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The optimal cut-off values of Klotho for predicting all-cause and cardiovascular mortality among chronic kidney disease: results from NHANES – Scientific Reports

Baseline characteristics of patients

A total of 2418 patients with stage 1–4 CKD were included in the current analysis. The mean age of our participants was 62.4 ± 10.7 years, 1169 (48.4%) participants were male, and 36.6% of them were non-Hispanic white. The mean Klotho was 826.8 ± 307.3 pg/ml. The mean eGFR was 69.3 ± 24.3 mL/min/1.73 m2, and the median ACR was 42.8 mg/g (IQR: 11.6, 103.8), and 25.2%, 26.3%, 45.4% and 3.1% of participants were at CKD stage 1–4, respectively. As shown in Table 1, patients with lower Klotho levels are more likely to be older patients, to be smokers, to have hypertension and CVD, to be less likely to meet suggested physical activity guidelines, and to have higher 25(OH)D levels and lower DBP, eGFR, fasting glucose or HbA1c levels. During follow-up, 535 deaths were recorded. A total of 188 patients died of CVD (Table 2).

Table 2 Hazard ratios (95% CIs) for the association between serum Klotho levels and all-cause and cardiovascular mortality.

Distribution of Klotho and trends across CKD stages

The Klotho concentration ranged from 152.5 pg/ml to 2927.3 pg/ml with a roughly normal distribution. Klotho concentrations gradually decreased with the CKD stages (Fig. 2).

Figure 2
figure 2

Distribution of Klotho in all patients and trends stratified by CKD stages. Note: CKD chronic kidney disease.

Klotho and outcomes

All-cause mortality

The mean follow-up for the cohort was 87.9 months. During follow-up, 535 deaths were recorded. Among those cases, 171 (28.3%), 138 (22.8%), 121 (20.0%), and 105 (17.4%) deaths were identified in Klotho quartiles 1, 2, 3, and 4, respectively (Table 2). When Klotho was examined in quartiles, the association with all-cause mortality remained significant and did not change significantly in all multivariable models, with the lowest quartile indicating a 34% higher risk of all-cause mortality compared with the highest quartile after adjustment for demographics, living habits, CVD risk factors, biomarkers of mineral metabolism, and CKD stages (Table 2).

Cardiovascular mortality

A total of 188 patients died of CVD. Among those cases, 67 (11.1%), 41 (6.8%), 45 (7.4%), and 35 (5.8%) were recorded across Klotho quartiles 1, 2, 3, and 4, respectively (Table 2). In categorical analyses, the lowest quartile of Klotho was associated with a higher risk of cardiovascular mortality (HR 1.56 (95% CI: 1.02, 2.38)) before adjusting for calcium, phosphate, 25(OH)D and CKD stages, compared with the highest quartile. However, this tendency did not reach statistical significance in stepwise multivariable models (Table 2).

The optimal cut-off values of Klotho

The ROC curves were utilized to choose the optimal cut-off values of Klotho for all-cause and cardiovascular mortality after including the above covariates in Model 3. Our results indicated that the optimal cut-off values of Klotho for predicting all-cause and cardiovascular mortality were 548.8 pg/ml and 660.9 pg/ml, respectively, and the sensitivity were 76% and 83%, respectively; the specificity were both 68%. The AUC were 0.78 and 0.82, respectively (Fig. 3).

Figure 3
figure 3

The optimal cutoff points of Klotho for all-cause and cardiovascular mortality using ROC curves. Note: “Label” represents the optimal cutpoint of Klotho, sensitivity and specificity. “Value” represents the youden index (i.e., sensitivity + specificity-1).

Cubic splines

To visualize the dose–response relationship of Klotho and all-cause and cardiovascular mortality, the restricted cubic splines were plotted with the optimal cutoff points as the reference. In spline analyses, we observed a consistent pattern for all-cause and cardiovascular mortality. The range of Klotho in the curve was restricted because the majority of patients (96.3%) had Klotho concentrations between 300 pg/ml and 1500 pg/ml. As shown in Fig. 4, a non-linear association was observed between Klotho and all-cause and cardiovascular mortality. The reference values for each hazard ratio were Klotho = 548.8 pg/ml and 660.9 pg/ml for all-cause and cardiovascular mortality, respectively (the red dotted line). When Klotho concentrations were below 700 pg/ml (the blue dotted line), the risk of all-cause and cardiovascular mortality gradually increased as the Klotho concentration decreased.

Figure 4
figure 4

Dose–response relationship between Klotho and all-cause and cardiovascular mortality. Note: The reference values for each hazard ratio were Klotho = 548.8 pg/ml and 660.9 pg/ml for all-cause and cardiovascular mortality, respectively (red dotted line). The solid line represents the estimated hazard ratio and the dotted line represents the 95% confidence interval. The range of Klotho in the curve was restricted because the majority of patients (96.3%) had Klotho concentrations that ranged between 300 pg/ml and 1500 pg/ml.

The optimal cutpoints of Klotho and outcomes

To verify the effects of the chosen cut-off values, we stratified patients into two groups (Klotho < 548.8 pg/ml, N = 366 (15.1%) vs. ≥ 548.8 pg/ml, N = 2052 (84.9%) and Klotho < 660.9 pg/ml, N = 408 (16.9%) vs. ≥ 660.9 pg/ml, N = 2010 (83.1%) for further analysis.

All-cause mortality

Among 535 deaths, 120 (32.8%) and 415 (20.2%) deaths were identified in patients with Klotho < 548.8 pg/ml and those ≥ 548.8 pg/ml, respectively. Kaplan–Meier survival curves were shown in Fig. 5a. Participants with serum Klotho in higher levels had significantly higher overall survival compared to those with lower Klotho levels (log-rank test, P < 0.001). Similar findings were observed when Klotho was examined in Cox regression, patients with Klotho < 548.8 pg/ml indicating a higher risk of all-cause mortality compared with those ≥ 548.8 pg/ml. This association remained significant in all multivariable models (HR and 95% CI 1.52 (1.23, 1.87), P < 0.0001) (Table 3).

Figure 5
figure 5

The Kaplan–Meier curves for the optimal cutoff points of Klotho and all-cause and cardiovascular mortality.

Table 3 Hazard ratios (95% CIs) for the associations between Klotho and all-cause and cardiovascular mortality.

Cardiovascular mortality

Among those cases, 48 (11.8%) and 140 (7.0%) cases were recorded in Klotho < 660.9 pg/ml and ≥ 660.9 pg/ml, respectively. Kaplan–Meier survival curves are shown in Fig. 5b. Participants with serum Klotho in lower level had significantly higher risk of cardivascualr mortality compared to those with higher Klotho levels (log-rank test, P < 0.001). In categorical analyses, the risk of cardiovascular mortality is higher in patients with Klotho < 660.9 pg/ml compared with those ≥ 660.9 pg/ml (HR and 95% CI: 1.58 (1.13, 2.22), P = 0.008) in the fully adjusted model (Table 3).

Sensitivity analysis

Eighty-eight participants were excluded from the analysis because they died during the first two years of follow-up, leaving 2330 participants available for the analysis. The association did not change significantly in the sensitivity analysis (Table 3).

Interaction and subgroup analysis

We did not observe the interactions between Klotho and age, sex, physical activity, smoking, obesity, diabetes, hypertension and CKD stages for all-cause and cardiovascular mortality (Fig. 6). However, a significant interaction between Klotho and CVD were identified for all-cause mortality (P for interaction < 0.001). Patients with CVD had a higher risk of all-cause mortality with a cut-off value of Klotho < 548.8 pg/ml compared to those without CVD (HR and 95% CI 2.07 (1.50, 2.84) vs. 1.15 (0.86, 1.53)). The association between serum Klotho concentration and all-cause and cardiovascular mortality was consistent in subgroups according to age (40–59 vs. 60–79 years old), sex, physical activity (meet the suggested guideline or not), smoking (never, former or current), obesity, diabetes, hypertension, CVD and CKD stages (stage 1–2 vs. stage 3–4).

Figure 6
figure 6

Subgroup analysis: Association between serum Klotho levels and all-cause mortality and cardiovascular mortality. Note: The cut-off values of Klotho are 548.8 pg/ml for all-cause mortality (a) and 660.9 pg/ml for cardiovascular mortality (b), respectively.