Alpha-klotho is an anti-aging protein involved in the regulation of oxidative stress, inflammation, cellular senescence, and multiple signaling pathways (1, 2). Preclinical studies on animal models had demonstrated that α-klotho possesses antioxidative and anti-inflammatory properties by decreasing reactive oxygen species (ROS) activity and suppressing interleukin (IL-) 6, IL-1β, and tumor necrosis factor alpha (TNF-α) levels, and was potentially protective against diseases such as contrast-induced acute kidney injury, pulmonary epithelial damage from hyperoxia, valvular fibrosis, and cardiac dysfunction in cardiorenal syndrome (3–6). In the field of cardiovascular disease, α-klotho was postulated to play roles in the regulation of myocardial remodeling and aging of the vasculature (7, 8). Such antioxidative and protective effect of α-klotho against cell injury was also supported by in vitro and in vivo human studies (9, 10). Among patients undergoing non-emergent coronary angiography, reduced soluble α-klotho level was found to correlate with the presence and severity of coronary artery diseases (11). Similarly, in patients with chronic kidney disease (CKD), an inverse correlation between soluble α-klotho level and left ventricular mass index had been observed (12). In cancer research, soluble α-klotho was proposed to act as a tumor suppressor by regulating cell metabolism and modulating multiple oncogenic pathways (13, 14). Therefore, research on the potential therapeutic roles of klotho in oncology, renal and cardiovascular diseases is emerging, and some beneficial effects of treating klotho deficiency have been observed in animal models (14, 15).

Several epidemiological studies had shown that in older community-dwelling adults, low serum α-klotho concentrations were associated with poor health outcomes including poor skeletal muscle strength (16), disability in activities of daily living (17), and increased risk of all-cause mortality (18). In addition, in older adults with CKD, higher serum α-klotho levels have been associated with slower decline in kidney function (19). Furthermore, among individuals with advanced kidney disease or those receiving renal replacement therapy, evidence suggested that lower α-klotho levels were linked to an increased risk of all-cause mortality (20).

Although previous studies have shown the associations between α-klotho levels and various health outcomes, evidence on the association of α-klotho with the risk of death in the general population is relatively scarce. A recent study using nationally representative data from the National Health and Nutrition Examination Survey (NHANES) in the United States (U.S.) reported a null association between serum α-klotho level and all-cause mortality in adults (21). However, individuals with marked impairment of kidney function were also included in that study. While poor kidney function significantly correlated with low levels of soluble α-klotho (22), the inclusion of these individuals in the analysis may have biased the study results. Additionally, the follow-up time in that study was relatively short with a mean of 57.7 months, which might fail to capture the long-term results. Accordingly, our study aims to investigate the association between soluble α-klotho level and long-term mortality risk among adults with estimated glomerular filtration rate (eGFR) ≥ 30 mL/min/1.73 m² using the updated data from the NHANES.

The population aged 56.1 ± 0.2 years (survey weighted mean ± standard error) and 47.8% of them were male. The race/ethnicity distribution included 73.1% White people, 9.1% Black people, 11.3% Hispanics, and 6.5% other races/ethnicities ( Table 1 ). In addition, three quarters of the population were overweight or obese, and nearly half were never smokers ( Table 1 ). Using weighted multiple linear regression, we found that females, non-Hispanic Black people (vs. non-Hispanic White people), individuals with higher eGFR, never or former smokers, and those with a history of HCV infection positively correlated to higher α-klotho levels. In contrast, individuals with higher BMI and older age were associated with lower α-klotho levels. Diabetes, hypertension, CVD, and previous stroke were not significantly associated with soluble α-klotho levels ( Table 2 ).

During a median follow-up of 99.0 months (interquartile range: 68.0-129.0 months), a total of 1,490 participants died (11.8 per 10000 person-months). By Kaplan-Meier method, we observed that individuals in the lowest septile of soluble α-klotho level had higher risk of all-cause mortality (shown in Figure 1 ). Using weighted Cox regression analysis with adjustment for age and sex (model 1 in Table 3 ), our results showed that group 2 (HR: 0.72, 95% CI 0.58-0.93, p<0.05), group 3 (HR: 0.68, 95% CI 0.52-0.90, p<0.01), group 4 (HR: 0.69, 95% CI 0.54-0.88, p<0.01), group 5 (HR: 0.70, 95% CI 0.55-0.89, p<0.01), and group 6 (HR: 0.64, 95% CI 0.47-0.86, p<0.01) were associated with lower all-cause mortality risk compared with group 1; however, we did not observe a significant difference in mortality risk between group 1 and group 7 ( Table 3 ). The association remained consistent after further adjustment for race/ethnicity, BMI, survey cycle, eGFR, diabetes, hypertension, CVD, stroke, HCV infection, smoking status, and family income-to-poverty ratio (model 2, model 3, and model 4 in Table 3 ).

Using group 4 as the reference group, we observed that both group 1 and group 7 were associated with 40% greater risk of death (shown in Figure 2A ). In addition, when we stratified our analysis by kidney function, we observed a significant biphasic association between soluble α-klotho level and mortality risk in individuals with eGFR ≥ 60 mL/min/1.73 m², but there was no significant association between α-klotho level and mortality risk in those with eGFR < 60 mL/min/1.73 m² (shown in Figures 2B, C ).

Using multinomial logistic regression analysis, we found that individuals in group 1 were more likely to be former or current smokers, Black people, and have lower BMI and lower eGFR compared with those in group 4 ( Table 4 ). On the other hand, individuals in group 7 were more likely to be female, Black people or Hispanics, have lower BMI and higher eGFR, have HCV infection, and were less likely to be former or current smokers compared with those in group 4 ( Table 4 ).

In this population-based cohort study, our results showed that male sex, current smoking, older age, higher BMI, and lower eGFR were associated with lower soluble α-klotho levels, while HCV infection significantly correlated to higher soluble α-klotho levels. In addition, we observed a non-linear relationship between soluble α-klotho levels and mortality risk, with individuals in the lowest and highest septile at increased risk of all-cause mortality compared to those in the middle septile. When stratified by kidney function, we showed that the U-shaped association between soluble α-klotho levels and mortality risk was significant in those with eGFR ≥ 60 mL/min/1.73 m², but not in those with eGFR < 60 mL/min/1.73 m².

Previous research on the association between α-klotho levels and mortality risk had yielded inconsistent results. Among patients with pre-dialysis CKD or end-stage kidney disease (ESKD) undergoing maintenance hemodialysis, several studies suggested that a low soluble klotho level correlated with an increased risk of all-cause mortality (23–27), while others reported no significant differences in mortality risk between individuals with low and high levels of klotho (28–31). Although a meta-analysis suggested that a lower serum klotho level significantly correlated with an increased risk of all-cause mortality in CKD patients (20), substantial heterogeneity and relatively small sample sizes in each study had precluded a robust conclusion. Our study showed that serum α-klotho level and the risk of death were not strictly inversely correlated, which might explain the inconsistent findings in previous studies. In addition, klotho is composed of three isoform categories (membrane klotho, secreted klotho, and soluble klotho), and the detection of klotho by immunostaining can be affected by cross-reactivity and may not always be specific due to inter-assay variability (32). The non-specificity and cross-reactivity may have led to measurement bias and the conflicting results in previous studies. Although the detection of soluble α-klotho in NHANES was limited to a single commercial ELISA kit, whether measurement bias has affected our findings remains to be clarified.

The U-shaped dose-response relationship in biology was commonly observed in clinical and public health studies, and it had been postulated that this biphasic response might reflect a combined agonist-antagonist effect of the biological substance on its receptor system or the result of compensation for a disruption of homeostasis (33). A cohort study using soluble α-klotho as a continuous variable similarly showed a U-shaped relationship between klotho level and mortality risk among diabetic population (34), which was consistent with our findings. In addition, in patients without or with early CKD, a U-shaped association between klotho level and risk of fracture had also been observed (35, 36). Given the complex roles of klotho in regulation of mineral and vitamin D metabolism (37), our findings may suggest that individuals at both extremes of soluble α-klotho may have disrupted physiological homeostasis and thus be at higher risk of mortality.

Our study showed that HCV infection was significantly associated with higher soluble α-klotho levels, whereas other comorbidities including diabetes, hypertension, CVD, and previous stroke were not. Previous studies had suggested that an increased level of klotho could represent a compensatory response to physiologic stress and inflammation (38, 39). In alcoholic cirrhotic patients, studies showed higher-than-normal levels of soluble α-klotho in association with liver function derangement, possibly reflecting an attempt to regulate increased inflammation (40). In addition, in alcoholic individuals, those with higher klotho levels were found to have a greater mortality risk compared to those with lower levels, and this correlation was even more pronounced in cirrhotic patients (39). Whether our observation that HCV infection was associated with higher soluble α-klotho levels involved similar mechanisms remains to be elucidated.

Our study using a nationally representative population added to the evidence for the complex association between soluble α-klotho levels and long-term mortality risk. However, there are some limitations that should be considered when interpreting our findings. First, serum α-klotho was measured using stored surplus serum and therefore sample quality may have introduced measurement bias. Second, sociodemographic factors and comorbidity status were determined by participants’ self-reports, which may have influenced the accuracy of the measurements. Finally, despite our effort to adjust for several potential confounders, residual confounding may still exist and affect our results. Given the observational nature of our study, the causality between soluble α-klotho levels and mortality risk cannot be confirmed.

In summary, among middle-aged and older U.S. adults, our study showed a U-shaped association between soluble α-klotho level and all-cause mortality risk, especially for those with eGFR ≥ 60 mL/min/1.73 m². While there is no established normal range for soluble α-klotho, our findings suggest that individuals with levels ≤ 575.8 or ≥ 1140.1 pg/ml are both at increased risk of death. The extremely high soluble α-klotho levels may represent a compensatory response to physiological stress and chronic inflammation and therefore correlate to a poor prognosis. Further research is warranted to see if our findings can be reproduced and to explore the underlying mechanisms. In addition, it is important to consider whether the increased mortality risk observed at the highest extreme of soluble α-klotho would affect the findings of studies exploring the therapeutic potential of klotho.

Publicly available datasets were analyzed in this study. This data can be found at: https://wwwn.cdc.gov/nchs/nhanes/Default.aspx.

The studies involving humans were approved by the research ethics review board of the NCHS. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

Research concept and study design: M-YJ and M-HC. Data acquisition: M-YJ. Data analysis/interpretation: M-HC, H-WW, Y-TH, and M-YJ. Statistical analysis: M-YJ. Supervision: M-YJ. Each author contributed intellectual content during the manuscript drafting/revision process and accepts accountability for the overall work and ensuring that questions pertaining to the accuracy or integrity of any portion of the work are appropriately investigated and resolved. M-YJ had full access to all study data and take responsibility for data integrity and data analysis accuracy. All authors contributed to the article and approved the submitted version.