This study investigated the effects of resistance training, endurance training, and exogenous irisin administration on plasma irisin levels, the expression of Klotho and HSP70 (at both mRNA and protein levels) in renal tissue, and long-term survival in NMRI mice. While the study employed young mice as a preventive model to simulate early-life interventions, our findings demonstrate that all interventions significantly increased plasma irisin concentrations, upregulated Klotho and HSP70 expression, and improved survival rates compared to the control group, highlighting their potential to enhance cellular resilience and systemic health. These results position irisin as a promising exercise-mimetic agent, particularly for individuals with limited mobility such as the elderly, post-surgical patients, or those with severe neuromuscular or orthopedic impairments.

Physical exercise is widely recognized as a potent intervention for promoting health and mitigating cellular stress, largely through the upregulation of protective factors like Klotho and HSP70, which are implicated in cellular resilience and longevity.¹⁴ Previous studies have consistently shown that exercise enhances the expression of these genes across various models. For instance, Mostafidi et al. reported that regular aerobic exercise increased plasma Klotho levels via PGC-1α activation in humans,¹⁵ while Ji et al. found that both intermittent and continuous aerobic exercise in Sprague Dawley rats significantly elevated Klotho mRNA and protein levels, reduced Reactive Oxygen Species (ROS), and improved survival, suggesting a role for Klotho in exercise-mediated longevity.¹⁶ More recently, a 2024 study demonstrated that 8 weeks of endurance and resistance exercise increased serum FGF23 and soluble Klotho levels, improving glucose metabolism in type 2 diabetic women.¹⁷ Additionally, Shamsi et al. reported that resistance training in rats enhanced HSP70 expression in skeletal muscle, supporting its role in cellular protection and stress response.¹⁸

Our results align with these findings, as both resistance and endurance training significantly upregulated Klotho and HSP70 expression in the renal tissue of NMRI mice (Figure 2). Resistance training exerted the most pronounced effect, increasing Klotho mRNA by 3.85-fold and HSP70 mRNA by 3.49-fold (p<0.05), compared to 2.12-fold and 2.53-fold increases, respectively, with endurance training (p<0.05). At the protein level, resistance training led to a 3.48-fold increase in Klotho and a 2.80fold increase in HSP70, while endurance training induced more modest elevations (1.50-fold for Klotho and 1.30fold for HSP70). This disparity likely stems from the broader mechanical and metabolic stimuli induced by resistance training, which activate a more diverse range of protective signaling pathways compared to endurance training, which is more associated with metabolic health and inflammation reduction.¹⁹ This distinction suggests that the type and intensity of physical activity may differentially influence anti-aging gene expression, with implications for designing targeted exercise interventions to support renal function and systemic health in populations at risk for kidney dysfunction.

A key finding of this study is the ability of exogenous irisin to replicate many of the beneficial effects of exercise, supporting its potential as a therapeutic alternative for individuals unable to engage in physical activity. Irisin injection increased plasma irisin levels by 62.1% (7.27±0.66 ng/mL), closely mirroring the 65.5% increase observed with resistance training (7.42±0.35 ng/mL) and surpassing the 46.7% increase with endurance training (6.38±0.65 ng/mL) (Figure 1). Irisin injection also significantly up-regulated Klotho and HSP70 expression at both mRNA and protein levels, with effects comparable to those of resistance and endurance training (Figure 2). These findings are consistent with prior studies reporting that irisin, a myokine released during exercise, can mimic exercise-induced benefits by enhancing metabolism and upregulating protective gene expression.^12,20,21 The mechanism underlying irisin’s effects likely involves the activation of PGC-1α and FNDC5 in skeletal muscle, leading to irisin production, which subsequently engages downstream pathways such as MAPK and PI3K/Akt in target tissues like the kidney.^22,25 These pathways may enhance mitochondrial function, reduce oxidative damage, and upregulate longevity-related genes, thereby protecting against renal stress and injury—a critical consideration given the kidney’s vulnerability to age-related decline. For instance, a recent study demonstrated that irisin administration in a mouse model of renal ischemia-reperfusion injury reduced levels of renal injury markers (e.g., serum creatinine, BUN, Kim-1, and NGAL) and preserved mitochondrial integrity by upregulating autophagy-related proteins (PINK1, PARK2, LC3) while downregulating mitochondrial membrane proteins (TOM20, TIM23), highlighting its renoprotective potential.²⁶

Correlation analyses further elucidate irisin’s regulatory role, revealing a strong positive correlation between plasma irisin levels and Klotho mRNA expression (R=0.9498, p<0.0001), suggesting that irisin may directly enhance Klotho expression, a protein with well-documented anti-aging and renoprotective properties (Figure 2). A more moderate but significant correlation was observed with HSP70 expression (R=0.7511, p<0.003), indicating that irisin also influences stress-protective pathways, albeit to a lesser extent (Figure 2). The coordinated upregulation of Klotho and HSP70 suggests a potential synergistic effect in mitigating oxidative stress and inflammation, key drivers of renal injury and aging. This is supported by Sugiura et al., who demonstrated that Klotho gene therapy in a mouse model of renal ischemia-reperfusion injury reduced apoptosis by upregulating HSP70, highlighting Klotho’s role in oxidative stress mitigation.²⁷ Additionally, a recent study found a positive correlation between hippocampal irisin levels and HSP70 expression in rats with high-fat, high-fructose diet-induced cognitive dysfunction, suggesting that irisin contributes to stress response pathways across different tissues.²⁸

Although our primary focus was on the molecular modulation of anti-aging pathways, we also conducted an exploratory long-term survival analysis to evaluate whether early-life interventions could influence longevity trajectories. Despite the lack of statistically significant differences among groups based on the Log-rank (Mantel-Cox) test (p=0.093), a significant linear trend was detected using the Log-rank test for trend (p=0.042), indicating a potential dose–response relationship between intervention intensity and survival benefit.

By the end of the 20-month follow-up, the control group exhibited the highest mortality (8/12), while the resistance training group showed the lowest (3/12), followed by irisin injection (4/12) and endurance training (5/12). Notably, the median survival time was reached only in the control group (480 days), whereas it remained undefined in all intervention groups—suggesting prolonged survival. These findings align with growing evidence that early-life molecular enhancement—particularly via Klotho and HSP70—may contribute to long-term physiological resilience. For instance, Feng et al. reported that three months of swimming exercise in young C57BL/6J mice significantly extended health span, though not lifespan, supporting the concept that early interventions confer persistent systemic benefits.²⁹

Our data complement this concept by showing a modest, yet consistent trend toward improved survival following early resistance training or irisin administration. Moreover, our results resonate with studies demonstrating that increased Klotho expression extends lifespan and attenuates age-related tissue degeneration, and that preconditioning exercise protocols can reduce mortality in disease models by modulating inflammation and organ integrity.6,30-33

It is worth emphasizing that our survival data were obtained in young, healthy animals without disease burden, and without repeated intervention post-treatment. Therefore, the observed differences in mortality likely reflect the long-term programming effects of transient early-life interventions, rather than continuous stimulation.

Taken together, this study supports the hypothesis that brief, early-life exercise or irisin treatment may enhance longevity potential by initiating protective molecular programs, particularly in tissues like the kidney, where Klotho expression is pivotal. These preliminary findings warrant further investigation in aged or disease-prone models and raise the prospect of developing early-life or one-time exercise-mimetic interventions to promote healthy aging trajectories.

This study provides compelling evidence that both physical exercise, particularly resistance training and exogenous irisin administration can significantly upregulate the renal expression of Klotho and HSP70, two critical regulators of cellular stress resistance and longevity. Our findings demonstrate that early-life interventions not only induce favorable molecular adaptations but may also confer long-term survival benefits, even in the absence of continuous stimulation. Notably, irisin mimicked many of the beneficial effects of exercise, supporting its potential as a promising exercise-mimetic candidate for populations unable to engage in physical activity.

Although the survival analysis did not yield statistically significant differences across all tests, a meaningful trend toward improved survival in the intervention groups was observed, reinforcing the hypothesis that early-life molecular enhancement may promote physiological resilience during aging. These findings highlight the translational potential of muscle-derived signaling molecules like irisin in targeting systemic aging pathways.

Future studies using aged or disease-prone models, as well as investigations into tissue-specific effects beyond the kidney, will be essential to fully elucidate the therapeutic potential of irisin and refine strategies for delaying age-related decline. Overall, our work adds to the growing body of evidence that skeletal muscle exerts far-reaching endocrine effects with implications for aging, renal health, and systemic homeostasis.