Mechanisms of IL-17A Neutralisation in Alleviating Renal Fibrosis and Inflammation in Spontaneously Hypertensive Rats.

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Yu Wang, Zhenzhen Chen, Meng Wu, and Weiyi Li. The first draft of the manuscript was written by Anshuai Ba and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Anshuai Ba and Zhenzhen Chen contributed equally to this work and are co‐first authors.

This article does not contain any studies with human participants performed by any of the authors. All procedures strictly adhered to the ARRIVE guidelines and the UK Animals (Scientific Procedures) Act 1986, following approval by the Biomedical Research Ethics Committee of Anhui University of Science and Technology (Approval No.: SZ2023‐027).

This work was supported by: (1) Medical Special Cultivation Project of Anhui University of Science and Technology (Grant No. YZ2023H1A007); (2) The Open Fund Project of Anhui Provincial Key Laboratory of Industrial Dust Deep Purification and Occupational Health Safety (Grant No. AYZJSGXLK202202005); (3) Innovative Teaching Team in Physiology and Pathophysiology (Grant No. 2023CXTDF142).

Based on our previous findings, we hypothesized that IL‐17A might be involved in renal fibrosis and inflammation development in SHRs. To test this hypothesis, we administered IL‐17A NAb to SHRs starting at 10 weeks of age and compared SBP and renal function between IL‐17A NAb‐treated and control groups at 30 weeks. Sustained SBP elevation can lead to glomerular hypertension, damage to the glomerular filtration barrier, proteinuria, and progression of renal vascular sclerosis, ultimately causing glomerular filtration rate decline and progressive renal impairment [ 11 ]. Results demonstrated that IL‐17A NAb intervention significantly reduced SBP in SHRs, with reductions becoming evident at 20 weeks and reaching maximal difference at 30 weeks (Figure  1A , p  < 0.0001). Serum urea, serum creatinine (Scr), and 24‐h urinary protein quantification—critical markers for evaluating renal function changes, particularly glomerular filtration and tubular reabsorption—are widely used in chronic kidney disease diagnosis, monitoring, and therapeutic assessment [ 12 ]. Compared with controls, the IL‐17A NAb‐treated group showed significantly reduced serum urea, Scr, and 24‐h urinary protein excretion (Figure  1B–D , all p  < 0.05). These results indicate that neutralising IL‐17A can delay hypertension progression and improve renal function in SHRs. IL‐17A neutralisation delayed hypertension progression and improved renal function in SHRs. (A) SBP dynamics in IL‐17A NAb‐treated versus control SHRs measured biweekly from 10 to 30 weeks of age. (B) Serum urea, (C) Scr, and (D) 24‐h urinary protein quantification in treatment and control groups at 30 weeks. Data are expressed as mean ± SD ( n  = 6). To further evaluate the protective effects of IL‐17A neutralisation on end‐stage renal damage in SHRs, we performed renal histopathological analyses. H&E staining revealed significant pathological changes in the control group compared to the IL‐17A NAb intervention group, including marked glomerular structural disorganisation, atrophy, tubular interstitial hyperplasia, and thickened microvascular walls with progressive luminal narrowing (Figure  2A ). Blinded assessments by two independent pathologists indicated significantly higher renal tissue injury scores in the control group versus the intervention group (Figure  2B p  < 0.01). Furthermore, Masson's staining showed that IL‐17A NAb intervention significantly reduced renal fibrosis in SHRs (Figure  2C,D , P  < 0.01). These histopathological findings confirm that neutralisation of IL‐17A effectively alleviates renal fibrotic lesions in SHRs. IL‐17A neutralisation ameliorated renal histopathology in SHRs. (A) Representative H&E‐stained sections showing renal tissue morphology. (B) Semiquantitative injury scores from blinded evaluation by two independent pathologists. (C) Representative Masson's trichrome‐stained sections (blue = collagen deposition). (D) Quantitative analysis of renal interstitial fibrosis area. Data are expressed as mean ± SD ( n  = 6). Chronic inflammation plays a critical role in hypertension pathogenesis, with macrophages serving as key inflammatory participants central to hypertension development and progression [ 13 ]. Notably, macrophage infiltration and phenotype polarisation (pro‐inflammatory M1 versus anti‐inflammatory/repair‐associated M2) are pivotal regulators of renal fibrosis. Previous studies demonstrate that IL‐17A activation promotes macrophage polarisation toward the M2 phenotype, driving secretion of pro‐fibrotic factors like TGF‐β1 that exacerbate fibrosis [ 14 ]. To investigate IL‐17A neutralisation effects on macrophage polarisation, we analysed macrophage phenotype marker expression in renal tissues of SHRs using IHC (Figure  3A,B ), Western blot (Figure  3C ), flow cytometry (Figure  3D ), and RT‐qPCR (Figure  3E ). Results indicated that IL‐17A neutralisation significantly inhibited M2 polarisation (all relevant markers p   0.05). Effects of IL‐17A neutralisation on macrophage polarisation in SHR renal tissues. (A) Representative IHC staining of M1 macrophage markers (iNOS and CD86) with quantitative analysis of their positive areas. (B) Representative IHC staining of M2 macrophage markers (Arg‐1 and CD163) with quantitative analysis of their positive areas. (C) Representative immunoblots and relative expression levels of iNOS, CD86, Arg‐1, and CD163 proteins. (D) Proportion of CD86 + and CD163 + cells among CD68 + macrophages. (E) mRNA expression levels of iNOS, CD86, Arg‐1, and CD163. Data are presented as mean ± SD ( n  = 6). Renal damage caused by hypertension is often accompanied by progressive fibrosis, ultimately leading to the loss of kidney function. EMT is considered a critical cellular biological mechanism in this process [ 15 ]. In chronic kidney injury, renal tubular epithelial cells, under the stimulation of inflammatory factors such as TGF‐β1 and IL‐17A, as well as mechanical stress, may undergo EMT. This process involves the loss of epithelial markers (e.g., E‐cadherin) and the acquisition of mesenchymal markers (e.g., α‐SMA and Collagen III), transforming into migratory myofibroblasts. These myofibroblasts promote renal interstitial fibrosis directly by secreting large amounts of ECM [ 16 ]. In this study, given that IL‐17A neutralisation significantly reduced the infiltration of M2 macrophages in renal tissue, we hypothesized that its antifibrotic effects might be associated with the inhibition of the EMT process. To investigate this, we assessed EMT‐related markers in SHR renal tissues using IHC (Figure  4A ), Western blot analyses (Figure  4B ), and RT‐qPCR (Figure  4C ). The results showed that after IL‐17A NAb treatment, the expression of the epithelial marker E‐cadherin was significantly increased ( p  < 0.01), whereas the expression of mesenchymal markers α‐SMA and Collagen III was markedly reduced (all p  < 0.05). IL‐17A NAb inhibited EMT in SHR renal tissues. (A) Representative IHC images showing the expression of E‐cadherin, α‐SMA, and Collagen III, with quantitative analysis of their positive areas. (B) Representative immunoblots and relative expression levels of E‐cadherin, α‐SMA, and Collagen III proteins. (C) mRNA expression levels of E‐cadherin, α‐SMA, and Collagen III. Data are expressed as mean ± SD ( n  = 6). TGF‐β1, a key pro‐fibrotic factor, activates the TGF‐β/Smad signalling pathway by binding to its type II receptor (TβRII) and phosphorylating the type I receptor (TβRI), which subsequently recruits and phosphorylates Smad2/3 [ 17 ]. Phosphorylated Smad2/3 forms a complex with Smad4 that translocates to the nucleus, directly regulating EMT‐related gene transcription. This process suppresses epithelial markers, induces mesenchymal markers, and enhances cellular migratory capacity [ 18 ]. Consequently, the TGF‐β/Smad pathway represents a central molecular mechanism driving EMT and renal fibrosis. To evaluate IL‐17A neutralisation effects on this pathway, we measured TGF‐β1, phosphorylated Smad2/3 (p‐Smad2/3), and inhibitory Smad7 expression in SHRs renal tissues via Western blot. Results demonstrated significantly reduced TGF‐β1, p‐Smad2, and p‐Smad3 protein expression in the IL‐17A NAb group versus controls (Figure  5 , p   0.05). IL‐17A neutralisation suppressed TGF‐β/Smad pathway activation in SHRs renal tissues. Representative immunoblots of proteins in the TGF‐β/Smad pathway are shown, along with quantitative analysis of the relative expression levels of TGF‐β1, p‐Smad2, p‐Smad3, and Smad7. Data are expressed as mean ± SD ( n  = 6). Notably, IL‐17A functions not only as an indirect EMT regulator but also as a key renal inflammation driver. Previous studies demonstrate IL‐17A directly stimulates renal resident and infiltrating immune cells to secrete pro‐inflammatory cytokines (e.g., IL‐6, TNF‐α, and IL‐1β), creating a chronic inflammatory microenvironment [ 19 ]. This microenvironment exacerbates renal damage via pathways like NF‐κB while significantly upregulating TGF‐β1 expression—a central mediator linking inflammation to fibrosis. TGF‐β1 subsequently activates Smad signalling to drive EMT progression. We therefore hypothesize that IL‐17A neutralisation improves EMT by suppressing upstream inflammatory responses. To investigate this, we measured inflammatory cytokines in SHRs plasma (Figure  6A ) and renal tissue (Figure  6B ). Results showed IL‐17A neutralising antibody significantly reduced IL‐6, TNF‐α, IL‐1β, MCP‐1, and IL‐21 levels in both SHRs plasma and renal tissues (all p  < 0.05). IL‐17A neutralising antibody alleviated systemic and renal inflammation in SHRs. (A) Plasma levels of IL‐6, IL‐1β, TNF‐α, MCP‐1, and IL‐21 measured by ELISA. (B) Representative immunoblots and relative expression levels of IL‐6, IL‐1β, TNF‐α, MCP‐1, and IL‐21 proteins. Data are expressed as mean ± SD ( n  = 6). Neutralisation of IL‐17A significantly reduced the expression levels of pro‐inflammatory cytokines such as IL‐6, TNF‐α, and IL‐1β, which act as central drivers of the inflammatory cascade and exacerbate renal injury through activation of specific signalling pathways. Previous studies have indicated that the pro‐inflammatory effects of IL‐17A depend on the coordinated activation of canonical signalling pathways, including JAK/STAT, PI3K/AKT, and NF‐κB [ 20 , 21 ]. Among these, the JAK/STAT pathway promotes the transcription of inflammatory genes via phosphorylation of STAT3 (pSTAT3), the PI3K/AKT pathway enhances inflammatory responses through phosphorylation of PI3K and AKT, and phosphorylation of P65 in the NF‐κB pathway further drives excessive release of inflammatory mediators, thereby aggravating tissue injury [ 22 ]. Our results demonstrate that IL‐17A neutralisation markedly suppressed the activation of these pathways, as evidenced by reduced phosphorylation‐to‐total protein ratios of JAK1, STAT3, PI3K, AKT, and P65 (Figure  7A,B , p  < 0.01)., consequently inhibiting the expression of pro‐inflammatory cytokines. Notably, in the present study, the expression of SOCS3, a negative regulator of the JAK/STAT pathway, remained unaltered (Figure  7B , p  > 0.05). Nevertheless, IL‐17A neutralisation effectively inhibited the phosphorylation of JAK1 and STAT3, suggesting that this intervention may modulate JAK/STAT signalling through mechanisms independent of SOCS3, either directly or indirectly, leading to decreased pro‐inflammatory cytokine production [ 23 ]. IL‐17A neutralisation attenuates renal injury by targeting the inflammatory network mediated by PI3K/AKT, JAK/STAT, and NF‐κB (P65) pathways. (A) Representative images and relative expression levels of proteins in the PI3K/AKT and P65 pathways. (B) Representative images and relative expression levels of proteins in the JAK1/STAT3 pathway and its regulator SOCS3. Data are expressed as mean ± SD ( n  = 6).

The roles of IL‐17A and pathways such as NF‐κB, PI3K/AKT, JAK/STAT, and TGF‐β/Smad in renal fibrosis have been established in CKD. However, their interplay in hypertensive nephropathy remains unclear. This study extends beyond mere observation of these factors in a new model by systematically analysing their changes following IL‐17A neutralisation in SHRs.” and “These effects are further linked to suppressed TGF‐β/Smad signalling and EMT. We demonstrate that IL‐17A blockade concurrently reduces multiple inflammatory pathways (JAK/STAT, PI3K/AKT, and NF‐κB) and specifically diminishes M2 macrophage polarisation. These effects are further linked to suppressed TGF‐β/Smad signalling and epithelial‐mesenchymal transition (EMT). Collectively, our results suggest that IL‐17A may act as a central coordinator, linking hypertensive stress to renal fibrosis by fostering a pro‐fibrotic immune environment via M2 macrophages and amplifying inflammatory networks. This integrated perspective reveals a multi‐level mechanism in hypertensive kidney injury, providing a clearer framework than single‐pathway studies and highlighting potential immunomodulatory strategies alongside blood‐pressure control. IL‐17A, a signature cytokine of Th17 cells, plays a pivotal role in bridging innate and adaptive immunity, particularly in inflammation, host defence, and autoimmunity [ 24 , 25 ]. Its signalling has been extensively investigated in various disease models. For example, intradermal IL‐17A injection in STAT3‐overexpressing mice induces psoriasis‐like dermatitis with upregulation of psoriasis‐associated genes and inflammatory mediators [ 26 ]. In allergic asthma, IL‐17A knockdown activates the xCT‐GSH‐GPX4 axis, inhibiting ferroptosis in airway epithelial cells and alleviating inflammation [ 27 ]. Similarly, in a rosacea‐like model, IL‐17A neutralisation reduces skin damage, inflammatory mediators, TLR4/NF‐κB signalling, angiogenesis, and TGF‐β1‐driven EMT and fibrosis [ 28 ]. These studies underscore the broad pathogenic influence of IL‐17A across inflammatory and fibrotic conditions. Hypertensive renal injury and its progression to fibrotic remodelling constitute a core pathology of CKD. Previous work has linked IL‐17A to CKD development [ 29 ] and its synergy with TGF‐β in promoting EMT [ 30 ]. In renal fibrosis, IL‐17A directly induces TGF‐β1 expression in tubular epithelial cells, activating Smad2/3 signalling and extracellular matrix deposition [ 31 ]. A similar mechanism operates in pulmonary fibrosis, where IL‐17 upregulates TGF‐β1 and Smad2/3 phosphorylation in alveolar epithelial cells [ 32 ]. This IL‐17‐initiated TGF‐β1/Smad cascade, often coordinated with ERK1/2 activation, underscores the cytokine's central role in fibrosis across organs. Building on this foundation, our study shows that IL‐17A neutralisation in SHRs exerts potent anti‐fibrotic effects, suppressing renal overexpression of α‐SMA and Collagen III, reducing TGF‐β1 levels, and inhibiting Smad2/3 phosphorylation. Future studies should further delineate the precise crosstalk between IL‐17A and TGF‐β signalling in hypertensive nephropathy progression. The TGF‐β/Smad pathway not only regulates EMT and fibrosis but also drives macrophage polarisation toward the M2 phenotype. TGF‐β via Smad2/3 upregulates M2 markers and anti‐inflammatory cytokines while suppressing M1 markers [ 33 , 34 ]. In diabetic wound healing, enhanced Smad2/3 phosphorylation promotes M2 polarisation and accelerates closure, whereas TGF‐β/Smad inhibition reverses these effects [ 33 ]. Similarly, in high‐altitude pulmonary hypertension, hypoxia‐activated TGF‐β–Smad2/3 signalling correlates with M2 accumulation and disease progression, attenuated by pathway blockade [ 34 ]. These findings highlight the TGF‐β/Smad axis as a key regulator of M2 polarisation in diverse pathologies. Emerging evidence indicates that IL‐17A also critically influences macrophage polarisation. Mechanistically, IL‐17A can directly drive M2 polarisation via NF‐κB activation, upregulating CD163, CD206, and related genes—an effect blocked by NF‐κB inhibitors [ 35 ]. In endometriosis, IL‐17A indirectly promotes M2 marker expression and macrophage recruitment [ 36 ]. In cervical cancer, IL‐17A enhances M2 polarisation and tumour‐promoting functions of macrophages [ 37 ]. Consistent with these reports, our study in hypertensive renal injury shows that IL‐17A neutralisation suppresses renal M2 polarisation. Thus, IL‐17A appears to steer macrophages toward an M2 phenotype across contexts, though its mode of action and functional outcomes may vary with the disease microenvironment, supporting its role as a context‐dependent regulator of macrophage plasticity. Conversely, IL‐17A can exacerbate local inflammation by inducing pro‐inflammatory cytokines and activating immune cells [ 38 ]. Sustained IL‐17A‐driven inflammation promotes renal fibrosis, tubular atrophy, and glomerulosclerosis, contributing to renal function loss [ 39 ]. IL‐17A amplifies inflammation through multiple pathways [ 40 , 41 ]. For instance, its neutralisation or silencing alleviates bleomycin‐induced pulmonary fibrosis by suppressing complement activation, epithelial apoptosis, and collagen deposition [ 42 ]. Our data corroborate that IL‐17A neutralisation effectively mitigates renal fibrosis and inflammation in SHRs. IL‐17A NAb treatment downregulated pro‐inflammatory cytokines (IL‐6, TNF‐α, IL‐1β, MCP‐1, and IL‐21) and inhibited activation of JAK/STAT, PI3K/AKT, and NF‐κB pathways. Notably, while JAK1 and STAT3 phosphorylation were reduced, SOCS3 expression remained unchanged, suggesting that IL‐17A NAb inhibits JAK/STAT signalling via SOCS3‐independent mechanisms. These may involve other SOCS family members, activation of protein tyrosine phosphatases, inhibition of upstream IL‐17 receptor signalling, or cross‐inhibition from concurrently downregulated pathways such as PI3K/AKT [ 43 , 44 , 45 ]. Further investigation of specific phosphatases or broader SOCS protein panels will clarify the precise mechanisms involved. The clinical relevance of targeting IL‐17A is supported by the approval of the anti‐IL‐17A monoclonal antibody secukinumab for Crohn's disease and psoriasis [ 46 ]. Moreover, elevated IL‐17A and STAT3 mRNA levels are reported in hypertension‐induced target organ damage [ 47 ]. Our study extends these observations by clarifying IL‐17A's role in hypertensive renal injury and demonstrating the dual anti‐inflammatory and anti‐fibrotic effects of IL‐17A NAb. Unlike earlier studies focusing on isolated pathways, we systematically show concurrent modulation of JAK/STAT, PI3K/AKT, and NF‐κB pathways upon IL‐17A neutralisation, offering a more integrated mechanistic understanding and underscoring IL‐17A's therapeutic potential. Regarding the experimental design, it is noted that a normotensive WKY rat group was not included in the main 20‐week interventional study. This is because the primary objective was to evaluate the therapeutic effect and mechanism of IL‐17A neutralisation within the context of established hypertensive renal injury, rather than to delineate differences between hypertensive and normotensive states. The use of both isotype (IgG) and vehicle (PBS) control groups within the SHR cohort was designed to specifically isolate the effects attributable to IL‐17A blockade. While the SHR model is a valuable tool for studying hypertension and target organ damage, its direct translational relevance to human hypertensive nephropathy requires careful consideration due to important species‐specific differences. The pathogenesis of hypertension in SHRs relies heavily on genetic drivers of sympathetic overactivity and renal sodium handling, which do not fully capture the complex, multifactorial aetiology seen in humans [ 48 ]. Additionally, renal injury in SHRs progresses more rapidly and uniformly compared to the slower, variable course in patients, potentially affecting therapeutic interpretations [ 49 ]. Furthermore, human hypertensive nephropathy is often complicated by comorbidities like diabetes and obesity, which are absent in the standard SHR model. Thus, while our findings in SHRs demonstrate a pathogenic role for IL‐17A and support its neutralisation as a therapeutic strategy within this model, they represent a preclinical proof‐of‐concept rather than a direct prediction of human outcomes. Clinical translation will require validation in more complex models that incorporate disease heterogeneity and, ultimately, in human patient populations. Several additional limitations of this study warrant consideration. First, the inclusion of only male SHRs, while avoiding the confounder of estrous cycle‐related hormonal fluctuations, limits the generalizability of our findings to females. Given the known immunomodulatory and renoprotective effects of oestrogen, therapeutic responses to IL‐17A neutralisation may differ by sex. Second, the experimental design does not fully disentangle the direct antifibrotic and anti‐inflammatory effects of IL‐17A blockade from its indirect benefits mediated through systemic blood pressure reduction. Although renal signalling pathways were modulated, part of the observed renoprotection could be secondary to improved hypertension. Furthermore, blood pressure was monitored using the tail‐cuff method, which is susceptible to stress‐induced variability and provides intermittent rather than continuous measurements, potentially affecting data accuracy. On a mechanistic level, systemic IL‐17A neutralisation cannot identify the precise cellular source (e.g., Th17 cells, γδ T cells, or neutrophils) responsible for the renal benefits. Although the anti‐IL‐17A monoclonal antibody used is well‐validated and an isotype control was included, we cannot formally exclude potential off‐target effects in vivo. Finally, while the SHR model recapitulates key features of human hypertensive nephropathy, its homogeneous genetic background, accelerated disease progression, and early therapeutic intervention differ from the heterogeneity, chronicity, and later diagnosis typical of human disease. Our findings thus require further validation in other chronic kidney disease models (e.g., diabetic nephropathy or unilateral ureteral obstruction) and reinforcement through genetic approaches or rescue experiments.

In conclusion, this study systematically elucidated the mechanisms by which IL‐17A NAb suppresses renal fibrosis and inflammation, highlighting its regulatory effects through the modulation of multiple signalling pathways. These findings provide new insights into potential therapeutic strategies for hypertension‐associated CKD.

Hypertension represents a major global public health issue, affecting over 1 billion people worldwide. Its detrimental effects extend beyond cardiovascular complications to encompass progressive target organ damage [ 1 ]. Of particular concern, hypertensive nephropathy has emerged as a leading cause of chronic kidney disease (CKD) and end‐stage renal disease (ESRD), imposing significant clinical and economic burdens on patients and healthcare systems [ 2 ]. Under sustained hypertensive conditions, the kidneys undergo a cascade of pathophysiological changes. Progressive renal fibrosis represents the central pathological process and final common pathway [ 3 ]. Histologically, renal fibrosis manifests as tubular atrophy/dilation, interstitial fibroblast activation/proliferation, inflammatory cell infiltration, and excessive deposition and dysregulated remodelling of ECM components—including collagens I, III, and IV, as well as fibronectin [ 4 ]. The resultant replacement of functional renal parenchyma with fibrotic scar tissue underlies the progressive decline in glomerular filtration rate and ultimately drives irreversible renal function loss [ 5 ]. Although RAS inhibitors—including ACE inhibitors and angiotensin receptor blockers—remain the cornerstone of current antihypertensive and renoprotective therapy, their efficacy is primarily limited to blood pressure control and early renal injury mitigation. These agents demonstrate notably restricted effectiveness against established or progressive fibrotic processes [ 6 ], often failing to prevent eventual progression to ESRD. Therefore, elucidating the underlying mechanisms of renal fibrosis and identifying novel therapeutic targets and strategies capable of effectively intervening in the fibrotic process are of paramount importance for improving the long‐term prognosis of patients with hypertensive nephropathy. Mounting evidence indicates that chronic low‐grade inflammation is a key driver of hypertension and its associated target organ damage [ 7 ]. Within the kidneys, immune cell infiltration and activation of pro‐inflammatory cytokine networks persist throughout the course of hypertensive renal injury and directly promote fibrogenesis. Among these mediators, IL‐17A—a potent pro‐inflammatory cytokine primarily produced by Th17 cells and others—has been observed to be significantly elevated in the serum and renal tissues of both hypertensive patients and animal models. Furthermore, its levels correlate closely with proteinuria, declining renal function, and the extent of renal fibrosis [ 8 , 9 ]. IL‐17A can directly stimulate intrinsic renal cells (such as fibroblasts and tubular epithelial cells), inducing them to secrete increased levels of pro‐inflammatory cytokines, chemokines, and pro‐fibrotic factors like TGF‐β. It also promotes ECM synthesis while inhibiting its degradation, thereby directly driving renal fibrosis [ 10 ]. Our previous animal studies demonstrated that SHRs exhibit significantly elevated levels of IL‐17A in both the spleen and kidneys compared with normotensive Wistar‐Kyoto (WKY) rats (Figure  S1 ). This finding prompted us to hypothesize that the IL‐17A signalling pathway may play a critical role in the pathogenesis of hypertensive nephropathy and the associated renal interstitial fibrosis. However, whether targeted neutralisation of IL‐17A can alleviate hypertensive renal injury and the underlying mechanisms involved remain elusive. Therefore, this study aims to systematically evaluate the effects of IL‐17A neutralisation on the progression of hypertension, renal function, and renal fibrosis in SHRs, and to further investigate whether its renoprotective role involves the coordinated modulation of multiple signalling pathways—including JAK/STAT, PI3K/AKT, NF‐κB, and TGF‐β/Smad—as well as potential crosstalk between macrophage polarisation and EMT. These investigations are expected to elucidate the role of IL‐17A as a central node within the inflammation‐fibrosis network in hypertensive nephropathy, thereby providing novel mechanistic insights and experimental evidence for immunomodulatory therapies targeting IL‐17A.

The authors declare no conflicts of interest.

Male SHRs were procured from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Licence No.: SCXK (Jing) 2021‐0006). To avoid potential confounding effects of hormonal fluctuations on hypertension and renal pathology, only male rats were included in the present study. All animals were confirmed healthy by physical examination and housed under specific pathogen‐free (SPF) conditions. Environmental parameters were strictly controlled: temperature (20°C ± 2°C), relative humidity (50% ± 5%), and a 12‐h light/dark cycle. Standard rodent diet and water were available ad libitum throughout the experimental period. SHRs were allowed to acclimate for a week before starting procedures. To assess the potential renoprotective effect of IL‐17A neutralisation against renal fibrosis, SHRs were treated beginning at 10 weeks of age (initial body weight 220–250 g). The rats were randomly allocated to three experimental groups ( n  = 6 per group): (1) the IL‐17A neutralisation antibody (NAb) group, receiving intraperitoneal (i.p.) injections of anti‐IL‐17A monoclonal antibody (Bio X Cell, USA, catalogue #BE0173) at 1 mg/kg twice weekly; (2) the isotype control group, administered an equivalent dose of control IgG (Bio X Cell, USA, catalogue #BE0088) following the same schedule and route; and (3) the vehicle control group, injected i.p. with an equal volume of sterile phosphate‐buffered saline (PBS). Treatment was maintained for 20 weeks. All animals were humanely euthanized within 24 h after the final injection for tissue collection. Euthanasia was performed under deep anaesthesia induced by intraperitoneal injection of sodium pentobarbital (150 mg/kg), followed by confirmation of cardiac and respiratory cessation. Spleens harvested from rats were minced under sterile conditions and gently homogenised in PBS. The homogenate was passed through a 70‐μm cell strainer, and cells were pelleted by centrifugation (1500× g , 5 min, 4°C). After discarding the supernatant, the pellet was resuspended in 2 mL of red blood cell (RBC) lysis buffer (Absin, China) to remove erythrocytes. A single‐cell suspension was then prepared, and total cells were counted with a haemocytometer. The cell concentration was adjusted to 1 × 10 7  cells/mL for subsequent steps. Splenic lymphocytes were stimulated for 4 h at 37°C in a 5% CO 2 incubator in the presence of a protein transport inhibitor cocktail (BD Pharmingen, USA). Cells were collected by centrifugation (1200× g , 5 min, 4°C), resuspended in 100 μL PBS, and incubated with APC‐conjugated anti‐rat CD4 antibody (Thermo Fisher Scientific, USA) for 30 min at 4°C in the dark for surface staining. After washing with PBS, intracellular IL‐17A staining was performed using a commercial fixation/permeabilization kit (BD Pharmingen, USA) according to the manufacturer's instructions. Briefly, cells were fixed, permeabilized, and incubated with PE‐conjugated anti‐IL‐17A antibody (Thermo Fisher Scientific, USA) for 30 min at 4°C in the dark. Finally, stained cells were resuspended in 1 mL flow cytometry staining buffer. Fluorescence data were acquired on a BD FACSCanto flow cytometer and analysed using FlowJo software (v10.8.1; Tree Star). The gating strategy for flow cytometry is shown in Figure  S2 . Single‐cell suspensions from renal tissue were prepared as described above. An aliquot of the cell suspension was incubated with a panel of fluorescently labelled antibodies for 40 min at 4°C in the dark. The antibody panel included APC‐conjugated anti‐CD68 (abcam, UK), PE‐conjugated anti‐CD86 (BD Biosciences, USA), and BB700‐conjugated anti‐CD163 (BD Biosciences, USA). After staining, cells were washed twice with PBS to remove unbound antibodies and finally resuspended in 1 mL of flow cytometry staining buffer. Data acquisition was performed on a flow cytometer, and analysis was conducted using FlowJo software (v10.8.1; Tree Star). The gating strategy for flow cytometry is shown in Figure  S3 . Systolic blood pressure (SBP) was measured non‐invasively in conscious rats using tail‐cuff plethysmography (XH‐200 Constant Temperature Non‐invasive Blood Pressure System; China). It is acknowledged that this method is susceptible to stress‐induced variability and provides intermittent rather than continuous data, with radiotelemetry considered the gold standard for continuous, stress‐free monitoring. Nevertheless, the tail‐cuff approach was selected for this study due to its practicality for long‐term, repeated measurements across multiple experimental cohorts. To mitigate the inherent limitations, rigorous acclimation protocols were employed: all animals underwent systematic acclimatisation to restraint over a 5‐day period prior to data collection. During each measurement session, the instrument was pre‐warmed for 30 min to maintain the tail chamber at 37°C. Rats were placed in restraints, their tails positioned through the cuff, and allowed to habituate until visibly calm (typically 5–10 min). Recording began only when the animals were quietly at rest. The cuff automatically inflated to occlude caudal artery flow (pulse signal disappearance) and then gradually deflated; SBP was recorded at the point of pulse signal reappearance. A minimum of 10 consecutive cycles were obtained per session. Following the exclusion of the highest and lowest readings, the mean SBP was calculated from the remaining stable values. Terminal blood samples were collected from all groups via the orbital plexus under deep anaesthesia immediately prior to euthanasia. Blood was transferred into serum separator tubes and clotted at room temperature for 30 min. Samples were centrifuged (4000× g , 15 min, 4°C), and serum was aliquoted and stored at −80°C. Renal function biomarkers were assayed using an AU5800 automated clinical chemistry analyser (Beckman Coulter, Brea, CA, USA). Renal tissue samples from each rat group were fixed in 10% neutral buffered formalin at 4°C for 48 h. Tissues were then dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin. Serial sections (4–5 μm thick) were cut from the paraffin blocks. Sections were deparaffinised in xylene, rehydrated through a graded ethanol series, and stained. Haematoxylin and eosin (H&E) staining was used to assess general histopathological changes, while Masson‘s trichrome staining specifically visualised collagen deposition. The extent of renal interstitial fibrosis was quantified by measuring the collagen‐positive area in Masson‘s trichrome‐stained sections using ImageJ software (v1.53; NIH). For each renal section, five non‐overlapping, cortical fields were randomly selected. After colour deconvolution to isolate the blue (collagen) stain, a consistent threshold was applied to all images to differentiate positive staining from background. The percentage of fibrosis area (blue‐positive area/total tissue area × 100%) was calculated for each field, and the mean value per animal was used for statistical analysis. Paraffin sections (4 μm thick) were deparaffinised and rehydrated. Antigen retrieval was performed using citrate buffer (pH 6.0) under high pressure. Endogenous peroxidase activity was blocked by incubating sections with 3% H 2 O 2 for 15 min at room temperature. Non‐specific binding was blocked with 5% bovine serum albumin (BSA) for 40 min at room temperature. Sections were then incubated overnight at 4°C with primary antibodies against: E‐cadherin (Boster, China), Collagen III (Boster, China), inducible nitric oxide synthase (iNOS) (Boster, China), CD86 (Boster, China), arginase‐1 (Arg‐1) (Boster, China), CD163 (Boster, China), α‐smooth muscle actin (α‐SMA) (Cell Signalling Technology, USA). After washing with Tris‐buffered saline (TBS; 3 × 5 min), sections were incubated with appropriate HRP‐conjugated secondary antibodies for 45 min at room temperature. Following TBS washes (3 × 5 min), immunoreactivity was visualised using diaminobenzidine (DAB) substrate. Sections were counterstained with haematoxylin, dehydrated, cleared in xylene, and mounted with neutral balsam. All IHC slides were analysed in a blinded manner regarding the treatment groups. Protein expression was quantified by calculating the percentage of positively stained area per field. Five non‐overlapping cortical fields per section were captured under a 40× objective lens (corresponding to a scale bar of 25 μm) using a light microscope with a calibrated digital camera under identical lighting conditions. Images were analysed using ImageJ software (v1.53; NIH). For each marker, a colour threshold was set to identify DAB‐positive (brown) staining, and the same threshold was applied uniformly across all images within the same experimental batch. The positive area percentage (positive pixel area/total tissue pixel area × 100%) was measured. The average value from the five fields represented the expression level for that sample. Total RNA from rat kidney tissues was extracted using the Trizol reagent (Sangon Biotech, Shanghai, China) according to the manufacturer‘s protocol. Reverse transcription to synthesise cDNA was performed at 42°C using the MightyScript Plus First‐Strand cDNA Synthesis Kit (Sangon Biotech, Shanghai, China). Gene expression quantification was conducted via qPCR using the SGExcel FastSYBR Master Mix (Sangon Biotech, Shanghai, China) in a final reaction volume of 10 μL. The qPCR amplification program was set as specified in the manufacturer‘s protocol: pre‐denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 5 s and annealing/extension at 60°C for 30 s. GAPDH was used as the internal reference gene, and the relative expression levels of target genes were calculated using the comparative cycle threshold (Ct) method (2 −∆∆Ct ). The sequences of the primers used for the analysis are listed in Table  S1 . Serum samples (1.5 mL per rat, collected as described in Section  2.3 ) were analysed for IL‐6, IL‐1β, TNF‐α, MCP‐1, and IL‐21 concentrations using commercial ELISA kits (Boster Biological Technology, China) according to manufacturer protocols. Absorbance at 450 nm (OD450) was measured with a microplate reader. Standard curves generated from reference standards were used to determine cytokine concentrations in all samples. Renal tissue samples (50 mg) were homogenised in 500 μL RIPA buffer on ice. Homogenates were incubated on ice for 40 min, then centrifuged (12 000× g , 10 min, 4°C). Supernatants were collected for protein quantification using a BCA assay kit with BSA standards. Protein concentrations were equalised, mixed with 5× SDS loading buffer, denatured (100°C, 10 min), and cooled on ice. Equal protein amounts underwent 10% SDS‐PAGE followed by PVDF membrane transfer. Membranes were blocked with 5% non‐fat milk in TBST for 1.5 h at room temperature and incubated overnight at 4°C with primary antibodies (detailed in Table  S2 ). After five 5‐min TBST washes, membranes were incubated with HRP‐conjugated goat anti‐rabbit secondary antibody (Boster Biological Technology, China, 1:10000) for 2 h at room temperature. Protein bands were visualised using enhanced chemiluminescence substrate and imaged with a ChemiDoc imaging system. Band densities were quantified using ImageJ software (v1.53; NIH). Data are presented as the mean ± standard deviation (SD). The normality of distribution was assessed using the Shapiro–Wilk test, and the equality of variances was evaluated with the F ‐test. For data satisfying both normality and homogeneity of variance, differences between two groups were analysed using an unpaired two‐tailed Student‘s t ‐test. When variances were unequal, Welch‘s correction was applied. Comparisons among three or more groups were performed by one‐way analysis of variance (ANOVA) followed by Tukey‘s post hoc test. For data that did not follow a normal distribution, the Mann–Whitney U test was used for two‐group comparisons, and the Kruskal–Wallis test followed by Dunn‘s multiple comparison test was applied for multi‐group comparisons. All statistical analyses were conducted using GraphPad Prism software (version 10.1.2). Statistical significance was determined at p  < 0.05.

Data S1: Supporting Information.