Forty healthy male C57BL/6J mice (8 weeks old, 20–22 g) were purchased from the Animal Experimentation Center of Xi’an Jiaotong University. All procedures conformed to the Guide for the Care and Use of Laboratory Animals (8th ed., ISBN-10: 0-309-15396-4) and were approved by the Animal Ethics Committee of Shaanxi Normal University. Mice were housed at 22 °C–25 °C with 50%–60% humidity under a 12-h light/dark cycle with free access to food and water. After 1 week of acclimation, they were randomly assigned to four groups (n = 10 each): sham (S), sham + exercise (SE), myocardial infarction (MI) and myocardial infarction + exercise (ME).

Normal rat kidney (NRK) cells were obtained from CytoCell. Cells were maintained in DMEM/F-12 supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin at 37 °C in a humidified 5% CO₂ incubator. When confluence reached 70%–80%, cells were exposed to 0.2 mmol L⁻¹ H₂O₂ for 4 h to induce acute oxidative stress, after which they immediately received pharmacological treatments. Interventions included recombinant human IGF-1 (rhIGF-1, PeproTech, 100 ng mL⁻¹), the AMPK agonist AICAR (Sigma-Aldrich, 500 µM), the IGF-1R inhibitor NVP-AEW541 (Selleckchem, 5 µM), the PI3K inhibitor LY294002 (Selleckchem, 10 µM), and freshly prepared H₂O₂ (0.2 mmol L⁻¹ in sterile PBS). All agents were added directly after modelling, and treatment durations (typically 12–24 h) were set according to subsequent protein or gene-expression assays to evaluate their protective effects and signalling impacts following oxidative stress (Figure 1).

Myocardial-infarction model construction: After induction of anaesthesia with inhaled isoflurane, each mouse was fixed supine on the surgical table. A mid - sternal thoracotomy was performed to expose the heart, and the left anterior descending coronary artery was ligated. ST-segment elevation or T-wave inversion on the postoperative electrocardiogram, together with visible blanching of the apical myocardium, was taken as evidence of successful modelling

Exercise protocol: One week after surgery, mice underwent adaptive incremental treadmill training _{(5–10 m min} ⁻¹ _{, 10–30 min d} ⁻¹ _{, 5 consecutive days)}. Formal training consisted of a 10-min warm-up at 5 m min⁻¹ _{(≈40–50% VO2max)}, followed by intermittent running: 3 min at 8 m min⁻¹ _{(50–60% VO2max)} alternated with 7 min at 12 m min⁻¹ _{(80–90% VO2max)} for a total of 50–60 min d⁻¹, 5 days week⁻¹ for 4 weeks. The VO_2max values were set according to previous measurements and were not determined individually for each animal. Exercise intensity was determined based on VO_2max values reported in prior studies using comparable mouse models rather than individual testing, to minimize stress and variability. This method has been widely applied to ensure reproducibility of moderate-intensity protocols (Huang et al., 2025; Mohebinejad et al., 2025; Li et al., 2022).

The day after the 4-week training period, mice were anaesthetised with isoflurane, fixed supine and depilated. M-mode echocardiography was performed with a small-animal probe to measure the left-ventricular internal diameter in systole (LVIDs) and diastole (LVIDd), and to calculate ejection fraction (EF) and fractional shortening (FS). For each parameter, the mean of six consecutive cardiac cycles was recorded.

Immediately after the final session, kidneys were rapidly excised, rinsed in ice-cold saline, and partly fixed in 4% paraformaldehyde for 48 h. Samples were dehydrated through a graded ethanol series, cleared in chloroform, embedded in paraffin and sectioned serially at 5 µm. Sections were stained with haematoxylin–eosin (HE; Shanghai Solarbio, G1120) and periodic-acid–Schiff (PAS; Shanghai Solarbio, G1280), dehydrated, cleared with xylene and mounted with neutral resin. Tubular epithelial swelling, cell desquamation, nucleocytoplasmic separation and tubular atrophy were examined and photographed under an Olympus BX51 light microscope, and quantitative analysis was performed with Image-Pro Plus software.

Total RNA from renal tissues and cells was extracted with an RNA Rapid Extraction Kit (Beijing Polymed Biosciences). Reverse transcription was carried out using the M5 Super Plus qPCR RT Kit with gDNA Remover in a 20 µL reaction to generate cDNA. Real-time quantitative PCR was performed with 2× M5 HiPer SYBR Premix EsTaq in a 20 µL system under the following programme: 95 °C for 3 min, then 40 cycles of 95 °C for 10 s and 51 °C for 30 s. GAPDH served as the internal control, and relative expression was calculated by the 2^-ΔΔCt method. All qPCR primers were designed by Beijing Qingke Biotechnology; sequences are listed in Table 1.

Approximately 50 mg of kidney tissue was homogenised on ice in lysis buffer (RIPA:PMSF: phosphatase inhibitor = 100:1:1). After centrifugation at 12,000 rpm for 15 min, supernatants were quantified by the BCA method, adjusted to 4 ng μL⁻¹ and denatured at 100 °C for 10 min. Samples were separated on SDS–PAGE gels and transferred to NC membranes at 300 mA under cooling. Membranes were blocked with 3% BSA for 2 h, then probed with primary antibodies against IGF-1 (Abcam, ab133542), IGF-1R (Abcam, ab182408), PI3K (CST, 4257S), AKT (CST, 9272S), TNF-α (CST, 11948S), IL-6 (Abcam, ab9324), Cleaved Caspase-3 (CST, 9661) and GAPDH (Abcam, ab181602). After 2 h at room temperature, membranes were incubated with HRP-conjugated secondary antibodies, washed five times with TBST and developed with ECL; band intensities were quantified using a multicolour imaging system.

For biochemical assays, reagents were added sequentially to 96-well plates, and absorbance was measured on a microplate reader at kit-specified wavelengths: SOD (Nanjing Jiancheng, A001-3, 550 nm), MDA (Nanjing Jiancheng, A003-1, 532 nm) and BUN (Nanjing Jiancheng, C013-2, 520 nm) were measured in kidney homogenates, while IGF-1 (R&D Systems, MIG100, 450 nm) was determined in serum.

Images were processed with ImageJ, Western blot data with Image Lab, and statistical analysis with GraphPad Prism 9. One-way ANOVA or linear regression was applied; multiple comparisons used Tukey’s test. Data are presented as mean ± SD. *p < 0.05 indicated statistical significance, **p < 0.01 high significance and ***p < 0.001 very high significance.

Myocardial echocardiography showed that ventricular systolic motion was weakened in the MI group compared with the S group, whereas the ME group displayed an improvement (Figure 2A). LVIDs (3.21 ± 0.15 mm) and LVIDd (4.58 ± 0.12 mm) were significantly increased, while EF (42.3% ± 3.5%) and FS (21.8% ± 2.1%) decreased compared with S mice (EF 68.5% ± 2.9%, FS 36.2% ± 1.8%; p < 0.001). In contrast, the ME group exhibited smaller LVIDs and LVIDd and higher EF and FS than the MI group (p < 0.001) (Figure 2B). These findings confirm successful construction of the MI model—cardiac function declined in MI mice—while intermittent exercise safely and effectively improved cardiac performance in the ME group.

Histopathology of the kidney revealed intact architecture with no evident injury in S and SE mice on both HE and PAS staining. In MI mice, tubular epithelial swelling and detachment, interstitial oedema and tissue damage were observed; In ME mice, pathological changes were attenuated, with relatively preserved tubular structure and less interstitial damage (Figure 3A). Biochemically, MI mice displayed significantly higher serum BUN and MDA levels and lower SOD activity and IGF-1 concentrations than controls (p < 0.001). Intermittent exercise reduced BUN and MDA while elevating SOD and IGF-1, all with high statistical significance (p < 0.001) (Figures 3B–E). Thus, MI induces renal injury and dysfunction, whereas intermittent exercise substantially alleviates kidney damage and restores renal function.

Pro-apoptotic genes, pro-inflammatory genes and the kidney-injury marker KIM-1 were significantly upregulated in the MI group versus the S group (p < 0.01 or p < 0.001), whereas the anti-apoptotic gene Bcl-2, antioxidant genes and genes in the IGF-1/IGF-1R/PI3K/AKT pathway were downregulated (p < 0.01 or p < 0.001). Compared with the MI group, the ME group showed reductions in pro-apoptotic, pro-inflammatory and injury-related transcripts (p < 0.01 or p < 0.001) and significant increases in anti-apoptotic, antioxidant and IGF-1-pathway transcripts (p < 0.01 or p < 0.001) (Figures 4A–D). These results indicate that MI suppresses renal IGF-1 expression and its downstream anti-inflammatory, anti-apoptotic and anti-oxidative signalling, whereas intermittent exercise re-activates the IGF-1 axis and mitigates MI-induced renal injury, thereby conferring renoprotection.

Protein analysis showed that IGF-1, IGF-1R, PI3K and AKT were reduced in MI mice compared with S mice (p < 0.001), whereas the inflammatory proteins TNF-α and IL-6 were significantly elevated (p < 0.001). In ME mice, IGF-1, IGF-1R, PI3K and AKT protein levels were significantly higher, and TNF-α and IL-6 lower, than in MI mice (p < 0.001) (Figures 5A,B). These findings confirm that MI depresses the renal IGF-1 pathway and its anti-inflammatory capacity, while intermittent exercise re-activates this pathway and alleviates MI-induced kidney damage. Western blot analysis was performed for both total and phosphorylated forms of pathway proteins where available. Total protein levels were used as an indirect indicator of signaling activation, consistent with previous studies reporting parallel changes in phosphorylation status.

Compared with the control group (C), the H₂O₂ group showed a marked reduction in p-PI3K and p-AKT expression, accompanied by significant elevations in the inflammatory factors IL-6 and Cleaved Caspase-3 (p < 0.01 or p < 0.001). Both the H₂O₂ + AICAR and H₂O₂ + rhIGF-1 treatments significantly increased p-PI3K and p-AKT levels while lowering IL-6 and Cleaved Caspase-3 (p < 0.01 or p < 0.001) (Figure 6). These findings indicate that H₂O₂ exposure in NRK cells mimics MI-related renal injury, whereas exogenous rhIGF-1 or AICAR enhances IGF-1 signalling and activates the downstream PI3K/AKT pathway, thereby attenuating H₂O₂-induced inflammation and cellular damage.

In the H₂O₂ + AICAR group, p-PI3K and p-AKT were strongly upregulated, whereas IL-6 and Cleaved Caspase-3 were suppressed (p < 0.01 or p < 0.001). When the IGF-1R inhibitor NVP-AEW541 was added (H₂O₂ + NVP-AEW541), p-PI3K and p-AKT levels fell sharply and IL-6 and Cleaved Caspase-3 rebounded (p < 0.01 or p < 0.001) (Figure 7). Thus, the cytoprotective effect of AICAR, which mimics exercise, depends largely on IGF-1R-mediated activation of the PI3K/AKT pathway to exert anti-inflammatory actions.

The H₂O₂ group suppressed p-IGF-1R and p-AKT while elevating IL-6 and Cleaved Caspase-3 (p < 0.01 or p < 0.001). In contrast, the H₂O₂ + AICAR group significantly increased p-IGF-1R and p-AKT and reduced IL-6 and Cleaved Caspase-3 (p < 0.01 or p < 0.001). When PI3K was blocked with LY294002 (H₂O₂ + LY294002), activation of p-AKT was impeded, IL-6 and Cleaved Caspase-3 rebounded, and p-IGF-1R was likewise suppressed (p < 0.01 or p < 0.001) (Figure 8). These results demonstrate that the PI3K/AKT pathway is indispensable for IGF-1-mediated anti-inflammatory protection in renal cells.