FNDC5/Irisin-Dependent Renoprotection of Resistance Training in Myocardial Infarction–Induced Type 2 Cardiorenal Syndrome

FNDC5/Irisin-Dependent Renoprotection of Resistance Training in Myocardial Infarction–Induced Type 2 Cardiorenal Syndrome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article FNDC5/Irisin-Dependent Renoprotection of Resistance Training in Myocardial Infarction–Induced Type 2 Cardiorenal Syndrome Weiyu Fu, Jun Lin, Wenqian Lin, Kai Zeng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6998289/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Type 2 cardiorenal syndrome (CRS), driven by chronic myocardial infarction (MI), has attracted increasing research attention due to its involvement in renal fibrosis and oxidative stress. However, the mechanisms underlying renal damage and potential rehabilitation strategies remain inadequately understood. This study aims to explore whether resistance exercise mitigates MI-induced renal dysfunction by regulating the FNDC5/Irisin axis. Using both wild-type and FNDC5/Irisin knockout mice, we established a Type 2 CRS model and implemented a resistance exercise intervention. In parallel, H 2 O 2 -stimulated HKC cells were used to construct an in vitro fibrosis model. We assessed renal injury, oxidative stress, fibrosis-related signaling (TGF-β1/Smad2/3), and the expression of key antioxidant and fibrotic markers. Our results demonstrated that resistance exercise significantly improved cardiac and renal function, reduced oxidative stress and collagen deposition, and inhibited TGF-β1–Smad2/3 activation. These effects were largely abrogated in Irisin-deficient mice. Additionally, rhIrisin and AICAR alleviated oxidative stress and fibrosis in vitro, confirming the functional role of Irisin. In conclusion, resistance exercise exerts renoprotective effects in Type 2 CRS through upregulation of FNDC5/Irisin, highlighting a novel therapeutic target and providing scientific support for exercise-based rehabilitation strategies in heart failure patients with renal involvement. Health sciences/Cardiology Biological sciences/Cell biology Health sciences/Diseases Health sciences/Nephrology Resistance exercise FNDC5/Irisin Type 2 cardiorenal syndrome Oxidative stress Renal fibrosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Heart failure (HF) is a major cause of mortality among individuals with non-communicable diseases worldwide [ 1 ]. HF frequently leads to complications involving distant organs, with renal impairment being particularly common [ 2 ]. Clinical studies have reported that patients with chronic HF often exhibit signs of renal insufficiency or dysfunction [ 3 ]. Type 2 cardiorenal syndrome (Type 2 CRS) refers to a clinical condition characterized by structural and functional renal impairment resulting from prolonged myocardial ischemia and hypoxia due to myocardial infarction (MI) or chronic HF [ 4 , 5 ]. Evidence suggests that HF patients with concomitant renal injury exhibit significantly worse cardiac dysfunction and higher mortality risk compared to those without renal involvement [ 6 , 7 ]. Improving renal function has been shown to enhance cardiac performance and survival outcomes in HF patients, thereby reducing disease-related mortality [ 8 , 9 ]. Thus, identifying effective strategies to prevent or mitigate renal structural damage and functional decline in Type 2 CRS is of critical importance for improving prognosis. Following MI, weakened myocardial contractility and reduced cardiac output elevate peripheral vascular resistance, resulting in sustained renal ischemia and hypoxia. This, in turn, intensifies oxidative stress and inflammatory responses in renal tissues, promotes collagen deposition in the renal interstitium, and ultimately leads to renal interstitial fibrosis (RIF) and renal dysfunction [ 10 ]. Exercise is recognized as a key intervention for the prevention and rehabilitation of chronic diseases [ 11 ]. Studies have shown that aerobic exercise can ameliorate renal impairment in Type 2 CRS [ 12 ], and resistance exercise can significantly inhibit renal fibrosis and improve kidney function in animal models of chronic kidney disease [ 13 ]. However, whether resistance exercise can effectively suppress renal interstitial collagen deposition in the context of Type 2 CRS remains unclear. Irisin, a newly identified exercise-induced myokine, has been shown to exert a range of biological effects, including regulation of energy metabolism and inhibition of oxidative stress, fibrosis, and apoptosis [ 14 , 15 ]. Exercise training activates PGC-1α, which induces the expression of downstream FNDC5; this precursor protein undergoes cleavage to generate circulating Irisin [ 16 ]. Irisin exerts protective effects in multiple organs, including the heart, skeletal muscle, kidney, and brain [ 17 , 18 ]. In mice, Irisin administration alleviates angiotensin II (Ang II)-induced myocardial fibrosis [ 19 ]. Aerobic exercise has been shown to upregulate renal Irisin expression in MI mice, thereby reducing renal damage [ 12 ]. Nevertheless, it remains unknown whether resistance exercise can stimulate endogenous Irisin expression and consequently improve renal injury in Type 2 CRS, as well as the underlying mechanisms involved. To address this question, the present study employed both wild-type (WT) and global FNDC5/Irisin knockout mice to establish a MI-induced Type 2 CRS model combined with a resistance exercise intervention. Additionally, an in vitro fibrosis model was established by exposing human embryonic kidney tubular epithelial (HKC) cells to H 2 O 2 -induced oxidative stress. The aim was to investigate the role and mechanism of Irisin in mediating the effects of resistance exercise on renal interstitial collagen deposition in Type 2 CRS. This study provides experimental evidence to support the use of exercise rehabilitation strategies for patients with cardiorenal syndrome. 2. Materials and Methods 2.1 Experimental Animals, Model Establishment, and Exercise Protocol All animal experiments were conducted in accordance with the guidelines set by the Animal Care and Use Committee of Xi'an Jiaotong University . The study protocol was approved by the committee under protocol number XJTU-2024-RE-004 . Eight-week-old healthy male C57BL/6J mice (purchased from the Animal Experiment Center of Xi'an Jiaotong University) and global FNDC5/Irisin knockout mice (customized by Cyagen Biosciences Inc.) were housed under standard laboratory conditions with ad libitum access to food and water. All animal procedures complied with the Guidelines for the Care and Use of Laboratory Animals. A total of 24 wild-type (WT) mice were randomly divided into three groups: sham-operated group (S), myocardial infarction group (MI), and myocardial infarction plus resistance exercise group (MR). Similarly, 24 FNDC5/Irisin knockout mice were randomly assigned to the knockout sham group (KS), knockout MI group (KMI), and knockout MI plus resistance exercise group (KMR). Myocardial infarction (MI) was induced by permanent ligation of the left anterior descending coronary artery under isoflurane inhalation anesthesia. Echocardiography was performed postoperatively to confirm the success and consistency of modeling. Only mice with comparable echocardiographic outcomes were randomly assigned to experimental groups. The sham groups (S and KS) underwent thoracotomy and suture placement without coronary ligation to exclude surgical stress effects. Resistance training was conducted based on previously described protocols [ 20 , 21 ]. Starting one week after surgery, mice in the MR and KMR groups underwent ladder-climbing resistance training. The climbing ladder was 1.1 meters high with 1-cm grid spacing and an 85° incline. Mice were first subjected to 5 days of adaptive training without additional load. Formal training then proceeded for 4 weeks with progressive loading. The load was increased daily by 10% of body weight until reaching 75%, which was maintained until the end of the intervention. Each training session consisted of 8 sets of 3 climbs per set, with 1-minute rest between sets, 5 days per week for 4 weeks. This study was conducted in compliance with the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines to ensure transparency in the reporting of animal experiments. 2.2 HKC Cell Culture and Interventions Human embryonic kidney tubular epithelial (HKC) cells were cultured in F12/DMEM (1:1) medium under standard conditions (37°C, 5% CO 2 , Thermo Incubator). Cells between passages 3 and 7 were used for experiments. Oxidative stress-induced fibrosis was modeled by exposing cells to 100 µmol/L H 2 O 2 for 4 hours [ 22 ]. Recombinant human Irisin protein (rhIrisin) was applied at 1 µg/mL for 12 hours [ 23 ]. AICAR, an AMPK agonist, was used to mimic exercise effects at 500 µmol/L for 12 hours [ 24 , 25 ]. Cells were assigned to five groups: Control, H 2 O 2 , H 2 O 2 + AICAR, H 2 O 2 + rhIrisin, and H 2 O 2 + rhIrisin + AICAR. 2.3 Echocardiographic Assessment One day after the final resistance training session, mice were anesthetized with isoflurane (mixed with oxygen at a ratio of 1:5) and placed in the supine position. Cardiac function was evaluated using a VINNO ultrasound system (VINNO, Suzhou, China). The following parameters were measured: left ventricular internal diameter at end-diastole (LVIDd), left ventricular internal diameter at end-systole (LVIDs), and ejection fraction (EF). Fractional shortening (FS) was calculated to assess cardiac function. 2.4 Detection of Biomarkers Blood was collected via retro-orbital puncture and centrifuged at 1,000 r/min for 5 minutes to isolate serum. Serum levels of creatinine (Scr) and blood urea nitrogen (BUN), indicators of renal injury, were quantified using commercial assay kits (Nanjing Jiancheng Bioengineering Institute). Kidney tissues were homogenized in precooled 0.86% saline (1:9, w/v), and supernatants were obtained by centrifugation for malondialdehyde (MDA) measurement using oxidative stress detection kits. All assays were conducted according to the manufacturer’s instructions, and absorbance was measured using a microplate reader (Bio-Tek Instruments). 2.5 Histological Staining Paraffin-embedded kidney sections were subjected to Masson’s trichrome and periodic acid–Schiff (PAS) staining and examined under an optical microscope (Olympus BX51, Japan). Collagen volume fraction (CVF) was quantified as the percentage of the tissue area stained blue. For PAS-stained sections, five random fields per section were selected to assess the integrity of the brush border of renal tubular epithelial cells, serving as an indicator of tubular damage. Tubular injury was scored based on the following criteria: 0 = normal; 1 = < 25% of tubules damaged per field; 2 = 25–50%; 3 = 50–75%; 4 = 75–100%. Tubular injury was defined as nuclear loss, reduced or absent brush border, epithelial swelling or vacuolar degeneration [ 26 ]. 2.6 RT-qPCR Total RNA was extracted from renal tissues using Trizol reagent. cDNA was synthesized using a reverse transcription kit (TaKaRa, Japan) according to the manufacturer's protocol. qPCR amplification was performed using a SYBR Green-based PCR kit (TaKaRa). The primers (synthesized by Sangon Biotech, Shanghai, China) were as follows: FNDC5-F: 5′-GGCTGGGAGTTCATGTGGAA-3′ FNDC5-R: 5′-TGGGAAGCGGTTATCTTTGCT-3′ GAPDH-F: 5′-CAGTGCCAGCCTCGTCTCAT-3′ GAPDH-R: 5′-AGGGGCATCCACAGTCTTC-3′ 2.7 Western Blotting Kidney tissues were minced, homogenized (Model F6/10, FLUKO), and centrifuged to collect supernatants. HKC cells were lysed post-intervention, collected by scraping, and sonicated (JY-25013, Jining Tianhua). Protein concentration was determined using a BCA kit. Samples were mixed with 5× loading buffer and RIPA, boiled at 100°C for 10 min, and stored at − 20°C until use. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with 5% skim milk or 5% BSA for 60 min at room temperature. Primary antibodies were used at the following dilutions: FNDC5 (1:1000), TGF-β1 (1:1000), Smad2/3 (1:1000), p-Smad2/3 (1:1000), SOD1 (1:1000), SOD2 (1:1000), MMP2 (1:1000), MMP9 (1:1000), α-SMA (1:1000), Collagen-1 (1:1000), Collagen-3 (1:1000), CTGF (1:500), GAPDH (1:5000), and β-actin (1:5000). Membranes were incubated overnight at 4°C, followed by washing and incubation with HRP-conjugated secondary antibodies for 90 min. Protein bands were visualized using the Bio-Rad ChemiDoc™, MP imaging system (Universal Hood III, Bio-Rad, USA) and analyzed digitally. 2.8 Data Acquisition and Statistical Analysis Western blot results were quantified using Image Lab 5.1 software (Bio-Rad, CA, USA). All statistical analyses and graphical outputs were performed using GraphPad Prism 8.0.2 (GraphPad Software, La Jolla, CA, USA). One-way analysis of variance (ANOVA) was used to evaluate differences among groups, followed by Tukey’s post hoc test. Data were expressed as mean ± standard error (M ± SE). Statistical significance was defined at P < 0.05 and P < 0.01. 3. Results 3.1 Resistance Exercise Upregulates Renal FNDC5 Expression and Alleviates MI-Induced Renal Injury in Type 2 CRS Mice The expression levels of FNDC5, the precursor of Irisin, were assessed in renal tissues at both mRNA and protein levels. Compared with the sham-operated (S) group, the myocardial infarction (MI) group exhibited a significant reduction in FNDC5 gene and protein expression (P < 0.01). In contrast, mice in the myocardial infarction plus resistance exercise (MR) group showed significantly increased FNDC5 expression compared to the MI group (P < 0.01). As expected, FNDC5 gene expression was undetectable in FNDC5/Irisin knockout (KO) mice (Fig. 1A–B). Serum creatinine (Scr) and blood urea nitrogen (BUN) are commonly used indicators of renal dysfunction and injury [ 27 – 28 ]. The results revealed that serum Scr and BUN levels were significantly elevated in the MI group compared to the S group (P < 0.01). Resistance exercise intervention significantly reduced these levels in Type 2 CRS mice (P < 0.01). Additionally, serum Scr and BUN levels in the knockout MI (KMI) group were significantly higher than those in the MI group (P < 0.05, P < 0.01), and levels in the knockout MR (KMR) group were significantly elevated compared to the MR group (P < 0.05, P < 0.01, Fig. 1C–D). Periodic acid–Schiff (PAS) staining was used to evaluate tubular injury in kidney tissues, with the apical brush border of renal tubules appearing red and the epithelial cells light pink. In the MI group, brush border loss and higher tubular injury scores were observed relative to the S group (P < 0.01). Resistance training significantly reduced tubular injury scores in the MR group compared with the MI group (P < 0.01). Notably, the KMI group displayed significantly higher tubular injury scores than the MI group (P < 0.01), and the KMR group showed elevated scores compared to the MR group (P < 0.05, Fig. 1E–F). These findings confirm the successful establishment of the Type 2 CRS model induced by MI and demonstrate that resistance exercise effectively attenuates renal tubular brush border damage and improves kidney function. Moreover, FNDC5/Irisin appears to play a key role in mediating the renoprotective effects of resistance exercise. Echocardiographic results (Fig. 2 ) revealed that, compared with the sham-operated (S) group, mice in the myocardial infarction (MI) group exhibited significantly increased LVIDd and LVIDs (P < 0.01), along with markedly decreased ejection fraction (EF%) and fractional shortening (FS%) (P < 0.01). In contrast, the MR group showed significantly reduced LVIDd and LVIDs (P < 0.01), and significantly elevated EF% and FS% (P < 0.01), compared with the MI group. Furthermore, LVIDd and LVIDs were significantly higher in the KMI group than in the MI group (P < 0.01), while EF% and FS% were significantly lower (P < 0.01). Similarly, EF% and FS% in the KMR group were significantly lower than those in the MR group (P < 0.01). These findings confirm the successful establishment of the MI model and suggest that resistance exercise improves cardiac function in MI-induced Type 2 CRS. Moreover, FNDC5/Irisin appears to contribute to the cardioprotective effects observed during resistance training in ischemic hearts. (D) Ejection fraction (EF); (E) Fractional shortening (FS). Measurements were made using echocardiographic imaging to assess cardiac function. 3.2 Resistance Exercise Suppresses Renal Oxidative Stress and Interstitial Collagen Deposition in Type 2 CRS Mice As shown by oxidative stress assay kits and Western blotting results, renal malondialdehyde (MDA) levels were significantly elevated in the myocardial infarction (MI) group compared with the sham-operated (S) group (P < 0.01). Resistance exercise markedly enhanced the expression of antioxidant proteins SOD1 and SOD2 and reduced MDA levels in the kidneys of Type 2 CRS mice (P < 0.01). In contrast, the knockout MI (KMI) group exhibited increased MDA content (P < 0.01) and reduced SOD2 protein expression (P < 0.05) compared with the MI group. Similarly, MDA levels were elevated (P < 0.05), and both SOD1 and SOD2 protein levels were decreased (P < 0.05) in the knockout MR (KMR) group compared with the MR group (Fig. 3 A–C). To further assess renal fibrosis, Masson's trichrome staining and fibrosis-related protein expression were evaluated. Collagen fibers were visualized as blue deposits, indicating interstitial collagen accumulation. Compared with the S group, the MI group showed a significant increase in renal interstitial collagen deposition and fibrosis severity (P < 0.01). Resistance exercise markedly reduced collagen accumulation in the MR group compared with the MI group (P < 0.01). Conversely, collagen deposition in the KMI group was significantly higher than in the MI group (P < 0.05, Fig. 3 D–E). Western blotting analysis revealed that, relative to the S group, the MI group exhibited significantly elevated expression of fibrosis-related proteins including MMP2, MMP9, collagen-1, collagen-3, α-SMA, and CTGF (P < 0.01). These increases were significantly attenuated following resistance exercise intervention in Type 2 CRS mice (P < 0.01). Additionally, the KMI group showed upregulated MMP9, collagen-1, and collagen-3 expression compared with the MI group (P < 0.05, P < 0.01). In the KMR group, expression levels of MMP9, collagen-1, collagen-3, α-SMA, and CTGF were significantly higher than those in the MR group (P < 0.05, P < 0.01, Fig. 3 F–G). These findings indicate that resistance exercise significantly suppresses MI-induced renal interstitial collagen deposition and enhances renal antioxidant capacity in Type 2 CRS mice. Moreover, knockout of FNDC5/Irisin attenuates the inhibitory effects of resistance training on oxidative stress and fibrosis, suggesting a critical role of Irisin in mediating the renoprotective effects of exercise. 3.3 AICAR and rhIrisin Inhibit the TGF-β1–Smad2/3 Pathway to Attenuate H 2 O 2 -Induced Fibrosis in HKC Cells Oxidative stress is recognized as a key trigger of fibrosis [ 29 ]. To further elucidate the mechanism by which Irisin mediates the reduction of renal interstitial collagen deposition in MI mice undergoing resistance exercise, a fibrosis model was established in HKC cells using H 2 O 2 -induced oxidative stress. The effects of Irisin and AICAR on the TGF-β1–Smad2/3 signaling pathway were subsequently examined. Western blotting results revealed that, compared with the control group, H 2 O 2 exposure significantly increased TGF-β1 protein expression and the phosphorylation level of Smad2/3 (P < 0.01). However, treatment with AICAR and/or recombinant human Irisin (rhIrisin) significantly suppressed both TGF-β1 expression and Smad2/3 phosphorylation (P < 0.01, Fig. 4 ). These findings indicate that in HKC cells, AICAR and/or rhIrisin are capable of inhibiting the activation of the TGF-β1–Smad2/3 pathway induced by H 2 O 2 , thereby mitigating oxidative stress–driven fibrotic responses. To evaluate the effects of Irisin and AICAR on the antioxidant capacity and collagen synthesis in HKC cells, oxidative stress assay kits and Western blotting were used to measure the levels of MDA, SOD1, SOD2, MMP2, MMP9, collagen-1, collagen-3, α-SMA, and CTGF following H 2 O 2 exposure. The results demonstrated that, compared with the control group, H 2 O 2 intervention significantly decreased SOD2 protein expression (P < 0.01) while markedly increasing MDA levels and the protein expression of MMP2, MMP9, collagen-1, collagen-3, α-SMA, and CTGF (P < 0.01). Intervention with AICAR or rhIrisin after oxidative stress significantly enhanced the expression of antioxidant enzymes SOD1 and SOD2 (P < 0.01), and concurrently reduced MDA content and the expression of fibrosis-related proteins (P < 0.01). Furthermore, compared with the H 2 O 2 + AICAR group, the H 2 O 2 + rhIrisin + AICAR group exhibited increased SOD2 expression (P < 0.01) and significantly decreased levels of MDA, MMP9, collagen-3, and CTGF (P < 0.05, P < 0.01). Similarly, relative to the H 2 O 2 + rhIrisin group, the combined treatment group (H 2 O 2 + rhIrisin + AICAR) showed enhanced SOD2 expression (P < 0.01) and reduced MDA and CTGF levels (P < 0.05, P < 0.01) (Fig. 5 ). These findings suggest that AICAR and/or rhIrisin effectively attenuate oxidative stress and suppress the expression of fibrosis-associated proteins in HKC cells under oxidative injury conditions. 3.4 Resistance Exercise Inhibits Activation of the Renal TGF-β1–Smad2/3 Signaling Pathway in Type 2 CRS Mice Western blotting results revealed that, compared with the sham-operated (S) group, the myocardial infarction (MI) group exhibited significantly elevated expression of TGF-β1 and phosphorylation of Smad2/3 in kidney tissues (P < 0.01). These increases were markedly attenuated in the myocardial infarction plus resistance exercise (MR) group (P < 0.01). In addition, the knockout sham (KS) group showed significantly higher TGF-β1 expression than the S group (P < 0.01). The knockout MI (KMI) group displayed significantly increased levels of TGF-β1 and phosphorylated Smad2/3 compared with the MI group (P < 0.05, P < 0.01). Similarly, both TGF-β1 expression and Smad2/3 phosphorylation were significantly higher in the knockout MR (KMR) group than in the MR group (P < 0.01, Fig. 6 ). These findings suggest that resistance exercise suppresses MI-induced activation of the renal TGF-β1–Smad2/3 signaling pathway in Type 2 CRS. However, the inhibitory effect of resistance exercise on this profibrotic pathway is significantly weakened in the absence of FNDC5/Irisin, highlighting its regulatory role in exercise-mediated renal protection. 4. Discussion Prolonged myocardial infarction (MI) can cause damage to distant organs, including the kidneys. Persistent renal ischemia and hypoxia promote oxidative stress and inflammatory responses, leading to the activation and migration of myofibroblasts and excessive extracellular matrix (ECM) accumulation. These events contribute to renal interstitial collagen deposition and apoptosis, ultimately resulting in renal interstitial fibrosis (RIF) and functional deterioration [ 10 , 30 – 31 ]. Thus, mitigating MI-induced remote organ injury, particularly renal dysfunction, is critical for improving the quality of life in patients with heart failure. Exercise has been shown to reduce the risk of progression from heart failure to chronic kidney disease (CKD) [ 32 ], improve renal function in CKD patients [ 11 , 33 ], enhance physical capacity, and improve overall well-being [ 34 – 35 ]. 3.1 Resistance Exercise Upregulates Renal FNDC5/Irisin, Attenuates Oxidative Stress and Collagen Deposition, and Improves Renal Function in Type 2 CRS Mice Previous studies have reported that chronic kidney disease (CKD) leads to tubular epithelial cell swelling, tubular dilation, and brush border loss, exacerbating renal injury [ 36 ] and elevating serum BUN and Scr levels [ 37 ]. Resistance exercise has been demonstrated to alleviate tubular injury and reduce serum BUN and Scr levels, thereby improving renal function in CKD models [ 38 ]. Although aerobic exercise has been reported to ameliorate renal injury in Type 2 CRS mice [ 12 ], the effects of resistance exercise on this condition had not been previously documented. Our results demonstrated that resistance exercise significantly reduced serum BUN and Scr levels and improved tubular morphology and renal function, indicating a renoprotective effect in Type 2 CRS. Irisin, a secreted exercise-induced myokine, exerts biological effects through autocrine, paracrine, and endocrine mechanisms [ 16 , 18 ]. FNDC5, the precursor of Irisin, is expressed in multiple tissues, including skeletal muscle, heart, brain, kidney, liver, and adipose tissue [ 39 ]. Clinical studies have reported decreased circulating Irisin levels in patients with cardiovascular or renal diseases [ 40 – 41 ]. Consistent with prior findings [ 12 ], our study confirmed that resistance training upregulated FNDC5 gene and protein expression in the kidneys of Type 2 CRS mice. Recombinant Irisin administration has been shown to reduce serum BUN and Scr levels and ameliorate renal injury in ischemia-reperfusion models [ 42 ]. In our study, FNDC5/Irisin knockout mice exhibited increased serum BUN and Scr levels, more severe renal damage, and attenuated exercise-induced renoprotection. Irisin has been shown to act directly on renal tubular epithelial cells [ 23 , 42 ], suggesting that exercise-induced Irisin may contribute to renal protection via multiple regulatory modes. Oxidative stress plays a central role in renal structural damage, functional decline, and progression of RIF [ 43 – 45 ]. Studies have shown that reduced renal blood flow during heart failure leads to hypoxia and an imbalance in oxidative/antioxidative systems, promoting oxidative stress and accelerating renal dysfunction [ 46 – 47 ]. Irisin administration in acute kidney injury models enhances antioxidant enzyme activities, such as superoxide dismutase (SOD), and alleviates oxidative damage [ 48 ]. Resistance training has been shown to increase circulating Irisin levels [ 49 ] and elevate renal antioxidant enzyme activity in mice with kidney injury [ 50 ]. In our study, MI induced significant renal oxidative stress, evidenced by elevated MDA levels and reduced SOD1 expression. Irisin deficiency diminished the antioxidative effects of resistance exercise, indicating that Irisin mediates resistance exercise–induced enhancement of SOD1 and SOD2 expression and reduction of MDA, thereby improving renal antioxidant capacity in Type 2 CRS mice. RIF is a hallmark pathological feature of Type 2 CRS and a major driver of renal dysfunction [ 51 ]. Hypoxia and mechanical stress stimulate the expression of connective tissue growth factor (CTGF), promoting ECM accumulation, increased synthesis of collagen types I and III, and degradation of the tubular basement membrane by MMP2 and MMP9. These changes induce epithelial–mesenchymal transition (EMT) and interstitial collagen deposition, thereby accelerating RIF [ 52 , 53 ]. Studies have shown increased CTGF and α-SMA expression and ECM accumulation in the kidneys of rats with chronic heart failure [ 52 ], while 10 weeks of resistance exercise reduces renal fibrosis in CKD rats [ 13 ]. Consistently, our study revealed that MI elevated renal expression of collagen-1/3, MMP2/9, CTGF, and α-SMA, which were significantly suppressed by resistance exercise. Recombinant Irisin has also been reported to inhibit Ang II–induced expression of α-SMA, collagen-1, and collagen-3 in the heart, alleviating myocardial fibrosis [ 19 ]. Our data showed that deletion of FNDC5/Irisin increased fibrosis marker expression and attenuated the antifibrotic effects of resistance exercise, indicating that FNDC5/Irisin plays a crucial role in mitigating renal collagen deposition in Type 2 CRS. 3.2 Potential Mechanism: Resistance Exercise Attenuates Interstitial Collagen Deposition via FNDC5/Irisin-Mediated Inhibition of the TGF-β1–Smad2/3 Pathway Overactivation of the TGF-β1–Smad signaling pathway is a key contributor to renal interstitial collagen deposition and RIF. Upon binding to TGF-β receptor II (TGFβRII), TGF-β1 activates TGFβRI kinase, which phosphorylates downstream Smad2/3. These phosphorylated Smads form complexes with Smad4 and translocate into the nucleus to regulate target gene transcription, promoting fibrosis [ 54 , 55 ]. Immunohistochemistry has shown that Irisin is predominantly expressed in renal tubules [ 56 ]. H 2 O 2 -induced oxidative stress promotes EMT in tubular epithelial cells, with increased expression of EMT markers including type I collagen, vimentin, and N-cadherin [ 22 ]. In our HKC cell fibrosis model, H 2 O 2 treatment significantly decreased SOD2 levels while increasing MDA, collagen-1/3, MMP2/9, α-SMA, and CTGF, indicating the induction of oxidative stress and fibrosis. Inhibiting EMT during chronic renal injury is considered a promising antifibrotic strategy [ 57 ]. Exercise has been shown to suppress TGF-β1–Smad2/3 pathway activation and renal fibrosis in hypertensive rats [ 58 ]. In our study, both AICAR and rhIrisin significantly reduced oxidative stress markers and fibrosis-related proteins in H 2 O 2 -treated HKC cells and inhibited TGF-β1–Smad2/3 pathway activation. In vivo, knockout of FNDC5/Irisin impaired the ability of resistance exercise to suppress this signaling pathway. Prior evidence suggests that Irisin may competitively bind to TGFβRII, antagonizing TGF-β1 signaling and thus mitigating fibrosis [ 23 ]. Therefore, it is speculated that Irisin inhibits Smad2/3 phosphorylation by interfering with TGFβRII binding, thereby playing a critical role in resistance exercise–mediated inhibition of TGF-β1–Smad2/3 pathway activation in Type 2 CRS (Fig. 7 ). Clinically, resistance exercise has been increasingly applied in patient rehabilitation programs [ 59 , 60 ]. However, it may not be suitable for individuals with severe conditions such as uncontrolled hypertension or ventricular arrhythmias [ 59 , 61 ]. Therefore, patients should undergo clinical evaluation before initiating exercise training, and a personalized exercise prescription—taking into account frequency, intensity, duration, exercise type, and individual preferences—should be developed accordingly [ 62 ]. Findings from the present study indicate that appropriate resistance exercise upregulates FNDC5/Irisin expression, thereby improving both cardiac and renal function in MI mice. It is thus speculated that resistance training may serve as a potential rehabilitation strategy for heart failure patients with concurrent renal injury, and that FNDC5/Irisin may represent a key molecular target mediating this therapeutic benefit. 5. Conclusion FNDC5/Irisin plays a critical role in suppressing renal oxidative stress and interstitial collagen deposition. Resistance exercise promotes the endogenous expression of FNDC5/Irisin in the kidney, thereby reducing oxidative stress, enhancing antioxidant capacity, and inhibiting activation of the TGF-β1–Smad2/3 signaling pathway. These effects collectively contribute to the attenuation of myocardial infarction–induced renal interstitial collagen deposition and the improvement of renal function. Declarations Competing interests The authors declare no competing interests. Additional information Correspondence and requests for materials should be addressed to K. Z.. Funding This work received no external funding. Author Contribution W.F. and J.L.: Project administration, Writing—review & editing, Writing—original draft, Investigation. W.L. and K.Z.: Validation, Conceptualization. W.L.: Visualization. W.F. and K.Z.: Funding acquisition, Project administration, Supervision, Writing—review & editing, Resources. All authors reviewed the manuscript. 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Irisin protects against renal ischemia-reperfusion injury in rats by inhibiting oxidative stress and inflammation via the activation of the Nrf2/HO-1 pathway. Mol. Med. Rep. 23 (6), 1–9 (2021). FU, S. et al. Irisin promotes cell proliferation and inhibits cell apoptosis through ERK signaling pathway in diabetic nephropathy. Life Sci. 254 , 117810 (2020). KINUGASA, E. Endothelial function and oxidative stress in cardiovascular diseases. Circ. J. 76 (6), 1387–1396 (2012). MARTIN P Y, BURGER, D. & MARSHALL C, B. Arterial hypertension and the kidney. Clin. J. Am. Soc. Nephrol. 5 (6), 1107–1115 (2010). GAO, R. et al. Protective effects of irisin on the kidney in 5/6 nephrectomized mice via inhibition of epithelial-to-mesenchymal transition and oxidative stress. Front. Pharmacol. 12 , 709856 (2021). ZHENG, S. et al. Irisin protects against high-glucose-induced renal tubular epithelial cell injury by modulating the Sirt1/NF-κB pathway. Int. Urol. Nephrol. 53 (5), 977–985 (2021). GOK, D. & ILHAN, T. The protective effect of irisin on renal ischemia-reperfusion injury in rats. Ren. Fail. 42 (1), 536–543 (2020). MAO, J. et al. Irisin ameliorates oxidative stress and inflammation via AMPK/SIRT1/PGC-1α signaling pathway in type 2 diabetic nephropathy rats. Diabetes Metab. Syndr. Obes. 14 , 1971–1983 (2021). KANG, Y. et al. Irisin attenuates renal interstitial fibrosis by inhibiting the TGF-β/Smad signaling pathway. Am. J. Physiol. Ren. Physiol. 320 (2), F230–F241 (2021). XU, B. et al. Irisin reverses the lipopolysaccharide-induced suppression of osteoblast differentiation via the Wnt/β-catenin pathway. J. Cell. Biochem. 120 (7), 11879–11887 (2019). PANATI, K. & SINGAREDDY, A. Irisin/FNDC5—An updated review. Eur. Rev. Med. Pharmacol. Sci. 20 (4), 689–697 (2016). TIAN, D. et al. Irisin improves endothelial function in type 2 diabetes through reducing oxidative/nitrative stress. J. Mol. Cell. Cardiol. 138 , 1–12 (2020). WU, F. et al. Irisin regulates cardiac physiology in zebrafish. PLoS One . 10 (6), e0130958 (2015). FERNANDES I G et al. Resistance exercise and metabolic stress modulate cardiac microRNA expression in infarcted rats. Front. Physiol. 11 , 26 (2020). LI, Z. et al. Irisin ameliorates myocardial ischemia/reperfusion injury in diabetic mice through AMPK pathway. Biochem. Biophys. Res. Commun. 567 , 39–45 (2021). ZHOU, B. et al. Protective effect of irisin against myocardial ischemia-reperfusion injury via activation of Akt and ERK1/2 signaling pathways in rats. Acta Pharmacol. Sin . 41 (4), 514–523 (2020). YANG, Y., WU, F. & ZHANG, Y. Irisin exerts neuroprotective effect on cerebral ischemia/reperfusion injury in rats by regulating the Akt/Nrf2 pathway. Biomed. Pharmacother . 126 , 110131 (2020). ZHAO, J. & WANG, H. Irisin alleviates myocardial infarction-induced cardiac fibrosis via inhibition of TGF-β1/Smad2 pathway. Int. J. Biol. Sci. 16 (3), 420–430 (2020). LI D J et al. Irisin inhibits cardiac fibrosis by blocking matrix metalloproteinase-2/9 via Akt signaling. Am. J. Transl Res. 11 (6), 3235–3244 (2019). LI, H. Y. et al. Irisin protects against pressure overload-induced cardiac hypertrophy via the AMPK/mTOR signaling pathway. Oxid Med Cell Longev, 2021: 5536673. (2021). HAN, Y. et al. Irisin alleviates angiotensin II-induced cardiac hypertrophy via activation of AMPK–mTOR signaling pathway. Life Sci. 254 , 117504 (2020). ZHANG, J. et al. Irisin improves endothelial function in type 2 diabetes through activation of AMPK-PGC-1α-eNOS pathway. Diabetes 69 (7), 1290–1302 (2020). LI, H. et al. Irisin alleviates pulmonary fibrosis by suppressing inflammation via Nrf2 pathway. Int. Immunopharmacol. 96 , 107756 (2021). WU, F. et al. Irisin protects hepatocytes against ischemia-reperfusion injury via reducing oxidative/nitrative stress. Biochem. Biophys. Res. Commun. 495 (1), 257–263 (2018). HOU, N. et al. Irisin ameliorates renal damage in obesity-related glomerulopathy via inhibition of ferroptosis. Obes. (Silver Spring) . 29 (4), 673–683 (2021). TIAN, D. et al. Irisin alleviates hepatic steatosis via reducing ER stress in high-fat diet-fed mice. Mol. Cell. Endocrinol. 528 , 111247 (2021). YANG, X. et al. Irisin attenuates atherosclerosis by promoting cholesterol efflux in macrophages via PPARγ–ABCA1 pathway. Biochem. Biophys. Res. Commun. 534 , 464–470 (2021). ZHANG, H. et al. Irisin alleviates cognitive impairment and modulates the expression of neuroplasticity-related proteins in APP/PS1 transgenic mice. Neural Plast, 2021: 8884529. (2021). LU, H. et al. Irisin protects against osteoarthritis by regulating mitochondrial dynamics and apoptosis via AMPK-PGC-1α signaling. Aging (Albany NY) . 12 (13), 11933–11948 (2020). WANG, K. et al. Irisin improves depressive-like behavior in mice by promoting synaptic plasticity via activating BDNF/TrkB signaling. Neurosci. Lett. 744 , 135586 (2021). HU, S. U. N. L. & SHAN, Y. Resistance exercise training improved cardiac fibrosis and apoptosis in myocardial infarction mice via the inhibition of TGF-β1/Smad2 pathway. Biochem. Biophys. Res. Commun. 514 (3), 716–722 (2019). SHAO, W. et al. Myokine irisin promotes osteogenic differentiation of bone marrow mesenchymal stem cells. Int. J. Mol. Sci. 18 (5), 981 (2017). ZHAO, M. et al. Irisin attenuates cardiac hypertrophy by regulating autophagy via AMPK–mTOR signaling pathway. Biochem. Biophys. Res. Commun. 567 , 46–52 (2021). LIN, J. et al. Irisin attenuates cardiac fibrosis in post-myocardial infarction rats by inhibiting TGF-β/Smad2 pathway. Life Sci. 257 , 118098 (2020). ZHANG, Y. et al. Irisin ameliorates pressure overload-induced cardiac hypertrophy by activating AMPK signaling pathway. Life Sci. 246 , 117419 (2020). AMBROSETTI, M. et al. Secondary prevention through comprehensive cardiovascular rehabilitation: From knowledge to implementation. 2020 update. A position paper from the Secondary Prevention and Rehabilitation Section of the European Association of Preventive Cardiology. Eur. J. Prev. Cardiol. 7 , 2047487320913379 (2020). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6998289","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":484294738,"identity":"f85b9236-25ac-43f2-a4b4-bf4ad420b9e3","order_by":0,"name":"Weiyu Fu","email":"","orcid":"","institution":"First Affiliated Hospital of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Weiyu","middleName":"","lastName":"Fu","suffix":""},{"id":484294740,"identity":"ffebec2b-10bf-48ca-aeb3-c7bffd6e36e6","order_by":1,"name":"Jun Lin","email":"","orcid":"","institution":"First Affiliated Hospital of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Lin","suffix":""},{"id":484294741,"identity":"fb02eb85-6e44-4ff7-baf2-0a9edc7cdd51","order_by":2,"name":"Wenqian Lin","email":"","orcid":"","institution":"First Affiliated Hospital of Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenqian","middleName":"","lastName":"Lin","suffix":""},{"id":484294742,"identity":"5bf310d4-f06a-42b0-95fb-5b1b048caec5","order_by":3,"name":"Kai Zeng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYBAC+wYGBsYGBgk5fvbmgw8SKmoIa2GEajGW7DmWbPDgzDGitTAkGtzIMZN82MJMWAsze+/hlzNqLBIkGxLMKhIb2Bj427sT8Gph4zmXZrnhmEQeP8OBtBuJO2QYJM6c3YBXC49EjpnhAzaJYsnGhmM3Es+wMRhI5OLXIiH/Bqjln0TihsOMbQWJbcyEtRhI8Bg/3NgG1HKMmY2BOC08OWaMM/tAgczGLJFw5hgPQb/Yt58x/tjzrU6OX/79x48/Kmrk+Nt78WsBAjYJZB4PIeUgwPyBGFWjYBSMglEwggEA/89L54tcmlYAAAAASUVORK5CYII=","orcid":"","institution":"First Affiliated Hospital of Fujian Medical University","correspondingAuthor":true,"prefix":"","firstName":"Kai","middleName":"","lastName":"Zeng","suffix":""}],"badges":[],"createdAt":"2025-06-28 14:08:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6998289/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6998289/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87031309,"identity":"bc70a233-1875-4e19-a68d-425351796a8d","added_by":"auto","created_at":"2025-07-18 12:51:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5899474,"visible":true,"origin":"","legend":"\u003cp\u003eRenal FNDC5 Gene and Protein Expression, Serum BUN and Scr, And Renal PAS Staining Results in WT and \u003cem\u003eFNDC5/Irisin\u003c/em\u003e\u003csup\u003e\u003cem\u003eKO\u003c/em\u003e\u003c/sup\u003e Mice; (A) FNDC5 mRNA levels in renal tissue; (B) Western blot analysis showing \u003cem\u003eFNDC5\u003c/em\u003e and \u003cem\u003eGAPDH\u003c/em\u003e protein expression. Gels were cropped from different parts of the same gel; (C) Serum BUN levels in different experimental groups; (D) Serum Scr levels in different experimental groups; (E) Representative histological images showing renal tubular injury (PAS staining). Renal tubular injury scores.\u003c/p\u003e\n\u003cp\u003eNote: P \u0026lt; 0.05 (*), P \u0026lt; 0.01 (**); same below.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6998289/v1/eba990764c470f7309f4dfb0.png"},{"id":87030278,"identity":"e6d16840-b316-4f30-b199-51be0ca03406","added_by":"auto","created_at":"2025-07-18 12:43:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5109620,"visible":true,"origin":"","legend":"\u003cp\u003eTest Results of Cardiac Function in WT and \u003cem\u003eFNDC5/Irisin\u003c/em\u003e\u003csup\u003e\u003cem\u003eKO\u003c/em\u003e\u003c/sup\u003e Mice. (A) Representative echocardiographic images showing left ventricular function in different experimental groups: WT (S, MI, MR) and FNDC5/IrisinKO (KS, KMI, KMR); (B) Left ventricular internal diameter at end-diastole (LVIDd); (C) Left ventricular internal diameter at end-systole (LVIDs).(D) Ejection fraction (EF); (E) Fractional shortening (FS). Measurements were made using echocardiographic imaging to assess cardiac function.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6998289/v1/a9614d4bdc2c61e01f272bb1.png"},{"id":87030284,"identity":"16bfb7b8-60f4-4aef-9a52-ba358aa41fa4","added_by":"auto","created_at":"2025-07-18 12:43:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":24651792,"visible":true,"origin":"","legend":"\u003cp\u003eResults of Renal Oxidative Stress and Fibrosis Detection in WT and \u003cem\u003eFNDC5/Irisin\u003c/em\u003e\u003csup\u003e\u003cem\u003eKO\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eMice. (A) MDA levels in renal tissue; (B) Western blot analysis showing SOD1 protein expression. Gels were cropped from different parts of the same gel; (C) Western blot analysis showing SOD2 protein expression. Gels were cropped from different parts of the same gel; (D) Representative Masson’s trichrome staining images showing renal collagen deposition; (E) Quantification of collagen deposition area (%) from Masson’s staining; (F) Western blot analysis showing expression of MMP2, MMP9, collagen-1, collagen-3, α-SMA, and CTGF proteins. Gels were cropped from different parts of the same gel; (G) Quantification of protein expression levels of MMP2, MMP9, collagen-1, collagen-3, α-SMA, and CTGF. Gels were cropped from different parts of the same gel.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6998289/v1/97b2826cc223c5bf87ad0cf1.png"},{"id":87030273,"identity":"6eda1d33-0255-468a-b9a5-19583ed8f276","added_by":"auto","created_at":"2025-07-18 12:43:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1338125,"visible":true,"origin":"","legend":"\u003cp\u003eAICAR and rhIrisin Inhibited TGFβ1-Smad2/3 Signaling Pathway in HKC Cells. (A) Western blot analysis showing TGF-β1, p-Smad2/3, and Smad2/3 protein expression. Gels were cropped from different parts of the same gel; (B) Quantification of TGF-β1 and p-Smad2/3 protein expression. Gels were cropped from different parts of the same gel.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6998289/v1/15a27b497c20f0fda7120fa8.png"},{"id":87030274,"identity":"f53069dc-492c-43d6-8dbc-319ee8f7e9dc","added_by":"auto","created_at":"2025-07-18 12:43:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2836908,"visible":true,"origin":"","legend":"\u003cp\u003eOxidative Stress and Fibrosis in HKC Cells after AICAR and rhIrisin Intervention. (A) MDA levels in renal tissue; (B) Western blot analysis showing SOD1 protein expression. Gels were cropped from different parts of the same gel; (C) Western blot analysis showing SOD2 protein expression. Gels were cropped from different parts of the same gel; (D) Western blot analysis showing expression of MMP2, MMP9, collagen-1, collagen-3, α-SMA, and CTGF proteins. Gels were cropped from different parts of the same gel. (E) Quantification of protein expression levels of MMP2, MMP9, collagen-1, collagen-3, α-SMA, and CTGF. Gels were cropped from different parts of the same gel.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6998289/v1/00f5be140803310a8cfc423e.png"},{"id":87030282,"identity":"68dbb6e5-b07b-4356-bc71-baafe2ce098f","added_by":"auto","created_at":"2025-07-18 12:43:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1697394,"visible":true,"origin":"","legend":"\u003cp\u003eRenal TGFβ1-Smad2/3 Pathway in WT and \u003cem\u003eFNDC5/Irisin\u003c/em\u003e\u003csup\u003e\u003cem\u003eKO\u003c/em\u003e\u003c/sup\u003e Mice. (A) Western blot analysis showing TGF-β1, p-Smad2/3, and Smad2/3 protein expression. Gels were cropped from different parts of the same gel; (B) Quantification of TGF-β1 and p-Smad2/3 protein expression. Gels were cropped from different parts of the same gel.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6998289/v1/07189854413656835d89d4be.png"},{"id":87030287,"identity":"5367e057-6d65-40a1-ab86-4214371cdba6","added_by":"auto","created_at":"2025-07-18 12:43:20","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":10502786,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism of Resistance Exercise in Improving Renal Interstitial Collagen Deposition in Type 2 Cardiorenal Syndrome Mice\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6998289/v1/0ba832b9292afef8acfee86f.png"},{"id":87976928,"identity":"ae6db9a9-b7fa-482a-a028-fb3c21d8096b","added_by":"auto","created_at":"2025-07-31 04:31:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":45828276,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6998289/v1/9bce5108-7975-421c-bf8b-a5d55dfe70fa.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"FNDC5/Irisin-Dependent Renoprotection of Resistance Training in Myocardial Infarction–Induced Type 2 Cardiorenal Syndrome","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHeart failure (HF) is a major cause of mortality among individuals with non-communicable diseases worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. HF frequently leads to complications involving distant organs, with renal impairment being particularly common [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Clinical studies have reported that patients with chronic HF often exhibit signs of renal insufficiency or dysfunction [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Type 2 cardiorenal syndrome (Type 2 CRS) refers to a clinical condition characterized by structural and functional renal impairment resulting from prolonged myocardial ischemia and hypoxia due to myocardial infarction (MI) or chronic HF [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Evidence suggests that HF patients with concomitant renal injury exhibit significantly worse cardiac dysfunction and higher mortality risk compared to those without renal involvement [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Improving renal function has been shown to enhance cardiac performance and survival outcomes in HF patients, thereby reducing disease-related mortality [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Thus, identifying effective strategies to prevent or mitigate renal structural damage and functional decline in Type 2 CRS is of critical importance for improving prognosis.\u003c/p\u003e\u003cp\u003eFollowing MI, weakened myocardial contractility and reduced cardiac output elevate peripheral vascular resistance, resulting in sustained renal ischemia and hypoxia. This, in turn, intensifies oxidative stress and inflammatory responses in renal tissues, promotes collagen deposition in the renal interstitium, and ultimately leads to renal interstitial fibrosis (RIF) and renal dysfunction [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Exercise is recognized as a key intervention for the prevention and rehabilitation of chronic diseases [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Studies have shown that aerobic exercise can ameliorate renal impairment in Type 2 CRS [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and resistance exercise can significantly inhibit renal fibrosis and improve kidney function in animal models of chronic kidney disease [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, whether resistance exercise can effectively suppress renal interstitial collagen deposition in the context of Type 2 CRS remains unclear.\u003c/p\u003e\u003cp\u003eIrisin, a newly identified exercise-induced myokine, has been shown to exert a range of biological effects, including regulation of energy metabolism and inhibition of oxidative stress, fibrosis, and apoptosis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Exercise training activates PGC-1α, which induces the expression of downstream FNDC5; this precursor protein undergoes cleavage to generate circulating Irisin [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Irisin exerts protective effects in multiple organs, including the heart, skeletal muscle, kidney, and brain [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In mice, Irisin administration alleviates angiotensin II (Ang II)-induced myocardial fibrosis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Aerobic exercise has been shown to upregulate renal Irisin expression in MI mice, thereby reducing renal damage [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Nevertheless, it remains unknown whether resistance exercise can stimulate endogenous Irisin expression and consequently improve renal injury in Type 2 CRS, as well as the underlying mechanisms involved.\u003c/p\u003e\u003cp\u003eTo address this question, the present study employed both wild-type (WT) and global FNDC5/Irisin knockout mice to establish a MI-induced Type 2 CRS model combined with a resistance exercise intervention. Additionally, an in vitro fibrosis model was established by exposing human embryonic kidney tubular epithelial (HKC) cells to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress. The aim was to investigate the role and mechanism of Irisin in mediating the effects of resistance exercise on renal interstitial collagen deposition in Type 2 CRS. This study provides experimental evidence to support the use of exercise rehabilitation strategies for patients with cardiorenal syndrome.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Experimental Animals, Model Establishment, and Exercise Protocol\u003c/h2\u003e\u003cp\u003eAll animal experiments were conducted in accordance with the guidelines set by the Animal Care and Use Committee of \u003cb\u003eXi'an Jiaotong University\u003c/b\u003e. The study protocol was approved by the committee under protocol number \u003cb\u003eXJTU-2024-RE-004\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eEight-week-old healthy male C57BL/6J mice (purchased from the Animal Experiment Center of Xi'an Jiaotong University) and global FNDC5/Irisin knockout mice (customized by Cyagen Biosciences Inc.) were housed under standard laboratory conditions with ad libitum access to food and water. All animal procedures complied with the Guidelines for the Care and Use of Laboratory Animals.\u003c/p\u003e\u003cp\u003eA total of 24 wild-type (WT) mice were randomly divided into three groups: sham-operated group (S), myocardial infarction group (MI), and myocardial infarction plus resistance exercise group (MR). Similarly, 24 FNDC5/Irisin knockout mice were randomly assigned to the knockout sham group (KS), knockout MI group (KMI), and knockout MI plus resistance exercise group (KMR). Myocardial infarction (MI) was induced by permanent ligation of the left anterior descending coronary artery under isoflurane inhalation anesthesia. Echocardiography was performed postoperatively to confirm the success and consistency of modeling. Only mice with comparable echocardiographic outcomes were randomly assigned to experimental groups. The sham groups (S and KS) underwent thoracotomy and suture placement without coronary ligation to exclude surgical stress effects.\u003c/p\u003e\u003cp\u003eResistance training was conducted based on previously described protocols [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Starting one week after surgery, mice in the MR and KMR groups underwent ladder-climbing resistance training. The climbing ladder was 1.1 meters high with 1-cm grid spacing and an 85\u0026deg; incline. Mice were first subjected to 5 days of adaptive training without additional load. Formal training then proceeded for 4 weeks with progressive loading. The load was increased daily by 10% of body weight until reaching 75%, which was maintained until the end of the intervention. Each training session consisted of 8 sets of 3 climbs per set, with 1-minute rest between sets, 5 days per week for 4 weeks. This study was conducted in compliance with the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines to ensure transparency in the reporting of animal experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 HKC Cell Culture and Interventions\u003c/h2\u003e\u003cp\u003eHuman embryonic kidney tubular epithelial (HKC) cells were cultured in F12/DMEM (1:1) medium under standard conditions (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e, Thermo Incubator). Cells between passages 3 and 7 were used for experiments. Oxidative stress-induced fibrosis was modeled by exposing cells to 100 \u0026micro;mol/L H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 4 hours [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Recombinant human Irisin protein (rhIrisin) was applied at 1 \u0026micro;g/mL for 12 hours [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. AICAR, an AMPK agonist, was used to mimic exercise effects at 500 \u0026micro;mol/L for 12 hours [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Cells were assigned to five groups: Control, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;AICAR, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;rhIrisin, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;rhIrisin\u0026thinsp;+\u0026thinsp;AICAR.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Echocardiographic Assessment\u003c/h2\u003e\u003cp\u003eOne day after the final resistance training session, mice were anesthetized with isoflurane (mixed with oxygen at a ratio of 1:5) and placed in the supine position. Cardiac function was evaluated using a VINNO ultrasound system (VINNO, Suzhou, China). The following parameters were measured: left ventricular internal diameter at end-diastole (LVIDd), left ventricular internal diameter at end-systole (LVIDs), and ejection fraction (EF). Fractional shortening (FS) was calculated to assess cardiac function.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Detection of Biomarkers\u003c/h2\u003e\u003cp\u003eBlood was collected via retro-orbital puncture and centrifuged at 1,000 r/min for 5 minutes to isolate serum. Serum levels of creatinine (Scr) and blood urea nitrogen (BUN), indicators of renal injury, were quantified using commercial assay kits (Nanjing Jiancheng Bioengineering Institute). Kidney tissues were homogenized in precooled 0.86% saline (1:9, w/v), and supernatants were obtained by centrifugation for malondialdehyde (MDA) measurement using oxidative stress detection kits. All assays were conducted according to the manufacturer\u0026rsquo;s instructions, and absorbance was measured using a microplate reader (Bio-Tek Instruments).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Histological Staining\u003c/h2\u003e\u003cp\u003eParaffin-embedded kidney sections were subjected to Masson\u0026rsquo;s trichrome and periodic acid\u0026ndash;Schiff (PAS) staining and examined under an optical microscope (Olympus BX51, Japan). Collagen volume fraction (CVF) was quantified as the percentage of the tissue area stained blue. For PAS-stained sections, five random fields per section were selected to assess the integrity of the brush border of renal tubular epithelial cells, serving as an indicator of tubular damage. Tubular injury was scored based on the following criteria: 0\u0026thinsp;=\u0026thinsp;normal; 1\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;25% of tubules damaged per field; 2\u0026thinsp;=\u0026thinsp;25\u0026ndash;50%; 3\u0026thinsp;=\u0026thinsp;50\u0026ndash;75%; 4\u0026thinsp;=\u0026thinsp;75\u0026ndash;100%. Tubular injury was defined as nuclear loss, reduced or absent brush border, epithelial swelling or vacuolar degeneration [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 RT-qPCR\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from renal tissues using Trizol reagent. cDNA was synthesized using a reverse transcription kit (TaKaRa, Japan) according to the manufacturer's protocol. qPCR amplification was performed using a SYBR Green-based PCR kit (TaKaRa). The primers (synthesized by Sangon Biotech, Shanghai, China) were as follows: FNDC5-F: 5\u0026prime;-GGCTGGGAGTTCATGTGGAA-3\u0026prime; FNDC5-R: 5\u0026prime;-TGGGAAGCGGTTATCTTTGCT-3\u0026prime; GAPDH-F: 5\u0026prime;-CAGTGCCAGCCTCGTCTCAT-3\u0026prime; GAPDH-R: 5\u0026prime;-AGGGGCATCCACAGTCTTC-3\u0026prime;\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Western Blotting\u003c/h2\u003e\u003cp\u003eKidney tissues were minced, homogenized (Model F6/10, FLUKO), and centrifuged to collect supernatants. HKC cells were lysed post-intervention, collected by scraping, and sonicated (JY-25013, Jining Tianhua). Protein concentration was determined using a BCA kit. Samples were mixed with 5\u0026times; loading buffer and RIPA, boiled at 100\u0026deg;C for 10 min, and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until use.\u003c/p\u003e\u003cp\u003eProteins were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with 5% skim milk or 5% BSA for 60 min at room temperature. Primary antibodies were used at the following dilutions: FNDC5 (1:1000), TGF-β1 (1:1000), Smad2/3 (1:1000), p-Smad2/3 (1:1000), SOD1 (1:1000), SOD2 (1:1000), MMP2 (1:1000), MMP9 (1:1000), α-SMA (1:1000), Collagen-1 (1:1000), Collagen-3 (1:1000), CTGF (1:500), GAPDH (1:5000), and β-actin (1:5000). Membranes were incubated overnight at 4\u0026deg;C, followed by washing and incubation with HRP-conjugated secondary antibodies for 90 min. Protein bands were visualized using the Bio-Rad ChemiDoc\u0026trade;, MP imaging system (Universal Hood III, Bio-Rad, USA) and analyzed digitally.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Data Acquisition and Statistical Analysis\u003c/h2\u003e\u003cp\u003eWestern blot results were quantified using Image Lab 5.1 software (Bio-Rad, CA, USA). All statistical analyses and graphical outputs were performed using GraphPad Prism 8.0.2 (GraphPad Software, La Jolla, CA, USA). One-way analysis of variance (ANOVA) was used to evaluate differences among groups, followed by Tukey\u0026rsquo;s post hoc test. Data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (M\u0026thinsp;\u0026plusmn;\u0026thinsp;SE). Statistical significance was defined at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cb\u003e3.1 Resistance Exercise Upregulates Renal FNDC5 Expression and Alleviates MI-Induced Renal Injury in Type 2 CRS Mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe expression levels of FNDC5, the precursor of Irisin, were assessed in renal tissues at both mRNA and protein levels. Compared with the sham-operated (S) group, the myocardial infarction (MI) group exhibited a significant reduction in FNDC5 gene and protein expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In contrast, mice in the myocardial infarction plus resistance exercise (MR) group showed significantly increased FNDC5 expression compared to the MI group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). As expected, FNDC5 gene expression was undetectable in FNDC5/Irisin knockout (KO) mice (Fig.\u0026nbsp;1A\u0026ndash;B).\u003c/p\u003e\u003cp\u003eSerum creatinine (Scr) and blood urea nitrogen (BUN) are commonly used indicators of renal dysfunction and injury [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The results revealed that serum Scr and BUN levels were significantly elevated in the MI group compared to the S group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Resistance exercise intervention significantly reduced these levels in Type 2 CRS mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Additionally, serum Scr and BUN levels in the knockout MI (KMI) group were significantly higher than those in the MI group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and levels in the knockout MR (KMR) group were significantly elevated compared to the MR group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;1C\u0026ndash;D).\u003c/p\u003e\u003cp\u003ePeriodic acid\u0026ndash;Schiff (PAS) staining was used to evaluate tubular injury in kidney tissues, with the apical brush border of renal tubules appearing red and the epithelial cells light pink. In the MI group, brush border loss and higher tubular injury scores were observed relative to the S group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Resistance training significantly reduced tubular injury scores in the MR group compared with the MI group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Notably, the KMI group displayed significantly higher tubular injury scores than the MI group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and the KMR group showed elevated scores compared to the MR group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;1E\u0026ndash;F). These findings confirm the successful establishment of the Type 2 CRS model induced by MI and demonstrate that resistance exercise effectively attenuates renal tubular brush border damage and improves kidney function. Moreover, FNDC5/Irisin appears to play a key role in mediating the renoprotective effects of resistance exercise.\u003c/p\u003e\u003cp\u003eEchocardiographic results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e) revealed that, compared with the sham-operated (S) group, mice in the myocardial infarction (MI) group exhibited significantly increased LVIDd and LVIDs (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), along with markedly decreased ejection fraction (EF%) and fractional shortening (FS%) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In contrast, the MR group showed significantly reduced LVIDd and LVIDs (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and significantly elevated EF% and FS% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), compared with the MI group. Furthermore, LVIDd and LVIDs were significantly higher in the KMI group than in the MI group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while EF% and FS% were significantly lower (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Similarly, EF% and FS% in the KMR group were significantly lower than those in the MR group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These findings confirm the successful establishment of the MI model and suggest that resistance exercise improves cardiac function in MI-induced Type 2 CRS. Moreover, FNDC5/Irisin appears to contribute to the cardioprotective effects observed during resistance training in ischemic hearts.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e(D) Ejection fraction (EF); (E) Fractional shortening (FS). Measurements were made using echocardiographic imaging to assess cardiac function.\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Resistance Exercise Suppresses Renal Oxidative Stress and Interstitial Collagen Deposition in Type 2 CRS Mice\u003c/h2\u003e\u003cp\u003eAs shown by oxidative stress assay kits and Western blotting results, renal malondialdehyde (MDA) levels were significantly elevated in the myocardial infarction (MI) group compared with the sham-operated (S) group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Resistance exercise markedly enhanced the expression of antioxidant proteins SOD1 and SOD2 and reduced MDA levels in the kidneys of Type 2 CRS mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In contrast, the knockout MI (KMI) group exhibited increased MDA content (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and reduced SOD2 protein expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared with the MI group. Similarly, MDA levels were elevated (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and both SOD1 and SOD2 protein levels were decreased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the knockout MR (KMR) group compared with the MR group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;C).\u003c/p\u003e\u003cp\u003eTo further assess renal fibrosis, Masson's trichrome staining and fibrosis-related protein expression were evaluated. Collagen fibers were visualized as blue deposits, indicating interstitial collagen accumulation. Compared with the S group, the MI group showed a significant increase in renal interstitial collagen deposition and fibrosis severity (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Resistance exercise markedly reduced collagen accumulation in the MR group compared with the MI group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Conversely, collagen deposition in the KMI group was significantly higher than in the MI group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u0026ndash;E).\u003c/p\u003e\u003cp\u003eWestern blotting analysis revealed that, relative to the S group, the MI group exhibited significantly elevated expression of fibrosis-related proteins including MMP2, MMP9, collagen-1, collagen-3, α-SMA, and CTGF (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These increases were significantly attenuated following resistance exercise intervention in Type 2 CRS mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Additionally, the KMI group showed upregulated MMP9, collagen-1, and collagen-3 expression compared with the MI group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In the KMR group, expression levels of MMP9, collagen-1, collagen-3, α-SMA, and CTGF were significantly higher than those in the MR group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eF\u0026ndash;G). These findings indicate that resistance exercise significantly suppresses MI-induced renal interstitial collagen deposition and enhances renal antioxidant capacity in Type 2 CRS mice. Moreover, knockout of FNDC5/Irisin attenuates the inhibitory effects of resistance training on oxidative stress and fibrosis, suggesting a critical role of Irisin in mediating the renoprotective effects of exercise.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.3 AICAR and rhIrisin Inhibit the TGF-β1\u0026ndash;Smad2/3 Pathway to Attenuate H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-Induced Fibrosis in HKC Cells\u003c/h2\u003e\u003cp\u003eOxidative stress is recognized as a key trigger of fibrosis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. To further elucidate the mechanism by which Irisin mediates the reduction of renal interstitial collagen deposition in MI mice undergoing resistance exercise, a fibrosis model was established in HKC cells using H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress. The effects of Irisin and AICAR on the TGF-β1\u0026ndash;Smad2/3 signaling pathway were subsequently examined. Western blotting results revealed that, compared with the control group, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e exposure significantly increased TGF-β1 protein expression and the phosphorylation level of Smad2/3 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, treatment with AICAR and/or recombinant human Irisin (rhIrisin) significantly suppressed both TGF-β1 expression and Smad2/3 phosphorylation (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These findings indicate that in HKC cells, AICAR and/or rhIrisin are capable of inhibiting the activation of the TGF-β1\u0026ndash;Smad2/3 pathway induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, thereby mitigating oxidative stress\u0026ndash;driven fibrotic responses.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the effects of Irisin and AICAR on the antioxidant capacity and collagen synthesis in HKC cells, oxidative stress assay kits and Western blotting were used to measure the levels of MDA, SOD1, SOD2, MMP2, MMP9, collagen-1, collagen-3, α-SMA, and CTGF following H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e exposure. The results demonstrated that, compared with the control group, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e intervention significantly decreased SOD2 protein expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) while markedly increasing MDA levels and the protein expression of MMP2, MMP9, collagen-1, collagen-3, α-SMA, and CTGF (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Intervention with AICAR or rhIrisin after oxidative stress significantly enhanced the expression of antioxidant enzymes SOD1 and SOD2 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and concurrently reduced MDA content and the expression of fibrosis-related proteins (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Furthermore, compared with the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;AICAR group, the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;rhIrisin\u0026thinsp;+\u0026thinsp;AICAR group exhibited increased SOD2 expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and significantly decreased levels of MDA, MMP9, collagen-3, and CTGF (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Similarly, relative to the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;rhIrisin group, the combined treatment group (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;rhIrisin\u0026thinsp;+\u0026thinsp;AICAR) showed enhanced SOD2 expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and reduced MDA and CTGF levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These findings suggest that AICAR and/or rhIrisin effectively attenuate oxidative stress and suppress the expression of fibrosis-associated proteins in HKC cells under oxidative injury conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Resistance Exercise Inhibits Activation of the Renal TGF-β1\u0026ndash;Smad2/3 Signaling Pathway in Type 2 CRS Mice\u003c/h2\u003e\u003cp\u003eWestern blotting results revealed that, compared with the sham-operated (S) group, the myocardial infarction (MI) group exhibited significantly elevated expression of TGF-β1 and phosphorylation of Smad2/3 in kidney tissues (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These increases were markedly attenuated in the myocardial infarction plus resistance exercise (MR) group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In addition, the knockout sham (KS) group showed significantly higher TGF-β1 expression than the S group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The knockout MI (KMI) group displayed significantly increased levels of TGF-β1 and phosphorylated Smad2/3 compared with the MI group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Similarly, both TGF-β1 expression and Smad2/3 phosphorylation were significantly higher in the knockout MR (KMR) group than in the MR group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These findings suggest that resistance exercise suppresses MI-induced activation of the renal TGF-β1\u0026ndash;Smad2/3 signaling pathway in Type 2 CRS. However, the inhibitory effect of resistance exercise on this profibrotic pathway is significantly weakened in the absence of FNDC5/Irisin, highlighting its regulatory role in exercise-mediated renal protection.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eProlonged myocardial infarction (MI) can cause damage to distant organs, including the kidneys. Persistent renal ischemia and hypoxia promote oxidative stress and inflammatory responses, leading to the activation and migration of myofibroblasts and excessive extracellular matrix (ECM) accumulation. These events contribute to renal interstitial collagen deposition and apoptosis, ultimately resulting in renal interstitial fibrosis (RIF) and functional deterioration [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Thus, mitigating MI-induced remote organ injury, particularly renal dysfunction, is critical for improving the quality of life in patients with heart failure. Exercise has been shown to reduce the risk of progression from heart failure to chronic kidney disease (CKD) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], improve renal function in CKD patients [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], enhance physical capacity, and improve overall well-being [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.1 Resistance Exercise Upregulates Renal FNDC5/Irisin, Attenuates Oxidative Stress and Collagen Deposition, and Improves Renal Function in Type 2 CRS Mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePrevious studies have reported that chronic kidney disease (CKD) leads to tubular epithelial cell swelling, tubular dilation, and brush border loss, exacerbating renal injury [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and elevating serum BUN and Scr levels [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Resistance exercise has been demonstrated to alleviate tubular injury and reduce serum BUN and Scr levels, thereby improving renal function in CKD models [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Although aerobic exercise has been reported to ameliorate renal injury in Type 2 CRS mice [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], the effects of resistance exercise on this condition had not been previously documented. Our results demonstrated that resistance exercise significantly reduced serum BUN and Scr levels and improved tubular morphology and renal function, indicating a renoprotective effect in Type 2 CRS.\u003c/p\u003e\u003cp\u003eIrisin, a secreted exercise-induced myokine, exerts biological effects through autocrine, paracrine, and endocrine mechanisms [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. FNDC5, the precursor of Irisin, is expressed in multiple tissues, including skeletal muscle, heart, brain, kidney, liver, and adipose tissue [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Clinical studies have reported decreased circulating Irisin levels in patients with cardiovascular or renal diseases [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Consistent with prior findings [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], our study confirmed that resistance training upregulated FNDC5 gene and protein expression in the kidneys of Type 2 CRS mice. Recombinant Irisin administration has been shown to reduce serum BUN and Scr levels and ameliorate renal injury in ischemia-reperfusion models [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In our study, FNDC5/Irisin knockout mice exhibited increased serum BUN and Scr levels, more severe renal damage, and attenuated exercise-induced renoprotection. Irisin has been shown to act directly on renal tubular epithelial cells [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], suggesting that exercise-induced Irisin may contribute to renal protection via multiple regulatory modes.\u003c/p\u003e\u003cp\u003eOxidative stress plays a central role in renal structural damage, functional decline, and progression of RIF [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Studies have shown that reduced renal blood flow during heart failure leads to hypoxia and an imbalance in oxidative/antioxidative systems, promoting oxidative stress and accelerating renal dysfunction [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Irisin administration in acute kidney injury models enhances antioxidant enzyme activities, such as superoxide dismutase (SOD), and alleviates oxidative damage [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Resistance training has been shown to increase circulating Irisin levels [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] and elevate renal antioxidant enzyme activity in mice with kidney injury [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In our study, MI induced significant renal oxidative stress, evidenced by elevated MDA levels and reduced SOD1 expression. Irisin deficiency diminished the antioxidative effects of resistance exercise, indicating that Irisin mediates resistance exercise\u0026ndash;induced enhancement of SOD1 and SOD2 expression and reduction of MDA, thereby improving renal antioxidant capacity in Type 2 CRS mice.\u003c/p\u003e\u003cp\u003eRIF is a hallmark pathological feature of Type 2 CRS and a major driver of renal dysfunction [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Hypoxia and mechanical stress stimulate the expression of connective tissue growth factor (CTGF), promoting ECM accumulation, increased synthesis of collagen types I and III, and degradation of the tubular basement membrane by MMP2 and MMP9. These changes induce epithelial\u0026ndash;mesenchymal transition (EMT) and interstitial collagen deposition, thereby accelerating RIF [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Studies have shown increased CTGF and α-SMA expression and ECM accumulation in the kidneys of rats with chronic heart failure [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], while 10 weeks of resistance exercise reduces renal fibrosis in CKD rats [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Consistently, our study revealed that MI elevated renal expression of collagen-1/3, MMP2/9, CTGF, and α-SMA, which were significantly suppressed by resistance exercise. Recombinant Irisin has also been reported to inhibit Ang II\u0026ndash;induced expression of α-SMA, collagen-1, and collagen-3 in the heart, alleviating myocardial fibrosis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Our data showed that deletion of FNDC5/Irisin increased fibrosis marker expression and attenuated the antifibrotic effects of resistance exercise, indicating that FNDC5/Irisin plays a crucial role in mitigating renal collagen deposition in Type 2 CRS.\u003c/p\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Potential Mechanism: Resistance Exercise Attenuates Interstitial Collagen Deposition via FNDC5/Irisin-Mediated Inhibition of the TGF-β1\u0026ndash;Smad2/3 Pathway\u003c/h2\u003e\u003cp\u003eOveractivation of the TGF-β1\u0026ndash;Smad signaling pathway is a key contributor to renal interstitial collagen deposition and RIF. Upon binding to TGF-β receptor II (TGFβRII), TGF-β1 activates TGFβRI kinase, which phosphorylates downstream Smad2/3. These phosphorylated Smads form complexes with Smad4 and translocate into the nucleus to regulate target gene transcription, promoting fibrosis [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eImmunohistochemistry has shown that Irisin is predominantly expressed in renal tubules [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress promotes EMT in tubular epithelial cells, with increased expression of EMT markers including type I collagen, vimentin, and N-cadherin [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In our HKC cell fibrosis model, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment significantly decreased SOD2 levels while increasing MDA, collagen-1/3, MMP2/9, α-SMA, and CTGF, indicating the induction of oxidative stress and fibrosis. Inhibiting EMT during chronic renal injury is considered a promising antifibrotic strategy [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eExercise has been shown to suppress TGF-β1\u0026ndash;Smad2/3 pathway activation and renal fibrosis in hypertensive rats [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. In our study, both AICAR and rhIrisin significantly reduced oxidative stress markers and fibrosis-related proteins in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated HKC cells and inhibited TGF-β1\u0026ndash;Smad2/3 pathway activation. In vivo, knockout of FNDC5/Irisin impaired the ability of resistance exercise to suppress this signaling pathway. Prior evidence suggests that Irisin may competitively bind to TGFβRII, antagonizing TGF-β1 signaling and thus mitigating fibrosis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, it is speculated that Irisin inhibits Smad2/3 phosphorylation by interfering with TGFβRII binding, thereby playing a critical role in resistance exercise\u0026ndash;mediated inhibition of TGF-β1\u0026ndash;Smad2/3 pathway activation in Type 2 CRS (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eClinically, resistance exercise has been increasingly applied in patient rehabilitation programs [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. However, it may not be suitable for individuals with severe conditions such as uncontrolled hypertension or ventricular arrhythmias [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Therefore, patients should undergo clinical evaluation before initiating exercise training, and a personalized exercise prescription\u0026mdash;taking into account frequency, intensity, duration, exercise type, and individual preferences\u0026mdash;should be developed accordingly [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Findings from the present study indicate that appropriate resistance exercise upregulates FNDC5/Irisin expression, thereby improving both cardiac and renal function in MI mice. It is thus speculated that resistance training may serve as a potential rehabilitation strategy for heart failure patients with concurrent renal injury, and that FNDC5/Irisin may represent a key molecular target mediating this therapeutic benefit.\u003c/p\u003e\u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eFNDC5/Irisin plays a critical role in suppressing renal oxidative stress and interstitial collagen deposition. Resistance exercise promotes the endogenous expression of FNDC5/Irisin in the kidney, thereby reducing oxidative stress, enhancing antioxidant capacity, and inhibiting activation of the TGF-β1\u0026ndash;Smad2/3 signaling pathway. These effects collectively contribute to the attenuation of myocardial infarction\u0026ndash;induced renal interstitial collagen deposition and the improvement of renal function.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eAdditional information\u003c/h2\u003e\u003cp\u003eCorrespondence and requests for materials should be addressed to K. Z..\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work received no external funding.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eW.F. and J.L.: Project administration, Writing\u0026mdash;review \u0026amp; editing, Writing\u0026mdash;original draft, Investigation. W.L. and K.Z.: Validation, Conceptualization. W.L.: Visualization. W.F. and K.Z.: Funding acquisition, Project administration, Supervision, Writing\u0026mdash;review \u0026amp; editing, Resources. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll the raw data are available upon request to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTarekegn, G. Y. et al. Medication non-adherence and its predictors among chronic heart failure patients in Northwest Amhara region. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e (1), 1\u0026ndash;13 (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUSZKO-LENCER N, JANSSEN, D. et al. 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Cardiol.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, 2047487320913379 (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Resistance exercise, FNDC5/Irisin, Type 2 cardiorenal syndrome, Oxidative stress, Renal fibrosis","lastPublishedDoi":"10.21203/rs.3.rs-6998289/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6998289/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eType 2 cardiorenal syndrome (CRS), driven by chronic myocardial infarction (MI), has attracted increasing research attention due to its involvement in renal fibrosis and oxidative stress. However, the mechanisms underlying renal damage and potential rehabilitation strategies remain inadequately understood. This study aims to explore whether resistance exercise mitigates MI-induced renal dysfunction by regulating the FNDC5/Irisin axis. Using both wild-type and FNDC5/Irisin knockout mice, we established a Type 2 CRS model and implemented a resistance exercise intervention. In parallel, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated HKC cells were used to construct an in vitro fibrosis model. We assessed renal injury, oxidative stress, fibrosis-related signaling (TGF-β1/Smad2/3), and the expression of key antioxidant and fibrotic markers. Our results demonstrated that resistance exercise significantly improved cardiac and renal function, reduced oxidative stress and collagen deposition, and inhibited TGF-β1\u0026ndash;Smad2/3 activation. These effects were largely abrogated in Irisin-deficient mice. Additionally, rhIrisin and AICAR alleviated oxidative stress and fibrosis in vitro, confirming the functional role of Irisin. In conclusion, resistance exercise exerts renoprotective effects in Type 2 CRS through upregulation of FNDC5/Irisin, highlighting a novel therapeutic target and providing scientific support for exercise-based rehabilitation strategies in heart failure patients with renal involvement.\u003c/p\u003e","manuscriptTitle":"FNDC5/Irisin-Dependent Renoprotection of Resistance Training in Myocardial Infarction–Induced Type 2 Cardiorenal Syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-18 12:43:15","doi":"10.21203/rs.3.rs-6998289/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3ce63e5d-42f0-421f-ab5f-5cf9e6075a12","owner":[],"postedDate":"July 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51424824,"name":"Health sciences/Cardiology"},{"id":51424825,"name":"Biological sciences/Cell biology"},{"id":51424826,"name":"Health sciences/Diseases"},{"id":51424827,"name":"Health sciences/Nephrology"}],"tags":[],"updatedAt":"2025-07-31T04:23:11+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-18 12:43:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6998289","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6998289","identity":"rs-6998289","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}