inhibiting the RAS in diabetic patients with kidney failure (Viberti, Wheeldon, & MicroAlbuminuria Reduction With, 2002). It is also being recognized now that many tissues have their own local RAS (Paul, Poyan Mehr, & Kreutz, 2006; Re, 2004). In particular, intrarenal Ang II is elevated in many forms of hypertension, implying a role in the development of hypertension and the associated kidney injury (Navar, Harrison‐Bernard, Nishiyama, & Kobori, 2002). Ang II overexpression specifically in the kidneys has been shown to increase blood pressure and the development of renal inflammation and fibrosis (Kobori et al., 2007). These studies illustrate an important role for Ang II in kidney disease.

Ang II is known to engage JAK and other non‐receptor TKs to elicit intracellular signalling (Doan, Ali, & Bernstein, 2001; Marrero et al., 1995). The JAK family of proteins are TKs and comprise four members in mammals: JAK1, JAK2, JAK3, and tyrosine kinase 2 (Schindler & Darnell, 1995). When JAK is activated, signal transducer and activator of transcription (STAT) proteins are recruited and phosphorylated. Following phosphorylation and activation, STATs form homodimers or heterodimers that translocate to the nucleus and activate the transcription of target genes (Murray, 2007). The JAK/STAT signalling pathway constitutes one of the primary regulatory pathways for cytokine expression (Schindler, 2002). Although Ang II has been documented to activate STAT in a number of cell types, the precise mechanisms involved in kidney cells are not fully clear. We have recently shown that Ang II directly binds myeloid differentiation protein 2 (MD2) to rapidly activate the toll‐like receptor 4 (TLR4; Han et al., 2017). Studies showing Ang II triggering TLR4 activation in the kidney are also emerging (Biancardi, Bomfim, Reis, Al‐Gassimi, & Nunes, 2017). Furthermore, Ang II has been reported to engage TLR4 to activate STAT3 in producing abdominal aortic aneurysms upon Ang II infusion in mice (Qin et al., 2015). These studies raise the intriguing possibility that Ang II engages TLR4 to activate STAT3 in the kidney, resulting in renal fibrosis.

In this study, we have tested the hypothesis that Ang II activates STAT3 through TLR4 in kidney epithelial cells and in kidney tissues of mice. We utilized renal epithelial cells to investigate the roles of STAT3 and TLR4 in Ang II‐induced renal dysfunction, because tubulointerstitial fibrosis, the downstream effect of epithelial dysfunction and loss, is a characteristic feature of hypertensive kidney disease. Furthermore, STAT3 activation in renal epithelial cells has been shown to induce extracellular matrix proteins (Ni et al., 2014). Hence, uncovering the mechanism by which Ang II induces epithelial dysfunction is important for the development of new therapeutic options. Our results show that Ang II causes rapid STAT3 activation via TLR4 and JAK2, but independent of AT₁ receptors in kidney epithelial cells. Activation of STAT3, downstream of Ang II, increased the expression of collagen and fibrogenic growth factors, causing renal fibrosis. We further show that both TLR4 deficiency and pharmacological inhibition of STAT3 effectively prevented Ang II‐induced kidney injury.

2. METHODS

2.1. Cell culture

Rat kidney proximal tubule epithelial line NRK‐52E was obtained from American Type Culture Collection (Manassas, VA; Cat# CRL‐1571; RRID:CVCL_0468). NRK‐52E cells were used as they are easily cultured and allow many downstream assays to be conducted. NRK‐52E cells also have an intact RAS (Alzayadneh & Chappell, 2015), which is critical to the objectives of this study. The cells were maintained in DMEM (Gibco, Eggenstein, Germany) containing 1.5‐g·L⁻¹ sodium bicarbonate and 4.5‐g·L⁻¹ glucose. The media was supplemented with 5% FBS (Gibco, Grand Island, NY), 100 U·ml⁻¹ of penicillin, and 100 mg·ml⁻¹ of streptomycin. Where indicated, IL‐6 and Ang II protein levels were measured in culture media, following all cell treatments and exposures, using commercial ELISA kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). Gene silencing in NRK‐52E was performed by transfecting cells with appropriate siRNAs using Lipofectamine (Invitrogen, Carlsbad, CA). Sequences of the siRNAs used are provided in Table S2. Briefly, cells were plated at a density of 5 × 10⁴ and cultured for 24 hr. Target siRNA or non‐targeting control siRNA were transfected at a final concentration of 50 pmol·ml⁻¹ using lipofectamine. Culture medium was replaced with fresh growth medium after 6 hr. Knockdown effectiveness was determined by western blotting.

2.2. Ang II‐induced kidney disease mouse model

All animal care and experimental procedures were approved by the Wenzhou Medical University Animal Policy and Welfare Committee (Approval Document No. wydw2016‐0130), and all animals received humane care according to the National Institutes of Health (USA) guidelines. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath, Drummond, McLachlan, Kilkenny, & Wainwright, 2010) and with the recommendations made by the British Journal of Pharmacology. Eight‐week‐old male C57BL/6 (stock #000664; RRID:IMSR_JAX:000664) and TLR4^−/− mice (B6.B10ScN‐Tlr4lps‐del/JthJ; Stock #007227; RRID:IMSR_JAX:007227) were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Animals were housed with a 12:12 hr light–dark cycle at a constant room temperature and fed a standard rodent diet. The animals were acclimatized to the laboratory for at least 2 weeks before initiating the studies. To develop the hypertensive model, mice were treated with Ang II by subcutaneous injection (1.4 mg·kg⁻¹·day⁻¹ in phosphate buffer, pH 7.2) for 1 month as described in our previous report (Skibba et al., 2016). The control animals received the same volume of buffer (100 μl). All animal experiments were performed and analysed by blinded experimenters. Treatment groups were assigned in a randomized fashion. Each mouse was assigned a temporary random number within the weight range. When mice were randomly divided in each group, they were given their permanent numerical designation in the cages. For each group, a cage was selected randomly from the pool of all cages.

Animal treatment (a): Adeno‐associated virus type/mutant 2 (AAV2) has been reported to produce robust expression in renal tissue (Qi et al., 2013). To knockdown STAT3 in mouse kidney, we injected AAV2 expressing STAT3 shRNA (Table S1; HanBio, BioTechnology Co, Ltd., Shanghai, China) by tail vein 1 week before Ang II injection (AAV‐shSTAT3). The control groups received the same volume of AAV2 vehicle expressing negative control sequence (AAV‐NC). Ten‐week‐old C57BL/6 mice (n = 28) were randomly allocated initially into two larger groups (n = 14 per group). The mice in AAV‐shSTAT3 group were further divided such that seven mice were randomized to receive daily subcutaneous injections of Ang II and seven to receive an equal volume of vehicle daily. Similarly, the AAV‐NC control animals were randomized either to receive daily injections of Ang II (n = 7) or vehicle (n = 7), resulting in four treatment groups in total: (a) AAV2‐NC‐treated control mice that received PBS (AAV2‐NC group, n = 7); (b) AAV2‐NC‐treated mice with Ang II (AAV2‐NC + Ang II group, n = 7); (c) AAV2‐shSTAT3‐treated mice that received PBS (AAV2‐shSTAT3, n = 7); and (d) AAV2‐shSTAT3‐treated mice with Ang II (AAV2‐shSTAT3 + Ang II, n = 7).

Animal treatment (b): Ten‐week‐old C57BL/6 mice (n = 21) were randomly allocated initially into two three groups: (a) WT mice were subcutaneously injected with PBS (WT Ctrl group, n = 7), (b) WT mice were challenged with Ang II (WT Ang II, n = 7), and (c) WT mice were challenged with Ang II and treated with STAT3 inhibitor S3I‐201 (WT Ang II + S3I‐201, n = 7). At the same time, 10‐week‐old TLR4^−/− mice with C57BL/6 background (n = 14) were randomized either to receive daily injections of Ang II or vehicle: (d) TLR4^−/− mice were subcutaneously injected with PBS (TLR4^−/− Ctrl, n = 7), and (e) TLR4^−/− mice were challenged with Ang II (TLR4^−/− Ang II, n = 7). The randomized animal division results in five treatment groups in total. STAT3 inhibitor S3I‐201 was dissolved in 0.5% CMC‐Na buffer for these studies and orally administrated at 5 mg·kg⁻¹ every other day in the 1‐month schedule. Mice in WT Ctrl, WT Ang II, TLR4^−/− Ctrl, and TLR4^−/− Ang II groups were orally administrated with the vehicle (0.5% CMC‐Na buffer) in the same schedule. Previous studies have used S3I‐201 at 10 mg·kg⁻¹ for a short, 14‐day period of time in obstructive nephropathy models (Matsui et al., 2017; Pang et al., 2010). Based on the expected level of fibrosis in Ang II‐challenged mice compared to obstructive kidney disease models, and the need for a longer term follow‐up, we treated the mice in this study with 5 mg·kg⁻¹ every other day. This dose was empirically determined to reduce STAT3 phosphorylation levels in mice.

At the indicated time points, blood pressure was measured by tail‐cuff using the telemetric blood pressure system (BP‐2010A, Softron Biotechnology). Measurements were performed during the day (1:00 p.m. to 5:00 p.m.) with previous 5 days of training. Blood pressure was measured each week, and 20 constant measurements were recorded at each point. After 1‐month treatment period, animals were killed using sodium pentobarbital anaesthesia. At 6 hr before killing, the spontaneous urine was collected using the following method: (a) the mouse was placed in a clean, dry, and empty cage and allowed to roam around; (b) the mouse is returned to its home cage and the urine was aspirated using a pipette and transferred to a collection tube as soon as the mouse urinates. We collected urine, blood, and kidney samples. Urine creatinine (Cr), albumin, and blood urea nitrogen (BUN) were detected using commercial kits (Nanjing Jiancheng Bioengineering Institute). ELISA kits were used to determine IL‐6 (Thermo Fisher) and Ang II protein (Nanjing Jiancheng Bioengineering Institute) in kidney tissue lysates.

2.3. Human kidney samples

Human tissues were obtained from the First Affiliated Hospital of Wenzhou Medical University. The procedure was approved by the Ethical Committee of First Affiliated Hospital of Wemzhou Medical University (Ethics Number: LCYJLS‐2018‐157) and was carried out in accordance with the Declaration of Helsinki. Archival kidney tissue from five patients with hypertension and five individuals without hypertension was examined. The tissue had been obtained at the time of nephrectomy for conventional renal carcinoma, with kidney tissue examined from regions of the kidney unaffected by tumour. The kidney biopsies were taken from roughly the same part of the kidney. Details of the patients are provided in Table S3. Sufficient frozen tissue was obtained to enable RNA isolation and qPCR, and protein detection.

2.4. Western blot analysis and immunoprecipitation

The antibody‐based procedures used in this study comply with the recommendations made by the British Journal of Pharmacology. Cell and tissue homogenates were prepared. Protein samples were subjected to 8–12% SDS‐PAGE and transferred onto PVDF membrane (Bio‐Rad Laboratory, Hercules, CA). Membranes were then probed with specific primary and secondary antibodies. Antigen–antibody complexes were visualized using enhanced chemiluminescence reagents (Bio‐Rad).

For immunoprecipitation, cell extracts were prepared and incubated with anti‐TLR4 or anti‐JAK2 antibody for 1 hr. Proteins were immunoprecipitated with protein G‐Sepharose beads at 4°C overnight. Samples were used for the detection of JAK2 or TLR4 as co‐precipitated proteins. The density of the immunoreactive bands was analysed using Image J software (NIH, Bethesda, MD; RRID:SCR_003070).

2.5. Reverse transcription and real‐time quantitative PCR

Total RNA was isolated from cells or tissues using TRIzol (Invitrogen). cDNA was synthesized from 1 μg total RNA with M‐MLV Reverse Transcriptase (Invitrogen) and random hexamer primers. Real‐time qPCR was carried out using the Eppendorf Real plex 4 instrument (Eppendorf, Hamburg, Germany). Primers sequences are presented in Table S4. The relative amount of each gene was normalized to the amount of β‐actin. The ΔΔ‐Ct algorithm was used for RT‐qPCR analysis.

2.6. Histology and immunostaining

Kidney specimens from mice were fixed in 10% neutral‐buffered formalin and embedded in paraffin. Tissue sections were subjected to haematoxylin–eosin staining and periodic acid‐Schiff (PAS) for routine histological analysis. Mesangial matrix expansion was evaluated from the examination of PAS staining. For PAS scoring, semiquantitatively on 30–50 glomeruli per kidney in each group (n = 7), using a scale from 0 to 4 score for description: 0 indicates no mesangial matrix expansion, 1 for minimal, 2 for mild, 3 for moderate, and 4 for diffuse (Melhem, Craven, Liachenko, & DeRubertis, 2002; Susztak, Raff, Schiffer, & Bottinger, 2006). Paraffin sections were also stained with 0.1% Sirius Red F3B (Solarbio Life Science, Beijing, China) and 1.3% saturated aqueous solution of picric acid to evaluate type IV collagen deposition. To quantify the level of fibrosis, 10 non‐overlapping fields in each tissue (n = 7) were scored on a semiquantitative scale (<5%, 5–10%, 10–25%, 25–50%, 50–75%, and 75–100%), relative to total tissue area in the field.

To detect phosphorylated STAT3 (p‐STAT3) in mouse kidneys, tissue sections were deparaffinized and hydrated. After performing heat‐induced antigen retrieval in citrate buffer (pH 6), endogenous peroxidase was blocked. Slides were then incubated with anti‐p‐STAT3 (phospho‐Tyr⁷⁰⁵) antibody; Cell Signaling Technology, Danvers, MA) overnight at 4°C. HRP‐conjugated secondary antibody and DAB chromogen were used for detection. Sections were counterstained with haematoxylin. p‐STAT3 in frozen human kidney tissues was detected by immunofluorescence staining. Tissues were fixed in acetone and blocked in 5% BSA (Sigma‐Aldrich, St Louis, MO; Cat# B2064). Fluorophore‐conjugated secondary antibodies were used, and slides were counterstained with DAPI (Abcam, Cambridge, MA, USA; Cat# ab104139).

To stain cultured cells, we fixed the cells in formalin and permeabilized by Triton. Primary p‐STAT3 antibody was applied overnight at 4°C. Fluorophore‐conjugated secondary antibody was then applied for 1 hr at room temperature. Cells were counterstained with DAPI. All images were captured using a Nikon microscope (Nikon, Japan).

2.7. Data and statistical analysis

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018). All experiments were randomized and blinded. In all in vitro experiments, data represent five independent experiments and expressed as means ± SEM. The exact group size (n) for each experimental group/condition is provided and “n” refers to independent values, not replicates. Statistical analysis was performed with GraphPad Prism 6.0 software (San Diego, CA, USA; RRID:SCR_002798). We used one‐way ANOVA followed by Dunnett's post hoc test when comparing more than two groups of data and one‐way ANOVA, non‐parametric Kruskal–Wallis test, followed by Dunn's post hoc test when comparing multiple independent groups. P values of ˂.05 were considered to be statistically significant. Post‐tests were run only if F achieved P < .05 and there was no significant variance inhomogeneity.

2.8. Materials

The suppliers of the antibodies used were as follows: antibodies to phospho‐STAT3 (Tyr⁷⁰⁵), Cat. #9145 and Stat3, Cat. #9139 were from Cell Signaling Technology: antibodies to phospho‐JAK2 (Tyr^{1007 + 1008}) Cat. #ab32101; JAK2, Cat. #ab108596; Lamin B, Cat. #ab16048; TLR4, Cat. #ab22048; AT₁ receptor, Cat. #ab18801; Collagen IV, Cat. #ab34710; TGF‐β1. Cat. #ab92486; GAPDH, Cat. #ab8245; donkey anti‐rabbit IgG H&L (Alexa Fluor® 647) Cat. #ab150075; rabbit anti‐human IgG H&L (TRITC), Cat. #ab6756; goat anti‐mouse IgG H&L (HRP) Cat. #ab6789 were from Abcam. Santa Cruz (Santa Cruz, CA) supplied the mouse anti‐rabbit IgG‐HRP, Cat. #sc2357. Other compounds were supplied as follows: S32‐201, (Sigma‐Aldrich, Cat# SML0330); human Ang II, (Aladdin, Shanghai, China; Cat# A107852); protein phosphatase inhibitors (Solarbio, Cat#P1260) and PMSF (Sangon Biotech, Cat# A100754). SC144 was supplied by Selleck Chemicals (Houston, TX)

2.9. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander, Christopoulos et al., 2017; Alexander, Fabbro et al., 2017a, 2017b; Alexander, Kelly et al., 2017).

3. RESULTS

3.1. Increased STAT3 activation in kidneys is associated with hypertension

To investigate the role of STAT3 in hypertension‐associated kidney injury, we examined archived renal biopsy specimens from normotensive and hypertensive human subjects. These samples had been obtained at the time of nephrectomy for renal carcinoma, but only tissues unaffected by tumour were analysed. We first stained the tissue sections for p‐STAT3(Tyr⁷⁰⁵) as a proxy for STAT3 activation, because phosphorylation at this residue is the main method of activating STAT3 by the upstream JAK (Levy & Darnell, 2002). Our results showed increased p‐STAT3 immunoreactivity in kidney tissues obtained from hypertensive subjects (Figure 1a), with no detectable staining in tissues from normotensive subjects. Lysates prepared from frozen tissues confirmed increased levels of p‐STAT3 proteins found in the samples from hypertensive patients (Figure 1b), along with increased levels of Ang II (Figure 1c). Although these results are derived from a limited number of samples, the data do indicate a potential link between hypertension, Ang II level, and STAT3 activation in human kidney tissues.

Figure 1.

Levels of STAT3 in human kidney tissues from hypertensive subjects. (a) Representative immunofluorescence staining images of human kidney samples, with a quantification graph for p‐STAT3/STAT3 immunofluorescence. Kidney tissues unaffected by tumour from five renal carcinoma patients without hypertension and five with hypertension were obtained and stained for the phosphorylated form of STAT3 (Tyr⁷⁰⁵) as well as total STAT3. Figure showing p‐STAT3 in red. Slides were counterstained with DAPI (blue). (Scale bar = 200 μm). (b) Representative western blot analysis of phospho‐STAT3 in lysates prepared from human renal tissue, as well as the densitometric quantification from five samples per group. STAT3 antibody detecting unphosphorylated forms was used as loading control. (c) Angiotensin II levels in human kidney tissue lysates as measured by ELISA. Data were normalized to total amount of proteins in the lysates. Data shown are means ± SEM; n = 5. *P < .05, significantly different from normotensive

3.2. Inhibition of STAT3 activation preserves renal function in mice challenged with Ang II

Recent studies have shown that administration of a JAK2 inhibitor prevented the development of renal injury in a renal ischaemia/reperfusion model (Yang et al., 2008). Furthermore, blocking AT₁ receptors or inhibiting JAK2 prevented the progression of proteinuria and hypertension in streptozotocin‐induced diabetic nephropathy (Banes et al., 2004). To test the role of STAT3 in hypertension‐associated kidney damage, we challenged mice with Ang II through subcutaneous administration. In this model, we inhibited STAT3 through injecting AAV2 (Qi et al., 2013) expressing STAT3 shRNA. Ang II administration in mice was associated with increased levels of Ang II in kidney tissues (Figure 2a). No changes were detected in AAV2‐STAT3 shRNA injected mice compared to those treated with negative control AAV2. Increased levels of kidney tissue Ang II were expected, as previous studies have demonstrated that Ang II infusion augmented levels of intrarenal angiotensinogen (Gonzalez‐Villalobos et al., 2008). Analysis of tissue lysates showed that Ang II administration in mice increased the levels of p‐STAT3 (Figure 2b). As expected, both STAT3 and p‐STAT3 levels were markedly reduced in mice injected with AAV2‐STAT3 shRNA, with or without Ang II challenge (Figure 2b). Furthermore, prevention of STAT3 activation in kidney tissues was not associated with any discernable changes in systolic blood pressure (Figure 2c). Importantly, blood pressure monitoring in mice validated the model of Ang II‐induced hypertension.

Figure 2.

Knockdown of STAT3 protects against angiotensin II‐induced renal injury. STAT3 was inhibited in mice by injecting adeno‐associated virus expressing STAT3 shRNA into 8‐week‐old C57BL/6 mice by tail vein. After 1 week, angiotensin II was administered by subcutaneous injection at 1.4 mg·kg⁻¹·day⁻¹ for 1 month. (a) Angiotensin II protein levels in mouse kidney tissues as detected by ELISA (AAV2‐NC = negative control shRNA expressing AAV; AAV2‐shSTAT3 = STAT3 shRNA expressing AAV. Data shown are means ± SEM; n = 7. *P < .05, significantly different as indicated; ns = not significant. (b) Representative western blot analysis of p‐STAT3 in mouse kidneys. Total STAT3 and GAPDH were used as controls. The graphs on the right show the summary data from western blots, as means ± SEM; n = 7. ^* P < .05, significantly different as indicated; ns = not significant. (c) Systolic blood pressure in mice showing values at indicated time points. Data shown are means ± SEM; n = 7. *P < .05, significantly different as indicated; ns = not significant. (d) Kidney tissue weights showing the effect of STAT3 knockdown. Ang II administration increased kidney tissue weight, which was not seen in mice injected with STAT3 shRNA expressing AAV. Data shown as ratio of kidney weight to body weight (means ± SEM; n = 7). *P < .05, significantly different as indicated; ns = not significant.. (e, f) Metabolic profile of angiotensin II‐induced mice showing urine albumin to creatinine ratio (e), and serum urea nitrogen (f). Data shown are means ± SEM; n = 7. *P < .05, significantly different as indicated; ns = not significant

We next examined parameters of kidney damage in these mice. The data show that Ang II administration decreased body weights but increased kidney tissue weights in the mice, after 1‐month injection (Figures S1 and 2d). This increase in kidney tissue weight was not seen in mice in which STAT3 was knocked down. Similar results were obtained when we examined urinary albumin : creatinine ratios or levels of BUN (Figure 2e,f). These findings suggest that Ang II increases STAT3 activation and that STAT3 may mediate Ang II‐induced kidney dysfunction.

3.3. STAT3 regulates Ang II‐induced fibrotic responses in mouse kidneys

We next examined the kidney tissues of mice following STAT3 knockdown. Histopathology showed that Ang II‐induced glomerular abnormalities, primarily showing disarrayed glomerular tufts and mesangial proliferation (Figure 3a). These histological abnormalities were reduced in mice with STAT3 knockdown. As the progress of kidney disease is characterized by the loss of renal cells and their replacement by extracellular matrix (Yu, Noble, & Border, 2002), we stained kidney sections with PAS to measure mesangial matrix expansion and Sirius Red staining to highlight collagen deposition. The data show global mesangial matrix expansion was observed in AAV2‐NC + Ang II group, while this change was attenuated in the AAV2‐shSTAT3 group (Figure 3b,d). Also, there was significant fibrosis in kidney tissues of mice challenged with Ang II, while only minimal staining was seen around glomeruli in kidney tissues of mice with STAT3 silencing (Figure 3c,e). We know from previous studies that Ang II modulates fibrosis by up‐regulating the expression of TGF‐β (Fogo, 2001). In fact, up‐regulation of TGF‐β1 has been demonstrated in nearly every type of fibrotic renal disease in animals and in humans (Bottinger & Bitzer, 2002; Zeisberg, Strutz, & Muller, 2001). Hence, we determined the levels of TGF‐β in kidney tissues of these mice. As shown in Figure 3f, Ang II‐induced TGF‐β and collagen I/collagen IV levels in kidney tissues of mice with intact STAT3 but not in mice in which STAT3 had been knocked down.

Figure 3.

Knockdown of STAT3 prevents renal fibrosis in mice with angiotensin II‐induced hypertension. (a) Representative haematoxylin and eosin (H&E) staining of kidney tissues harvested from mice with hypertension induced by angiotensin II (Ang II). The lower panel showed the zoomed in images of glomerulus from each group (upper scale bar = 200 μm; lower scale bar = 10 μm). (b) Representative images of PAS staining (scale bar = 10 μm). (c) Representative images of kidney tissues showing Sirius Red staining (scale bar = 200 μm). (d) Quantification of mesangial matrix score in panel b. Data shown are means ± SEM; n = 7. *P < .05, significantly different as indicated; ns = not significant. (e) Quantification of fibrotic areas in panel c. Data shown as percent total area. Data shown are means ± SEM; n = 7. *P < .05, significantly different as indicated; ns = not significant.. (f) Western blot analysis of TGF‐β and type I and IV collagen in kidney tissues from mice. GAPDH was used as loading control

3.4. Rapid activation of STAT3 in renal tubular cells is mediated through TLR4, independent of IL‐6/gp130 and angiotensin AT₁ receptors

To tease out the mechanisms of STAT3 activation by Ang II in kidneys, we utilized kidney NRK‐52E epithelial cells. We exposed the cells to Ang II for different periods and examined the levels of p‐STAT3. Our results show rapid STAT3 phosphorylation in response to Ang II and this was maintained for 90 min in kidney epithelial cells (Figure 4a). We confirmed the phosphorylation of STAT3 by staining NRK‐52E cells following Ang II stimulation. The results show Ang II increased immunoreactivity in cells (Figure 4b). Analysis of JAK2, which has been shown to activate STAT3 in kidney mesangial cells (Marrero, Banes‐Berceli, Stern, & Eaton, 2006) and vascular cells (Marrero et al., 1997), also showed that JAK2 was activated at 5–30 min after Ang II stimulation (Figure 4c). These results showed that JAK2 may be involved in Ang II‐induced STAT3 activation in kidney epithelial cells.

Figure 4.

Angiotensin II activates STAT3 phosphorylation in kidney epithelial cells, independent on IL‐6 pathway. (a) Western blot analysis of p‐STAT3 levels in rat kidney epithelial line NRK‐52E following exposure to angiotensin II (Ang II). Cells were exposed to 1‐μM angiotensin II for the indicated time points and p‐STAT3 levels in cell lysates were probed. Total STAT3 and GAPDH were used as loading control. (b) Representative immunofluorescence images showing levels and localization of p‐STAT3 (red) in NRK‐52E. Cells were exposed to 1‐μM angiotensin II for the indicated time points. Cells were counterstained with DAPI (blue; scale bar = 20 μm). (c) Western blot analysis of phosphorylated JAK2 (Tyr^{1007 + 1008}) levels in NRK‐52E cells following exposure to angiotensin II. Cells were treated as in panel a. Total JAK2 and GAPDH were used as loading control. (d) Levels of IL‐6 in NRK‐52E conditioned culture media as determined by ELISA. Cells were exposed to angiotensin II and IL‐6 levels in media were measured at indicated times (ns = not significant compared to 0 min). (e) Effect of inhibiting IL‐6/gp130 by SC144 on angiotensin II‐induced STAT3 phosphorylation. NRK‐52E were pretreated with SC144 for 1 hr before exposure to 1‐μM angiotensin II STAT3 phosphorylation was determined by immunoblotting. Total STAT3 and GAPDH were used as controls

An interesting finding in our study was the rapid activation of STAT3 by Ang II in cultured kidney epithelial cells. Previous studies have shown that Ang II induces rapid phosphorylation of STAT1 and STAT2 with delayed phosphorylation of STAT3 in cardiomyocytes. The delayed activation of STAT3 in cardiac cells is believed to be due to enhanced synthesis and release of IL‐6 (Sano et al., 2000). Therefore, we measured IL‐6 production and release by kidney epithelial cells exposed to Ang II for 0–120 min. Surprisingly, no changes were noted in IL‐6 levels (Figure 4d). At least at the early phase of Ang II stimulation, Ang II did not seem to involve IL‐6‐mediated STAT3 activation. To confirm this, we inhibited IL‐6 signalling by treating NRK‐52E cells with the IL‐6/gp130 inhibitor SC144. Our results confirm that pretreatment with SC144 did not inhibit Ang II‐induced STAT3 phosphorylation in kidney epithelial cells, indicating that Ang II‐induced rapid STAT3 activation was not mediated by IL‐6/gp130.

To identify the signalling mechanism engaged by Ang II to cause STAT3 activation, we performed a series of candidate protein knockdowns. We transfected NRK‐52E cells with siRNA targeting AT₁ receptors and another for JAK2. Silencing of the AT₁ receptors failed to inhibit either JAK2 or STAT3 phosphorylation in response to Ang II (Figure 5a). As expected from our results described above, JAK2 silencing prevented Ang II‐induced STAT3 phosphorylation (Figure 5b). We then silenced TLR4 and probed for STAT3 activation. Our results show that TLR4 deficiency reduces both JAK2 and STAT3 phosphorylation (Figure 5c). This result supported our hypothesis of the involvement of TLR4 in Ang II‐induced STAT3 activation. Co‐immunoprecipitation studies indeed showed a rapid protein–protein interaction between TLR4 and JAK2 in response to Ang II stimulation of NRK‐52E cells (Figure 5d). We next performed gene knockdown for STAT3 to determine whether there exists a feedback system regulating JAK2 and/or TLR4 in kidney epithelial cells. As shown in Figure 5e, no alterations in the levels of p‐JAK2 and TLR4 were observed upon STAT3 knockdown. Collectively, these findings show that Ang II causes rapid STAT3 activation in kidney epithelial cells through engaging TLR4‐JAK2, independent of AT₁ receptors.

Figure 5.

Angiotensin II phosphorylates STAT3 through toll‐like receptor4 (TLR4)‐JAK2 interaction. (a–c) The effect of knocking down AT₁ receptors (a), JAK2 (b), and TLR4 (c) on JAK and STAT3 phosphorylation induced by angiotensin II (Ang II). NRK‐52E epithelial cells were transfected with siRNAs which target candidate signalling proteins involved in STAT3 activation for 6 hr. After 6‐hr transfection, cells were exposed to 1‐μM angiotensin II for 5 min and then p‐JAK/JAK was probed by immunoblotting; or cells were exposed to 1‐μM angiotensin II for 60 min and then p‐STAT3/STAT3 and AT₁ receptors (or TLR4) were probed by immunoblotting. (d) Lysates prepared from NRK‐52E cells stimulated with 1‐μM angiotensin II for indicated time points were subjected to immunoprecipitation with TLR4 or JAK2 antibody (IP) and subsequent detection of JAK2 or TLR4 (IB) respectively. (e) NRK‐52E epithelial cells were transfected with siRNA which targets STAT3 for 6 hr. After 6‐hr transfection, cells were exposed to 1‐μM angiotensin II for 5 min and then p‐JAK/JAK, STAT3, and TLR4 proteins were probed by immunoblotting. All blots are representative from five independent experiments

3.5. Ang II‐induced STAT3 mediates fibrotic responses in kidney epithelial cells

We next determined the functional consequence of Ang II‐induced STAT3 activation in kidney epithelial cells. We silenced STAT3 in NRK‐52E cells and examined for induction of fibrotic proteins in response to Ang II. Transfection of cells with siRNA against STAT3, which significantly reduced STAT3 proteins (Figure 5e), showed no induction of collagen or TGF‐β in response to Ang II (Figure 6a). To confirm these results, we utilized a pharmacological inhibitor of STAT3, S3I‐201. S3I‐201 specifically prevents STAT3 phosphorylation and subsequent complex formation, and STAT3‐DNA binding activity (Siddiquee et al., 2007). To validate this inhibitor in our system, we treated NRK‐52E cells with increasing concentrations of S3I‐201 before challenging the cells with Ang II. S3I‐201 reduced the phosphorylation of STAT3 without altering the levels of STAT3 protein (Figure S2a, b). Analysis of nuclear fractions also revealed that S3I‐201 reduced Ang II‐induced STAT3 activation and nuclear translocation (Figure S2b,c). Furthermore, S3I‐201 did not alter JAK2 levels (Figure S2a,b), consistent with our gene silencing data (Figure 5e). Using the lowest concentration from these studies, we show that inhibition of STAT3 by S3I‐201 at 2.5 μM prevents Ang II‐induced collagen and TGF‐β protein levels (Figure 6b). These findings show that Ang II‐mediated STAT3 activation is involved in fibrotic responses in kidney cells.

Figure 6.

Inhibition of STAT3 prevents angiotensin II‐induced fibrotic responses in kidney epithelial cells. (a) NRK‐52E cells were transfected with siRNA targeting STAT3 or negative control siRNA. Cells were then exposed to 1‐μM angiotensin II (Ang II) for 24 hr. Lysates were probed for the levels of type IV collagen and TGF‐β. GAPDH was used as loading control. Blots are representative from five independent experiments. (b) Effect of STAT3 inhibitor S3I‐201 on angiotensin II‐induced fibrogenic protein levels in NRK‐52E cells. Cells were pretreated with S3I‐201 for 1 hr before being exposed to angiotensin II for 24 hr. Type IV collagen and TGF‐β levels were determined by immunoblotting. GAPDH was used as loading control. All blots are representative from five independent experiments

3.6. TLR4 deficiency and STAT3 inhibition protects kidneys from Ang II‐induced unregulated remodelling and fibrosis

Our last objective was to provide empirical evidence linking Ang II‐TLR4‐STAT3 to renal fibrosis. To achieve this goal, we challenged TLR4‐deficient mice with Ang II. In addition, we inhibited STAT3 in wild‐type mice using oral administration of S3I‐201 to bolster the results obtained using AAV2‐STAT3 shRNA. Ang II increased systolic blood pressure in mice (Figure S3). Neither TLR4 deficiency nor STAT3 inhibition in wild‐type mice prevented Ang II‐mediated increased blood pressure.

Administration of Ang II increased kidney Ang II levels in both wild‐type and TLR‐deficient mice (Figure 7a). The STAT3 inhibitor or TLR4 deficiency had no effect on kidney Ang II levels. Analysis of tissue lysates clearly showed increased p‐STAT3 and JAK2 in wild‐type mice upon Ang II administration (Figure 7b). As expected, S3I‐201 treatment of mice reduced p‐STAT3 but showed no effect on JAK2 phosphorylation levels. TLR4‐deficient mice failed to increase JAK2 and STAT3 phosphorylation in response to Ang II challenge. We confirmed STAT3 phosphorylation by Ang II by staining kidney tissues from these mice. Ang II‐induced p‐STAT3 immunoreactivity was reduced by S3I‐201 treatment, and not seen in TLR4‐deificient mice (Figure 7c).

Figure 7.

TLR4 knockout mice are protected against angiotensin II‐induced STAT3 activation and renal dysfunction. Angiotensin II (Ang II) was administered to 8‐week‐old TLR4^−/− and wild‐type (WT) littermates by subcutaneous injection for 1 month. One experimental group of WT mice receiving Ang II were treated with STAT3 inhibitor S3I‐201. (a) Angiotensin II levels in mouse kidney tissues as detected by ELISA. Data shown are means ± SEM; n = 7. *P < .05, significantly different as indicated; ns = not significant. (b) Western blot analysis of TLR4 and phosphorylated JAK2 and STAT3 in kidney tissues of mice challenged with angiotensin II . Total JAK2, STAT3, and GAPDH were used as internal controls. Representative blot is shown from n = 7 per group. (c) Representative immunohistochemical staining for p‐STAT3 (brown) in kidney tissue harvested from WT and TLR4‐deificent mice. Tissues were counterstained with haematoxylin (blue; scale bar = 400 μm). (d–f) Kidney/body weight ratio (d), levels of urine albumin to creatinine ratio (e), and serum BUN (f) were determined in each group. Data shown are means ± SEM; n = 7 per group. *P < .05, significantly different as indicated; ns = not significant

STAT3 inhibition by S3I‐201 and TLR‐deficiency protected mice from Ang II‐mediated kidney dysfunction as observed by urinary albumin to creatinine ratios and BUN (Figure 7d,e). Histopathology also showed reduced structural abnormalities and fibrosis upon STAT3 inhibition and TLR‐deficiency (Figure 8a–e). Finally, western blot analysis showed that TLR4 knockout mice do not exhibit Ang II‐mediated increases in collagen and TGF‐β levels (Figure 8f). STAT3 inhibitor S3I‐201 also suppressed collagen I, IV, and TGF‐β levels. Interestingly, the inhibitory effect on collagen by S3I‐201 was quite robust compared to TGF‐β. This may be due to different cell types or signalling pathways contributing to Ang II‐mediated TGF‐β induction. Taken together, our studies show that Ang II activates STAT3 through TLR4, and increased STAT3 activity causes renal fibrosis and dysfunction.

Figure 8.

TLR4‐deficiency and STAT3 inhibition protects mice from angiotensin II‐induced renal fibrosis. (a) Representative haematoxylin and eosin (H&E) stained kidney tissues from mice challenged with Ang II. The lower panel showed the zoomed in images of glomerulus from each group (upper scale bar = 200 μm; lower scale bar = 10 μm). (b) Representative images of PAS staining showing mesangial expansion in the glomeruli (scale bar = 10 μm). (c) Representative images of kidney tissues showing Sirius Red staining for types I, III, and V collagen. (d) Mesangial matrix expansion score in panel b. Data shown as mean ± SEM; n = 7. *P < .05, significantly different as indicated; ns = not significant. (e) Quantification of fibrotic areas in panel c. Data (means ± SEM) are shown as percent total area; n = 7. *P < .05, significantly different as indicated; ns = not significant. (f) Western blot analysis of type I and IV collagen and TGF‐β in lysates prepared from kidney tissues of mice. GAPDH was used as loading control. Representative blot is shown from n = 7 per group

4. DISCUSSION

The key findings of our study include the discovery that Ang II activates STAT3 in kidney epithelial cells through engaging TLR4 and JAK2, independent of IL‐6/gp130 and AT₁ receptors. Ang II‐mediated STAT3 activation in kidney epithelial cells increased the expression of collagen and TGF‐β. Chronic Ang II challenge increased fibrotic proteins and resulted in renal dysfunction in mice. As observed in our in vitro studies, inhibition of STAT3 activation, by a low MW inhibitor, normalized structural and functional alterations in mice caused by Ang II. Finally, we showed that mice deficient in TLR4 did not activate STAT3 in response to Ang II and were protected against Ang II‐induced kidney dysfunction.

Among the various regulatory systems that affect blood pressure, the RAS plays a significant role. A large body of experimental evidence shows that Ang II is able to induce the pathological hallmarks of hypertensive kidney disease. Kidneys have their own intact RAS and intrarenal angiotensinogen is produced in renal proximal tubular cells (Kobori, Harrison‐Bernard, & Navar, 2001), compatible with significant role in hypertensive kidney disease. Ang II administration has also been reported to increase intrarenal angiotensinogen expression (Gonzalez‐Villalobos et al., 2008). This intrarenal Ang II facilitates the progression of hypertension and renal injury (Gonzalez‐Villalobos et al., 2009). Our analyses show that chronic Ang II administration in mice results in increased Ang II protein levels in kidney tissues of mice. Recently, Marrero and colleagues showed that Ang II induces rapid phosphorylation of JAK2 in aortic smooth muscle cells (Bottinger & Bitzer, 2002). Although these important studies resulted from cardiovascular cells, Ang II‐induced activation of the JAK–STAT pathway has been demonstrated in renal proximal tubular cells and mesangial cells (Marrero et al., 2006; Zhang, Guo, Moini, & Ingelfinger, 2004). These later studies implicated the JAK/STAT pathway in hypertensive kidney disease. In the present study, analysis of human kidney samples from hypertensive and normotensive patients showed increased levels of both Ang II and STAT3 phosphorylation. Although we are unable to confirm the cellular source of Ang II and activated STAT3 in human kidney tissues, our results do implicate these two proteins in hypertensive kidney disease. It is interesting to note that p‐STAT3 was elevated in kidney samples from hypertensive subjects compared with normotensive patients, despite the fact that these patients were younger (mean 39.8 years for hypertensive group compared to 63.2 years for normotensive group). Another limitation is that the numbers in two groups were too small to be compared with each other. Previous studies also showed increasing levels of STAT3 with increasing age in epithelial compartments of the renal cortex (O'Brown, Van Nostrand, Higgins, & Kim, 2015). Further, STAT3 knockdown by AAV2 significantly attenuated Ang II‐induced renal fibrosis in mice. We noted that the AAV2 method may cause STAT3 down‐regulation in other tissues. Whether STAT3 inhibition in other tissues (e.g., in liver) plays a role in renal protection was not investigated in this study.

STAT3 inhibition has been reported to protect renal injuries. For example, STAT3 inhibition has been shown to ameliorate damage induced by unilateral ureteral obstruction (Pang et al., 2010), ischaemia/reperfusion (Arany, Megyesi, Nelkin, & Safirstein, 2006; Neria et al., 2009), oxidative stress (Neria et al., 2009), and diabetes (Banes et al., 2004). Very recently, a STAT1/3/5 inhibitor, nifuroxazide, was shown to inhibit diabetes‐induced kidney fibrosis (Said, Zaitone, Eldosoky, & Elsherbiny, 2018). In our kidney epithelial cell cultures, we showed that inhibiting STAT3 reduced Ang II‐mediated expression of collagen and TGF‐β. In vivo, both gene knockdown and pharmacological inhibition of STAT3 protected kidney function and reduced fibrosis in mice challenged with Ang II. These results show that STAT3 inhibition provides significant protection against Ang II‐induced extracellular matrix production and fibrosis, both in an acute setting (cell culture studies) and chronic setting (mouse studies). In the cell cultures, cells were challenged with Ang II for a short time period. These challenged cells exhibited rapid but transient increase in p‐STAT3. Inhibition of STAT3 in this setting reduced the expression of collagen and TGF‐β. Although, continuous challenge of mice with Ang II may result in a long‐term situation of STAT3 activation, STAT3 blockade in mice also lead to the similar result: inhibition of fibrosis in kidneys. These findings warrant further studies on S3I‐201 to determine whether this inhibitor has clinical utility in treating hypertensive kidney disease.

We found that Ang II activated the JAK/STAT pathway through TLR4. In NRK‐52E cells, Ang II causes rapid STAT3 activation via inducing TLR4 and JAK2 interaction, independent of AT₁ receptors. When TLR4 was knocked down in kidney epithelial cells, Ang II failed to activate JAK2 and STAT3. TLR4 knockout mice were also found to be protected against Ang II‐mediated JAK2 and STAT3 phosphorylation. These findings suggest that TLR4 plays a significant role in Ang II responsiveness. Previously, it was reported that TLR4 mutant mice are protected from renal fibrosis in a partial nephrectomy with Ang II infusion model (Souza et al., 2015). Others have shown that decreased renal fibrosis in TLR4‐deficient mice upon unilateral ureteral obstruction (Braga et al., 2012). Our previous studies showed that Ang II rapidly binds to a co‐receptor of TLR4, MD2 protein, which suggests that Ang II, at least in early state, may directly activate TLR4 (Han et al., 2017). MD2 knockout mice are also protected against Ang II‐mediated renal fibrosis and dysfunction (Xu et al., 2017). In the delayed phase, continuous cell damage and oxidative stress induced by Ang II or hypertension generate the release of many potential danger signals which may be recognized by, and activate, the TLR4.

One of the limitations in this work is that we are unable to confirm which cells are responsible for Ang II and STAT3 phosphorylation in mouse kidney tissues and human biopsy tissues. We performed the mechanistic studies only in cultures of kidney epithelial cells. Although the NRK‐52E cells have some advantages mentioned above, we fully aware that analysis of other kidney cell types is important. We anticipate that other cell types in kidney tissues including mesangial cells and podocytes may also utilize this signalling pathway. However, this possibility requires further work in confirmation. Another area of research is to examine the role of Ang II‐TLR4‐STAT3 axis in the epithelial‐to‐mesenchymal transition in kidney injuries, as reduction of Ang II‐induced renal fibrosis has been associated with a reduction of the epithelial‐to‐mesenchymal transition (He et al., 2018; Hu et al., 2018; Wang et al., 2018). Such experiments are important to better understand how STAT3 mediates epithelial loss and phenotype change, as well tubular atrophy.

In conclusion, our study has uncovered potentially important mechanisms underlying Ang II‐induced renal dysfunction. Our finding that Ang II activates STAT3 in kidney epithelial cells through engaging TLR4 and JAK2, independent of IL‐6/gp130 and AT₁ receptors is significant. A unique interplay between Ang II, hypertension, and TLR4 is emerging from recent studies. We showed that TLR4 deficiency does not regulate hypertension, but hypertension‐associated changes may regulate TLR4 to activate STAT3. We also demonstrated that Ang II‐mediated STAT3 activation in kidney epithelial cells is important for the induction of TGF‐β and collagen IV, two key drivers of fibrosis. Inhibiting STAT3 by S3I‐201 normalized Ang II‐induced structural and functional alterations in kidney tissues of mice, indicating a translational significance of STAT3 inhibitor as potential therapeutic agents for hypertensive kidney disease.

AUTHOR CONTRIBUTIONS

Z.X., C.Z., W.Y., S.X., and L.H. are responsible for the collection and analysis of data. Z.X., C.Z., W.Y., S.X., L.H., Z.K., J.W., G.L., and Y.W. are responsible for the interpretation of data. Z.K., J.W., G.L., and Y.W. are responsible for the conception and design. Z.K., J.W., G.L., and Y.W. are responsible for writing the manuscript. Z.K., J.W., G.L., and Y.W. are responsible for the manuscript revision.

CONFLICT OF INTERESTS

The authors declare no conflicts of interest.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design & Analysis, Immunoblotting and Immunochemistry, and Animal Experimentation, and as recommended by funding agencies, publishers and other organisations engaged with supporting research.

Supporting information

Table S1: Information of reagents used in the study.

Table S2: siRNA sequences used in this study

Table S3: Patient information

Table S4: Primer sequences for real‐time qPCR assay.

Figure S1: STAT3 knockdown attenuated Ang II‐induced mice body weight decrease. Body weight was determined at indicated times. Data were represented as mean ± SEM. n = 7; *P < 0.05 according to one‐way ANOVA.

Figure S2: S3I‐201 suppresses angiotensin II‐induced STAT3 phosphorylation. (A, B) NRK‐52E cells were pretreated with increasing concentrations of STAT3 inhibitor S3I‐201 (S3I) for 1 h before challenging with Ang II for indicated times. DMSO was used as vehicle control. STAT3 and JAK2 phosphorylation levels were then determined by western blotting. Representative blot is shown in panel A. Densitometric quantification is shown in panel B [n = 5; ns = not significant compared to Ang II; *P < 0.05 compared to Ang II; #P < 0.05 compared to Ctrl]. (C) Immunofluorescence staining of NRK‐52E for p‐STAT3. Cells were treated as indicated in panel A. p‐STAT3 immunoreactivity is shown in red. Cells were counterstained with DAPI (blue) [scale bar = 20 μm].

Figure S3: STAT3 inhibition and TLR4 deficiency does not alter systolic blood pressure in mice. Effect of subcutaneous infusion of Ang II on mice blood pressure. All measurements were made during day time (1:00 to 5:00 pm). Data are represented as mean ± SEM. n = 7; *P < 0.05 according to one‐way ANOVA.

Xu Z, Zou C, Yu W, et al. Inhibition of STAT3 activation mediated by toll‐like receptor 4 attenuates angiotensin II‐induced renal fibrosis and dysfunction. Br J Pharmacol. 2019;176:2627–2641. 10.1111/bph.14686