In this cross-sectional analysis of a nationally representative sample of U.S. adults aged 40 to 59 years (NHANES 2007–2016), we first reported a negative dose-dependent association between cannabis use frequency and Klotho, which was a specific effect independent of smoking behavior, although smoking had a similar effect on Klotho (Yao et al. 2022). Notably, this association exhibited pronounced effect modification in individuals with sedentary lifestyles, higher vitamin D, participants with cancer.

The growing societal normalization of cannabis underscores the urgency of clarifying its long-term health impacts, particularly on aging-related biomarkers. The Klotho protein, a pleiotropic regulator of aging pathways, exists in two functionally distinct isoforms: membrane-bound Klotho, which modulates fibroblast growth factor signaling, and soluble Klotho, a circulating form linked to antioxidative and anti-inflammatory properties (Kuro-o et al. 1997). Our findings align with emerging evidence of environmental influences on Klotho regulation. While Pedersen et al. reported no sex differences in serum Klotho concentrations across 120 healthy adults aged 19–66 using standardized immunoassays (Pedersen et al. 2013), Wang et al.’s NHANES-based analysis revealed significantly higher Klotho levels in females after adjusting for demographics, BMI, and tobacco exposure (Wang et al. 2024). This discrepancy may reflect methodological differences in population selection or confounder adjustment. Wang et al. further identified tobacco use and C-reactive protein (CRP) as male-specific Klotho suppressants, potentially attributable to males’ higher smoking prevalence and tobacco-associated chronic inflammatory states (Wang et al. 2024). Our findings revealed a similar sex dimorphism in Klotho levels among cannabis users. The amplified Klotho reduction in male cannabis users suggests shared biological pathways with tobacco toxicity, possibly involving androgen-mediated oxidative stress or inflammation.

Emerging evidence highlights bidirectional relationships between physical activity patterns and Klotho regulation (Vázquez-Lorente et al. 2024; Avin et al. 2014; Wu & Lu 2024; Hajare et al. 2025). In middle-aged sedentary adults, structured exercise interventions elevated plasma Klotho levels, suggesting activity-dependent Klotho upregulation (Vázquez-Lorente et al. 2024). Conversely, aging blunts this response: older adults (≥ 65 years) exhibit diminished Klotho increases after acute aerobic exercise compared to younger counterparts (25–45 years) (Avin et al. 2014). Notably, sedentary behavior modifies environmental influences on Klotho—a dose-dependent inverse association between caffeine intake and serum Klotho was exclusively observed in adults aged 40–79 with prolonged sedentariness (> 480 min/day; P for interaction < 0.05) (Wu & Lu 2024). We also found a correlation between lifestyle and Klotho levels in sedentary populations. In adults aged 40–59 who were long-term sedentary (> 240 min/day), we observed a dose-dependent negative correlation between cannabis use frequency and serum Klotho levels.

Klotho serves as a pivotal regulator of kidney homeostasis through its modulation of fibroblast growth factor 23 (FGF-23) signaling, vitamin D metabolism, and phosphate balance (Haussler et al. 2012). Vitamin D upregulates Klotho expression, while Klotho itself exerts negative feedback control over vitamin D synthesis (Haussler et al. 2012), forming a critical endocrine axis for calcium-phosphate homeostasis. Dysregulation of this axis elevates cardiovascular disease risk and mortality (de Borst et al. 2011; John et al. 2011). Our interaction analysis indicated that vitamin D status acted as an effect modifier of the association between cannabis use and serum Klotho levels. Lower vitamin D levels were typically associated with lower Klotho levels. Due to the ceiling effect, the decline in Klotho among individuals in lower vitamin D tertiles was less significant, whereas the decline in Klotho was more significant among those in the highest vitamin D tertile.

Klotho’s dual role as an aging suppressor and nephroprotective agent is well-established (Hajare et al. 2025). Epidemiologic studies demonstrate positive correlations between serum Klotho and eGFR, particularly in high-risk populations (elderly, obese, or diabetic individuals), while preclinical models link Klotho deficiency to oxidative stress, inflammation, and accelerated chronic kidney disease (CKD) progression (Zhang et al. 2022; Shikida et al. 2021). We found that frequent cannabis users in the highest eGFR tertile exhibited profound Klotho depletion (β = −46.9, P = 0.003). This results can be partially explained by the ceiling effect—the individuals with chronic diseases already have low Klotho levels, so further reduction due to cannabis use is limited. Given Klotho’s capacity to mitigate oxidative damage and fibrotic pathways (Yamamoto et al. 2005), its decline could create a vicious cycle—reduced Klotho accelerates renal injury, while kidney dysfunction further suppresses Klotho synthesis (Shikida et al. 2021). Clinically, serial Klotho monitoring in cannabis users may enable early identification of subclinical nephron stress preceding eGFR decline.

In addition to cancer, individuals without comorbidities had a greater reduction in Klotho from cannabis use compared to those with comorbidities, which was consistent with the subgroup analysis results of eGFR. Even though these results can be partly explained by the ceiling effect, it is still advisable for healthy individuals to minimize the use of recreational cannabis to mitigate potential risks. Similarly, for male and people with sedentary behavior, it is necessary to strengthen the publicity and education on the hazards of tobacco use such as cigarettes and cannabis, reduce the incidence of chronic diseases and malignant tumors, reduce passive smoking and cross-generational health effects, and finally reduce social medical expenses and social disease burden, which can achieve double improvement of health benefits and economic efficiency.