Klotho is a single-pass transmembrane protein predominantly expressed in the kidney. The extracellular domain of Klotho is subject to ectodomain shedding and is released into the circulation as a soluble form. Soluble Klotho is also generated from alternative splicing of the
Klotho is a single-pass transmembrane protein with a long extracellular domain and short cytoplasmic tail that appears to modulate aging [
The study population consisted of male and female chronic HD patients treated at Japanese Red Cross Koga Hospital, Ibaraki, Japan, recruited for a prospective single-center study. The rationale, design, and data collection procedures of the study have been described elsewhere [
Demographic and medical data, including age, gender, smoking history, and comorbid conditions, were obtained from the subjects’ medical records in addition to standardized interviews. The body mass index (BMI) was calculated from the weight and height measurements as the weight (kg) divided by the square of the height (m2). Blood samples were obtained before HD on the first dialysis day of the week. Patients had been in the supine position for at least 10 minutes before blood collection. Aliquots of serum were obtained immediately at study entry and stored at −80°C until further use. Laboratory data included the levels of hemoglobin (Hb), serum albumin (sAlb), total cholesterol, serum calcium (sCa), serum phosphorus (sPi), intact parathyroid hormone (iPTH), and 1,25-dihydroxyvitamin D [1,25(OH)2D]. The normalized protein catabolism rate (nPCR) and the
The primary outcomes were all-cause mortality and the first episode of fatal or nonfatal cardiovascular events, which were defined as angina or myocardial infarction (according to the Third Universal Definition of Myocardial Infarction) [
The data are expressed as either the number of participants or the percentage of the study population. The remaining data are expressed as the mean ± standard deviation (SD), or median and interquartile range (IR) for variables with a skewed distribution. The groups were compared using a one-way analysis of variance for normal distributions and the Kruskal-Wallis rank test for skewed distributions. The chi-square test was used to evaluate the proportional differences in categorical variables. Prior to conducting the survival analysis, the 25th, 50th, and 75th percentiles of serum sKlotho and serum FGF23 levels in the current study population were determined. The subjects were categorized as follows: low-Klotho group (low-KL), subjects with a serum sKlotho level under the 25th percentile; middle-Klotho group (middle-KL), subjects with a serum sKlotho level equal or over the 25th percentile and under the 75th percentile; high-Klotho group (high-KL), subjects with a serum sKlotho level of equal to or over the 75th percentile; low-FGF23 group (low-FGF23), subjects with a serum FGF23 level under the 25th percentile; middle-FGF23 group (middle-FGF23), subjects with a serum FGF23 level equal to or over the 25th percentile and under the 75th percentile; high-FGF23 group (high-FGF23), subjects with a serum FGF23 level of equal to or over the 75th percentile. We then assessed the impact of the serum sKlotho and FGF23 levels on the cumulative patient and cardiovascular event-free survival rates according to the Kaplan-Meier method combined with the log-rank test. Cox regression models were used to analyze the relationships between the primary outcomes and the serum sKlotho levels. Multivariate models were also applied to adjust for age, gender, diabetes mellitus, sPi, sAlb, and Hb, which are known to be associated with the risk of mortality in patients under chronic HD treatment [
Soluble Klotho was detectable in the serum of all 63 chronic HD patients, with serum concentrations of sKlotho ranging from 167 to 720 pg/mL (median 371, IR 309–449). No significant difference in serum sKlotho levels was observed between males and females. The baseline clinical and demographic profiles of the subjects with the three varied degrees of serum sKlotho levels are summarized in Table
Demographic and clinical characteristics of patients according to study group.
Characteristics | Total | Low-Klotho |
Middle-Klotho |
High-Klotho |
|
---|---|---|---|---|---|
Number | 63 | 14 | 35 | 14 | |
Age (year) | 64.2 ± 13.0 | 66.1 ± 11.9 | 63.8 ± 13.7 | 63.0 ± 13.0 | 0.80 |
Male gender (%) | 50.8 | 42.9 | 54.3 | 50.0 | 0.77 |
BMI (kg/m2) | 21.3 ± 3.2 | 20.3 ± 3.0 | 21.8 ± 3.2 | 20.9 ± 3.2 | 0.29 |
Diabetes mellitus (%) | 33.3 | 42.9 | 37.1 | 14.3 | 0.18 |
Duration of dialysis (year) | 6.7 ± 5.4 | 6.2 ± 4.0 | 6.5 ± 5.2 | 7.6 ± 7.1 | 0.77 |
History of cardiovascular disease (%) | 12.7 | 7.1 | 11.4 | 21.4 | 0.52 |
Smoking (%) | 62.9 | 64.3 | 58.8 | 71.4 | 0.70 |
Serum creatinine | 10.6 ± 2.8 | 9.9 ± 0.9 | 10.7 ± 2.8 | 10.9 ± 0.8 | 0.63 |
|
1.36 ± 0.28 | 1.47 ± 23 | 1.33 ± 0.28 | 1.31 ± 0.29 | 0.23 |
nPCR (g/kg/day) | 0.95 ± 0.18 | 0.97 ± 13 | 0.94 ± 0.20 | 0.94 ± 0.17 | 0.91 |
Cardiothoracic ratio (%) | 50.0 ± 5.3 | 50.8 ± 6.5 | 49.5 ± 4.8 | 50.1 ± 5.6 | 0.79 |
Systolic blood pressure (mmHg) | 154.1 ± 20.0 | 150.9 ± 20.4 | 156.3 ± 21.7 | 151.9 ± 15.5 | 0.63 |
Hemoglobin (g/dL) | 9.7 ± 1.3 | 9.5 ± 1.2 | 9.8 ± 1.4 | 9.5 ± 1.2 | 0.64 |
Serum albumin (g/dL) | 3.74 ± 0.36 | 3.61 ± 0.39 | 3.76 ± 0.34 | 3.84 ± 0.39 | 0.23 |
Total cholesterol (mg/dL) | 158 ± 1.3 | 153 ± 32 | 165 ± 36 | 143 ± 0.35 | 0.12 |
Serum calcium (mg/dL) | 8.9 ± 0.2 | 8.6 ± 1.3 | 9.0 ± 1.2 | 8.9 ± 1.3 | 0.60 |
Serum phosphorus (mg/dL) | 5.0 ± 0.2 | 4.6 ± 1.2 | 4.9 ± 1.4 | 5.6 ± 1.0 | 0.11 |
Intact PTH (pg/mL) | 99 (45–250) | 96 (47–210) | 94 (42–250) | 185 (79–298) | 0.35 |
Klotho (pg/mL) | 371 (309–449) | 245 (220–283) | 371 (341–401) | 550 (468–692) | <0.0001 |
Serum FGF23 (pg/mL) | 4511 (1184–15134) | 2115 (609–4422) | 4844 (779–17492) | 9036 (2815–21678) | 0.02 |
1,25(OH)2D (pg/mL) | 8.6 ± 4.4 | 7.7 ± 3.6 | 8.4 ± 4.5 | 9.7 ± 4.9 | 0.46 |
Vitamin D sterols (%) | 46.0 | 50.0 | 48.6 | 35.7 | 0.67 |
Phosphate binders (%) | 93.7 | 100 | 88.6 | 100 | 0.09 |
ESA (%) | 91.9 | 100 | 88.2 | 92.9 | 0.23 |
ARBs/ACEIs (%) | 47.6 | 21.4 | 48.6 | 71.4 | 0.03 |
CCBs (%) | 50.8 | 42.9 | 51.4 | 57.1 | 0.75 |
Statin (%) | 9.5 | 0 | 11.4 | 14.3 | 0.20 |
Variables are presented as numbers of patients (percentage), as mean ± SD, or as median (interquartile range), as appropriate. Klotho: serum soluble Klotho; BMI: body mass index; nPCR: normalized protein catabolism rate; PTH: parathyroid hormone; FGF23: fibroblast growth factor 23; ESA: erythropoiesis stimulating agent; ARBs: angiotensin receptor blockers; ACEIs: angiotensin converting enzyme inhibitors; CCBs: calcium channel blockers.
Twelve patients (19%) died within the median follow-up period of 65 months (IR 63–67). Four patients in the low-KL group, three in the middle-KL group, and two in the high-KL group died of cardiovascular events, while one patient in the low-KL group died of infectious disease. Only two patients in the low-KL group died of carcinoma, versus one patient in the middle-KL group. The serum Klotho levels of the nonsurvivors at study entry were numerically lower (median 319 pg/mL, IR 254–426) than those of the survivors (median 380 pg/mL, IR 337–449). The Kaplan-Meier analysis also showed that the cardiovascular event-free survival and mortality rates did not differ significantly between the three groups, whereas the cumulative survival rate was significantly lower in the low-KL group (Figure
Univariate Cox regression analyses of cardiovascular events, cardiovascular mortality, and all-cause mortality.
Parameter | Cardiovascular events | Cardiovascular mortality | All-cause mortality | |||
---|---|---|---|---|---|---|
Hazard ratio |
|
Hazard ratio |
|
Hazard ratio |
|
|
Age (year) | 1.06 (1.02–1.11) | 0.004 | 1.13 (1.04–1.26) | 0.002 | 1.09 (1.02–1.18) | 0.005 |
Male gender | 1.52 (0.65–3.68) | 0.33 | 2.02 (0.53–9.57) | 0.31 | 2.01 (0.63–7.53) | 0.24 |
BMI (kg/m2) | 1.11 (0.98–1.25) | 0.10 | 0.99 (0.79–1.20) | 0.93 | 1.00 (0.83–1.18) | 0.97 |
Diabetes mellitus | 1.42 (0.59–3.30) | 0.42 | 1.70 (0.42–6.41) | 0.43 | 2.12 (0.66–6.77) | 0.20 |
Duration of dialysis (year) | 0.95 (0.85–1.03) | 0.23 | 0.94 (0.77–1.07) | 0.40 | 0.94 (0.81–1.06) | 0.36 |
Serum creatinine | 0.97 (0.83–1.12) | 0.64 | 0.95 (0.74–1.19) | 0.65 | 0.90 (0.72–1.10) | 0.30 |
|
0.23 (0.06–1.00) | 0.05 | 0.69 (0.08–7.40) | 0.75 | 0.80 (0.12–6.29) | 0.83 |
nPCR (g/kg/day) | 0.05 (0.005–0.61) | 0.02 | 1.72 (0.05–62.78) | 0.77 | 1.54 (0.06–39.72) | 0.79 |
Cardiothoracic ratio (%) | 1.09 (1.01–1.18) | 0.02 | 1.15 (1.02–1.29) | 0.02 | 1.09 (0.98–1.21) | 0.10 |
Systolic blood pressure (mmHg) | 1.00 (0.98–1.02) | 0.93 | 1.00 (0.97–1.03) | 0.95 | 1.00 (0.98–1.03) | 0.75 |
Hemoglobin (g/dL) | 0.90 (0.65–1.26) | 0.54 | 0.35 (0.01–11.63) | 0.55 | 0.95 (0.62–1.45) | 0.80 |
Serum albumin (g/dL) | 0.55 (0.18–1.68) | 0.29 | 1.11 (0.19–7.42) | 0.91 | 1.14 (0.25–5.81) | 0.87 |
Total cholesterol (mg/dL) | 0.99 (0.97–1.00) | 0.06 | 0.98 (0.96–1.00) | 0.10 | 0.99 (0.98–1.01) | 0.47 |
Serum calcium (mg/dL) | 0.83 (0.59–1.18) | 0.30 | 0.72 (0.40–1.23) | 0.23 | 0.85 (0.52–1.34) | 0.48 |
Serum phosphorus (mg/dL) | 0.83 (0.61–1.14) | 0.25 | 0.94 (0.57–1.54) | 0.80 | 0.82 (0.54–1.26) | 0.36 |
Intact PTH (pg/mL) | 1.00 (0.99–1.00) | 0.91 | 1.00 (0.998–1.004) | 0.35 | 1.00 (0.997–1.003) | 0.79 |
Klotho (Low) | 2.17 (0.83–5.18) | 0.11 | 3.50 (0.86–13.26) | 0.08 | 4.38 (1.37–14.04) | 0.014 |
Serum FGF23 (High) | 1.33 (0.57–3.22) | 0.51 | 1.95 (0.52–9.25) | 0.33 | 0.98 (0.31–3.15) | 0.98 |
1,25(OH)2D (pg/mL) | 1.00 (0.90–1.09) | 0.96 | 1.07 (0.92–1.21) | 0.34 | 1.04 (0.90–1.17) | 0.96 |
Vitamin D sterols (No) | 0.65 (0.27–1.50) | 0.31 | 0.41 (0.09–1.56) | 0.19 | 0.59 (0.17–1.84) | 0.36 |
Phosphate binders (No) | 1.48 (0.24–5.09) | 0.61 | 2.01 (3.14–3.14) | 0.25 | 2.01 (2.29–2.29) | 0.19 |
ESA (No) | 0.94 (0.27–1.50) | 0.93 | 1.94 (2.42–2.42) | 0.19 | 1.94 (1.76–1.76) | 0.13 |
ARBs/ACEIs (No) | 1.57 (0.68–3.82) | 0.29 | 3.66 (0.88–24.60) | 0.08 | 3.15 (0.94–14.22) | 0.06 |
BMI: body mass index; nPCR: normalized protein catabolism rate; PTH: parathyroid hormone; Klotho: serum soluble Klotho; FGF23: fibroblast growth factor 23; ESA: erythropoiesis stimulating agent; ARBs: angiotensin receptor blockers; ACEIs: angiotensin converting enzyme inhibitors; CCBs: calcium channel blockers.
Kaplan-Meier plots of cardiovascular event-free survival (a), cardiovascular mortality (b), and cumulative survival (c). The patients were categorized into low-KL (<309 pg/mL), middle-KL (309 to <449 pg/mL), and high-KL (≥449 pg/mL) groups.
Cardiovascular event-free survival (a), cardiovascular mortality (b), and cumulative survival (c) by the 25th percentile of serum soluble Klotho. Note that the patients categorized into the low-KL (<309 pg/mL) group had poorer cardiovascular (
Significance of the serum FGF23 levels with respect to cardiovascular event-free survival (a), cardiovascular mortality (b), and cumulative survival (c). The patients were categorized into low-FGF23 (<1184 pg/mL), middle-FGF23 (1184 to <15134 pg/mL), and high-FGF23 (≥15134 pg/mL) groups. The Kaplan-Meier analysis with the log-rank test failed to demonstrate any significant differences between the groups.
In the current study, we demonstrated that chronic HD subjects have worse cardiovascular and all-cause mortality rates with low serum sKlotho levels than those without low serum sKlotho levels. Moreover, our results showed that a reduced level of serum sKlotho is associated with all-cause mortality, even after adjusting for confounding variables, implying that the circulating sKlotho is a potential predictive indicator of overall mortality among patients with end-stage renal disease (ESRD). Previous findings have demonstrated that low serum sKlotho is associated with adverse kidney disease outcomes and/or arterial stiffness in some subsets of CKD patients and is related to all-cause mortality in older community-dwelling adults [
Information regarding the kinetics of circulating sKlotho has been poorly understood [
The soluble form of Klotho may target multiple remote tissues and organs, comprising a wide range of biological activities, including antiaging properties [
Several studies in CKD patients have shown that circulating FGF23, a hormone involved in phosphorous and vitamin D homeostasis, predicts adverse outcome [
In this study, we demonstrate for the first time that chronic hemodialysis subjects with low serum soluble Klotho levels have worse cardiovascular and all-cause mortality rates than those without low soluble Klotho levels. Moreover, we found that reduced serum soluble Klotho is associated with all-cause mortality, even after adjusting for confounding variables, implying that the level of serum soluble Klotho plays a role as a predictive indicator of overall mortality in patients with ESRD.
The authors declare that they have no competing interests.
This study was supported in part by a Grant-in-Aid for Research on Advanced Chronic Kidney Disease, Practical Research Project for Renal Diseases from Japan Agency for Medical Research and Development, AMED, and the Japan Dialysis Outcome Research (2006-013). The authors thank Gary Baley for academic editing of the paper. The authors also thank the dialysis center staff in the Japanese Red Cross Koga Hospital for their excellent technical assistance.