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.