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The aging-suppressor gene klotho encodes a single-pass transmembrane protein that in mice is known to extend life span when overexpressed and resemble accelerated aging when expression is disrupted. It is not known whether there is a relationship between plasma levels of secreted klotho protein and longevity in humans.
We measured plasma klotho in 804 adults, greater than or equal to 65 years, in the InCHIANTI study, a longitudinal population-based study of aging in Tuscany, Italy.
During 6 years of follow-up, 194 (24.1%) of the participants died. In a multivariate Cox proportional hazards model, adjusting for age, sex, education, body mass index, physical activity, total cholesterol, high-density lipoprotein cholesterol, cognition, 25-hydroxyvitamin D, parathyroid hormone, serum calcium, mean arterial pressure, and chronic diseases, participants in the lowest tertile of plasma klotho (<575 pg/mL) had an increased risk of death compared with participants in the highest tertile of plasma klotho (>763 pg/mL; hazards ratio 1.78, 95% confidence interval 1.20–2.63).
In older community-dwelling adults, plasma klotho is an independent predictor of all-cause mortality. Further studies are needed to elucidate the potential biological mechanisms by which circulating klotho could affect longevity in humans.
THE aging-suppressor gene klotho encodes a single-pass transmembrane protein that is predominantly expressed in the distal tubule cells of the kidney, parathyroid glands, and choroid plexus of the brain. The klotho gene, named after the Greek goddess who spins the thread of life, was originally identified in a mutant mouse strain that could not express klotho, developed multiple disorders resembling human aging, and had a shortened life span (1). The aging phenotypes included arteriosclerosis, decreased bone mineral density, sarcopenia, skin atrophy, and impaired cognition (2). Overexpression of klotho in transgenic mice resulted in a significant extension of life span compared with wild-type mice (3). These observations have supported the idea that klotho plays an important role in longevity.
There are two forms of klotho, membrane and secreted, and each has different functions. Membrane klotho acts as an obligate coreceptor for fibroblast growth factor (FGF)-23, a bone-derived hormone that induces phosphate excretion into urine (4). Secreted klotho is involved in calcium homeostasis in the kidney (5,6) and inhibition of intracellular insulin and insulin-like growth factor-1 (IGF-1) signaling (3). Klotho gene transcripts for a putative secreted form of klotho protein were described in 1998 (7). In 2004, Imura and colleagues (8) demonstrated that klotho protein was present in both human sera and cerebrospinal fluid. The relationship of circulating klotho with clinical phenotypes in human aging has not been studied because of the lack of a sensitive and reliable assay for measurement of secreted klotho protein in the blood. For example, whether low plasma klotho concentrations are related to mortality is not known. Recently, a sensitive and specific assay was developed for the measurement of soluble α-klotho in humans (9).
We hypothesized that low plasma klotho levels were an independent risk factor for mortality. To address this hypothesis, we measured plasma klotho levels in a large, longitudinal population-based study of aging.
The study participants consisted of men and women, aged 65 years and older, who participated in the Invecchiare in Chianti, “Aging in the Chianti Area” (InCHIANTI) study, conducted in two small towns in Tuscany, Italy. The rationale, design, and data collection have been described elsewhere, and the main outcome of this longitudinal study is mobility disability (10). Briefly, in August 1998, 1,270 people aged 65 years and older were randomly selected from the population registry of Greve in Chianti (population 11,709) and Bagno a Ripoli (population 4,704), and of 1,256 eligible participants, 1,155 (90.1%) agreed to participate. Participants received an extensive description of the study and participated after written informed consent. The study protocol complied with the Declaration of Helsinki and was approved by the Italian National Institute of Research and Care on Aging Ethical Committee and by the Institutional Review Board of the Johns Hopkins University School of Medicine.
Participants were evaluated again for a 3-year follow-up visit from 2001 to 2003 (n = 926), 6-year follow-up visit from 2004 to 2006 (n = 844), and 9-year follow-up visit from 2007 to 2009 (n = 768). Of the 926 participants seen at the 3-year follow-up visit, 804 (86.8%) had blood drawn and plasma available for analysis. There were no significant differences in age, sex, other demographic factors, or subsequent mortality between those who did or did not participate in the blood drawing. Plasma klotho was measured at the 3-year follow-up visit and not the enrollment visit because of the greater availability of archived plasma samples from the 3-year visit. The 3-year visit will be referred to as the baseline visit for the present study of klotho and mortality.
At the end of the field data collection, mortality data of the original InCHIANTI cohort were collected using data from the Mortality General Registry maintained by the Tuscany Region. Analyses include those who refused to participate in the follow-up after baseline or those who moved away but were known to be alive at the time of censoring of this analysis. Causes of death were not available for all participants who died because cause-specific data have not yet been released by the Tuscany regional authorities. Therefore, the analysis in the present study is based upon all-cause mortality.
Demographic information and information on smoking and medication use were collected using standardized questionnaires. Smoking history was determined from self-report. Daily alcohol intake, expressed in grams per day, was determined based upon the European Prospective Investigation into Cancer and Nutrition food frequency questionnaire that had been validated in the Italian population. Education was recorded as years of school.
All participants were examined by a trained geriatrician. Diseases were ascertained according to standard, preestablished criteria and algorithms based upon those used in the Women’s Health and Aging Study for diabetes mellitus, coronary heart disease, chronic heart failure, stroke, and cancer (11). The diagnostic algorithm for the diagnosis of diabetes was based upon the use of insulin, oral hypoglycemic agents, and a questionnaire administered to the primary care physician of the study participant (11). Those who did not have diabetes by the algorithm but had a fasting plasma glucose more than 125 mg/dL (12) were also considered to have diabetes.
Systolic and diastolic blood pressures were calculated from the mean of three measures taken with a standard mercury sphygmomanometer during the physical examination. Weight was measured using a high-precision mechanical scale. Standing height was measured to the nearest 0.1 cm. Body mass index (BMI) was calculated as weight/height2 (kilograms per square meter). Mini-Mental State Examination was administered at enrollment, and a Mini-Mental State Examination score less than 24 was considered consistent with cognitive impairment (13). Chronic kidney disease was defined as estimated glomerular filtration rate of less than 60 mL/min/1.73 m2 using the four-variable Modification of Diet in Renal Disease Study equation of Levey and colleagues (14).
Blood samples were collected in the morning after a 12-h fast. Aliquots of serum and plasma were immediately obtained and stored at −80°C. Soluble α-klotho was measured in EDTA plasma using a solid-phase sandwich enzyme-linked immunosorbent assay (Immuno-Biological Laboratories, Takasaki, Japan) (9). The minimum level of detectability of the assay is 6.15 pg/mL. The minimum level is below the plasma concentrations that were found in our study. The intra-assay and interassay coefficients of variation were 4.1% and 8.9%, respectively, for klotho measurements in the investigator’s (R.D.S.) laboratory. The designation α-klotho is used to describe the original klotho gene and its product (6) and to distinguish it from a homolog, which was named β-klotho (15). Throughout this article, the term “klotho” refers to α-klotho. Commercial enzymatic tests (Roche Diagnostics, Mannheim, Germany) were used for measuring serum total cholesterol, triglycerides, and high-density lipoprotein cholesterol concentrations. Low-density lipoprotein cholesterol was calculated by the Friedewald formula (16). Serum 25(OH)D was measured using a radioimmunoassay (DiaSorin, Stillwater, MN) with intra-assay and interassay coefficients of variation of 8.1% and 10.2%, respectively (17). Serum intact parathyroid hormone (PTH) levels were measured with a two-site immunoradiometric assay kit (N-tact PTHSP; DiaSorin) with intra-assay and interassay coefficients of variation of less than 3.0% and 5.5%, respectively.
Variables are reported as medians (25th, 75th percentiles) or as percentages. Characteristics of the participants were compared across tertiles of plasma klotho and by vital status using Wilcoxon rank sum tests for continuous variables and chi-square tests for categorical variables. Cox proportional hazards models were used to examine the relationship between plasma klotho and all-cause mortality over 6 years of follow-up. Multivariate Cox proportional hazards models were adjusted for age, sex, BMI, and then other variables that were significant in the univariate analyses. Interaction terms were used to evaluate the relationship between age, plasma klotho, and mortality. Survival curves were compared using log-rank tests. All analyses were performed using SAS (v. 9.1.3; SAS Institute, Inc., Cary, NC) with a type I error of 0.05.
Overall, the mean (SD) plasma klotho concentrations were 697 (325) pg/mL. The characteristics of the participants across tertiles of plasma klotho concentrations are shown in Table 1. Lower plasma klotho concentrations were associated with older age, lower calcium, lower high-density lipoprotein cholesterol, high triglycerides, and greater cognitive impairment. There were no significant differences across the tertiles of plasma klotho by sex, education, alcohol intake, current smoking, BMI, physical activity, mean arterial pressure, 25(OH)D, PTH, total cholesterol, low-density lipoprotein cholesterol, or by prevalence of chronic diseases. The proportion of participants who died was highest in the lowest tertile and lowest in the highest tertile of plasma klotho (p = .0002). Plasma klotho decreased with increasing age, as shown in a scatterplot (Figure 1).
During 6 years of follow-up, 194 (24.1%) of 804 participants died. The demographic and health characteristics of participants who lived or died are shown in Table 2. Median plasma klotho concentrations were significantly lower in adults who died from all causes compared with adults who survived. Participants who died from all causes were more likely to be older; less educated; with lower mean arterial pressure; with lower total, high-density lipoprotein, and low-density lipoprotein cholesterol; and with Mini-Mental State Examination score less than 24; lower 25(OH)D; higher PTH; lower serum calcium; coronary heart disease; heart failure; peripheral artery disease; stroke; diabetes mellitus; cancer; and chronic kidney disease. There were no significant differences between participants who survived or died from all causes by sex, current smoking, BMI, and triglycerides. Survival curves for all-cause mortality in participants by tertile of plasma klotho are shown in Figure 2.
The relationship between plasma klotho and all-cause mortality was examined using multivariate Cox proportional hazards models (Table 3). The lowest and middle tertiles of plasma klotho were compared with the highest tertile (reference). The lowest tertile of plasma klotho was significantly associated with all-cause mortality after adjusting for age, sex, education, BMI, physical activity, total and high-density lipoprotein cholesterol, Mini-Mental State Examination score, 25(OH)D, PTH, serum calcium, mean arterial pressure, and chronic diseases (Table 3). In additional alternative models that adjusted for all covariates in the final model, we added interaction terms between plasma klotho and age, and these interaction terms were not significant.
In order to determine whether the relationship between klotho and all-cause mortality was relatively consistent in older versus the oldest old in this study, we stratified by age. In adults 65 to less than 80 years, the lowest and middle tertiles, respectively, of plasma klotho were associated with all-cause mortality compared with the upper tertile (hazards ratio 1.58, 95% confidence interval 0.88–2.86; hazards ratio 1.65, 95% confidence interval 0.93–2.93), adjusting for the same covariates as the final model in Table 3. In adults greater than or equal to 80 years, the lowest and middle tertiles, respectively, of plasma klotho were associated with all-cause mortality compared with the upper tertile (hazards ratio 2.53, 95% confidence interval 1.42–4.52; hazards ratio 1.85, 95% confidence interval 1.00–3.46). There was less power when the analyses were stratified, but the relationship between klotho and all-cause mortality went in the same direction in both age strata.
The present study demonstrates that older community-dwelling men and women with low plasma klotho are at a greater risk of dying, or, in other words, low plasma klotho is an independent predictor of mortality. To our knowledge, this is the first study to confirm that low plasma klotho is associated with longevity in humans—a hypothesis proposed by many previous authors but never empirically tested. These findings support the idea from the klotho mouse studies that secreted klotho is a factor that may play a role in longevity. Klotho may be of special interest as a risk factor for mortality in humans because life span has been shown to be related to the level of expression of klotho in mice (1,3). The homology between the klotho gene in mouse and humans is extremely high; in humans, the secreted form of klotho is more dominant than the membrane form (7).
There are several potential mechanisms by which circulating klotho could affect health and survival, including regulation of growth factor signaling pathways, protection against endothelial dysfunction, regulation of ion channels, and suppression of oxidative stress (2). Reduced signaling or action of insulin, IGF, and related hormones increases the life span of a wide range of organisms (18). Klotho-deficient mice are hypoglycemic and extremely sensitive to insulin (19), whereas mice that overexpress klotho show moderate resistance to insulin and IGF-1 but maintain normal fasting blood glucose levels and are not diabetic (3). These findings suggest that klotho may inhibit the insulin/ IGF-1 signaling pathway.
Endothelial dysfunction promotes atherosclerosis and is a major risk factor for adverse cardiovascular events (20). In klotho-deficient mice, endothelium-dependent vasodilatation is impaired and associated with reduced nitric oxide synthesis (21). In addition, parabiosis (surgical connection of vasculature) between klotho-deficient and wild-type mice restored the endothelial function of klotho-deficient mice. The delivery of the klotho gene via an adenoviral vector increased endothelial-dependent nitric oxide synthesis in a rat model of multiple atherogenic risk factors (22). The mechanism by which klotho regulates the synthesis of nitric oxide in the endothelium is not known.
Circulating klotho regulates two major ion channels in the kidney, transient receptor potential vanilloid 5 (TRPV5) ion channel, and renal outer medullary potassium channel (ROMK1). TRPV5 is expressed on the apical side of renal tubule cells and functions as the entry gate for transepithelial calcium reabsorption in the kidney. Circulating klotho increases the number of TRPV5 on the cell surface and thus increases renal calcium reabsorption (5). ROMK1 is a potassium channel located on the apical side or renal tubular cells and is responsible for potassium excretion in the urine. Circulating klotho increases the number of ROMK1 on the plasma membrane and increases potassium excretion into urine (23).
The present study showed that plasma klotho concentrations were positively associated with higher serum calcium concentrations. These findings are consistent with what is known about the relationship of circulating klotho with increasing calcium reabsorption in the kidney. The differences in serum concentrations across tertiles of klotho were small but statistically significant. The present study showed no significant relationship between plasma klotho and 25(OH)D and PTH. These results corroborate findings from a previous study, conducted in a convenience sample of infants through older adults, which showed no significant correlation between circulating klotho and PTH (9). Our previous studies in the same cohort have shown that older adults with lower 25(OH)D concentrations are at a higher risk of death (17). The results of the present study suggest that plasma klotho is an independent predictor of mortality in multivariate models that adjusted for 25(OH)D, PTH, and calcium, as well as other potential confounders.
Longevity is associated with an increased resistance to oxidative stress (24). Transgenic mice that overexpress klotho have reduced levels of urinary 8-OHdG, a marker for oxidative DNA damage (25). Klotho appears to increase resistance to oxidative stress through inhibition of insulin/IGF-1 signaling, decrease in phosphorylated forkhead (FOXO) transcription factors, and upregulation of antioxidant enzymes such as catalase and superoxide dismutase (25).
Genetic variants of klotho have been associated with longevity and health in humans. An allele termed KL-VS contains six sequence variants in complete linkage disequilibrium, two of which result in amino acid substitutions F352V and C370S in the klotho gene (26). The KL-VS variant was significantly associated with reduced longevity in three distinct populations (Bohemian Czechs, Baltimore Caucasians, and Baltimore African-Americans) with a combined odds ratio of 2.59 (27). In Ashkenazi Jews, the KL-VS variant of klotho was also associated with reduced longevity (27). Klotho polymorphisms are also associated with coronary artery disease (28–30), stroke (31), and bone density (32–35).
A limitation of the study is that the specific causes of death were not yet available for all the subjects who died during follow-up; thus, the analyses were limited to all-cause mortality. Further studies are needed in the future that examine the relationship between circulating klotho and cardiovascular disease mortality and cancer mortality. Another limitation is that there may be residual confounding in the multivariate models due to measurement error and incomplete characterization of variables that were included in the models, given that only one set of measurements was used to determine baseline status.
Although the present study provides the first evidence that circulating klotho is related with longevity in humans, there are many areas that need to be addressed in future studies. In humans, the relationship between circulating klotho and IGF-1 has not been described. It is not known whether circulating klotho is related to endothelial function. Whether adults with low plasma klotho levels have increased biomarkers for oxidative stress needs further investigation. It is not known whether genetic variants of klotho are associated with higher or lower levels of circulating klotho. Whether plasma klotho levels are an independent predictor of incident adverse aging-related outcomes, such as cardiovascular disease, osteoporosis, and renal disease, need to be examined in large longitudinal aging cohorts. Further insights are needed into the potentially important role of klotho in the biology of human aging.
This work was supported by the National Institute on Aging (NIA) (R01 AG027012), the Italian Ministry of Health (ICS110.1/RF97.71), and NIA contracts 263 MD 9164, 263 MD 821336, N.1-AG-1-1, N.1-AG-1-2111, and N01-AG-5-0002, the Intramural Research Program of NIA, National Institutes of Health, Baltimore, Maryland.