Study design and participants
For this study, data were obtained from the National Health and Nutrition Examination Survey. NHANES is a large-scale cross-sectional survey of all levels of the U.S. population, which is composed of two parts: interviews and physical examinations. The survey includes such items as nutrition, health and sociology of American families, and gathers representative sample data to reflect the population as a whole. It has been conducted since 1990, and a nationally representative sample of about 5000 people is surveyed annually, every 2 years is a survey cycle. As part of the investigation, NHANES provided written informed consent to all individuals participating, which was reviewed by the Centers for Disease Control and Prevention (CDC) and the National Center for Health Statistics (NCHS). During the survey period from 2007 to 2016, the NHANES surveyed 50,588 people in five cycles (2007–2008, 2009–2010, 2011–2012, 2013–2014, 2015–2016). After a series of screenings, 12,441 people qualified for data analysis as subjects of this study. The population screening exclusion criteria are as follows: (1) subjects without plasma S-Klotho data (n = 36,824); (2) subjects aged < 40 years or > 80 years (n = 95); (3) subjects missing blood pressure, long-term health information, important biochemical indicators, disease history or other data (n = 1171); (4) eGFRCKD–EPI < 15 ml/min/1.73m2 (n = 57). Details of the study design and exclusions are provided in the flowchart (Fig. 1).
Laboratory measurements
Blood samples were obtained from the venous blood of participants who had fasted for more than 9 h. The S-Klotho blood samples were stored in a (-80◦C) environment for data analysis in the University of Washington research laboratory. Researchers using a commercially available ELISA kit from Japan's IBL International, and using the sandwich ELISA to test S-Koltho after receiving the blood samples on dry ice [20]. To ensure the accuracy of the measured data, two quality control samples of low concentration and high concentration of Klotho were analyzed in duplicate in every ELISA board, and the final value was determined by calculating the average of the two results. The analysis results are automatically transmitted from the instrument to the laboratory Oracle management system, and the regional supervisor evaluates the results. Samples with repetitive results of more than 10% are marked as repetitive analysis. If the value of the quality control sample is not within the 2SD range of the specified value, the entire analysis run will be rejected and the sample analysis will be repeated. As a result of measuring and evaluating the samples of 114 obviously healthy individuals, S-Klotho ranged from 285.8 to 1638.6 pg/ml with a mean of 698.0 pg/ml. Biochemical indicators Blood samples are stored at (− 30 °C), and are analyzed by researchers using two methods: Beckman Synchron LX20 and Beckman UniCel® DxC800 Synchron. These biochemical indicators include urea, creatinine, serum uric acid, blood lipids, and fasting blood glucose levels. Hyperuricemia is defined as serum uric acid levels higher than or equal to 420 mmol/l in men and 360 mmol/l in women [21, 22].
Covariates measurements
Blood pressure and body mass index
Mercury sphygmomanometers calibrated with Bowman meters were used to measure the blood pressure of all the subjects. Following 5 min of sitting, the trained examiner asked the subjects to take three consecutive blood pressure measurements on their right arm, with a 30 s interval between every measurement. Based on three blood pressure measurements, a mean blood pressure was calculated, and then either hypertensive or non-hypertensive classification was assigned. Hypertension was defined as systolic blood pressure greater than or equal to 140 mmHg, or diastolic blood pressure greater than or equal to 90 mmHg, or both. The body mass index was calculated based on height and weight, and the formula was: (Kg)/(m2).
Smoking, drinking and physical activity
A questionnaire survey was used to gather information on smoking and drinking for all participants. Using a computer-assisted personal interview (CAPI) system, the interviewer asked questions to the subjects, and the drinking and smoking conditions were assessed based on their responses. The specific evaluation criteria are as follows:
Question1. Smoked at least 100 cigarettes in life?
Answer No: means no smoking.
Yes: represents smoking or has ever smoked.
(1) How long since quit smoking cigarettes?
No: Delegate still smoking.
Yes: Quit smoking.
Question2. Had at least 12 alcohol drinks/1 year?
Answer Yes: drink a lot.
No: little or no alcohol consumption.
(1) Had at least 12 alcohol drinks/lifetime?
No: hardly drink alcohol.
Yes: occasional drinking.
Considering low physical activity is directly related to low levels of S-Klotho, especially in middle-aged and elderly people, therefore, we include the variable of physical activity (PA) in the final model [42, 43]. The physical activity data obtained from the physical activity questionnaire (PAQ) filled out by all participants, which asked whether they had engaged in vigorous-intensity sports, fitness, or recreational activities for at least 10 min, resulting in large increases in breathing or heart rate; and whether they were involved in moderate-intensity sports, fitness or recreational activities cause small increases in breathing or heart rate and was done for at least 10 min continuously.
Personal medical history
During home interviews, the NHANES interviewer collected the subject's personal medical history in the form of a questionnaire. As part of the interview, the subjects were asked whether they had been told by a doctor or other professional health personnel that they had congestive heart failure, coronary heart disease, stroke, and diabetes. Accordingly, if the subject answers yes to any of the above-mentioned diseases, he is required to provide the date and medication status of the first diagnosis of the disease, to better collect personal disease history data. Renal function was assessed by eGFR using the Chronic Kidney Disease–Epidemiology Collaboration (CKD–EPI) equation, and those with an EGFR < 15 ml/min/1.73 m2 were excluded based on the results of the calculation.
Statistical analysis
The NHANES used a complex multistage probability sampling design to represent the health and nutrition status of the population at all levels within the United States, with oversampling in some subgroups. To avoid biased results in analysis and exaggeration of significance, the analyses were conducted in accordance with guidelines provided by the US Centers for Disease Control and Prevention and using the sample weights recommended by the guidelines. We calculated the sample weights of the two periods from 2007 to 2016, and completed the weight combination. The specific calculation formula is: MEC10YR = 1/5*WTMEC2YR. For continuous variables with a normal distribution, the data can be represented by the weighted mean ± SD; assuming the data have a skewed distribution, the weighted median is used to characterize the data mean, while the interquartile range describes the degree of dispersion; Comparing quantitative data between groups requires applying the Kruskal Wallis rank-sum test. The categorical variables were described by their rates or constituent ratios, and comparisons between groups were made with the chi-square test or Fisher's exact test. To test the correlation between the level of S-Klotho and the risk of hyperuricemia, a multiple logistic regression model was established based on the results of the univariate analysis, and the covariates with significant effects (P < 0.05) in the univariate analysis were included in the adjustment model.
The following models were developed: model 1, without adjusting for covariates; model 2, validated again after including age, sex, and race variables; model 3, with model 2 adjustment variables, as well as variables, such as blood pressure, smoking, BMI, urea, creatinine, triglyceride, high density cholesterol, low density cholesterol, fasting blood glucose, glycosylated hemoglobin, eGFRCKD–EPI, physical activity (PA), past disease history, etc. Considering that drinking may influence uric acid metabolism, drinking variables were included in the final model. Moreover, we conducted subgroup analyses by age and gender based on the model's final results, and the subgroup stratification variables were not included in their corresponding adjusted models. Finally, smooth curve fitting is used to show relationships between S-Klotho and the risk of hyperuricemia in middle-aged and elderly people. All data analysis was done by the statistical software package R (The R Foundation; http://www.r-project.org; version 3.5.3) and EmpowerStats (www.EmpowerStats.com; X&Y Solutions Inc.). P value of < 0.05 (double) was considered as statistically significant.