Serum klotho is inversely associated with metabolic syndrome in chronic kidney disease: results from the KNOW-CKD study

Background Metabolic syndrome (MS) is prevalent in chronic kidney disease (CKD). Klotho, a protein linked to aging, is closely associated with CKD. Each component of MS and klotho has an association. However, little is known about the association between klotho and MS per se. We investigated the association between serum klotho levels and MS using baseline cross-sectional data obtained from a large Korean CKD cohort. Methods Of the 2238 subjects recruited in the KoreaN Cohort Study for Outcome in Patients With Chronic Kidney Disease (KNOW-CKD) between 2011 and 2016, 484 patients with missing data on serum klotho and extreme klotho values (values lower than the detectable range or > 6000 pg/mL) or with autosomal dominant polycystic kidney disease patients were excluded. The data of the remaining 1754 subjects were included in the present study. MS was defined using the revised National Cholesterol Education Program Adult Treatment Panel (NCEP-ATP) III criteria. Serum klotho levels were measured using an enzyme-linked immunosorbent assay. Results Mean patient age was 54.9 ± 12.1 years and 1110 (63.3%) were male. The prevalence of MS among all study subjects was 63.7% (n = 1118). The median serum klotho level was 527 pg/mL (interquartile range [IQR]: 418–656 pg/mL). Serum klotho level was significantly lower in MS patients than patients without MS (Median [IQR]; 521 pg/mL [413, 651] vs. 541 pg/mL [427, 676], respectively; P = 0.012). After adjusting for age, sex, estimated glomerular filtration rate, and overt proteinuria, serum klotho was independently associated with MS (adjusted odds ratio [OR], 0.44; 95% confidence interval, 0.23–0.82; P = 0.010). Furthermore, the adjusted OR for MS was found to be significantly increased at serum klotho levels of < 518 pg/mL (receiver operating characteristic curve cut-off value). Conclusions Serum klotho was inversely associated with the presence of MS in patients with CKD. Trial registration This trial was registered on ClinicalTrials.gov on 26 June 2012 (https://clinicaltrials.gov;NCT01630486).


Background
Metabolic syndrome (MS) is a cluster of risk factors for diabetes and cardiovascular (CV) disease and is characterized by hypertension, hyperglycemia, hypertriglyceridemia, decreased high-density lipoprotein (HDL) cholesterol, and abdominal obesity [1,2]. Furthermore, MS is common in chronic kidney disease (CKD) patients [3]. Previous studies showed that MS was independently associated with an increased risk for CKD and vice versa [4,5].
Klotho, a protein linked to aging, is closely associated with CKD. In a previous study, klotho knock-out mice exhibited similarities with CKD patients, such as, hyperphosphatemia, ectopic soft tissue calcification, and arteriosclerosis [6], which suggested CKD might result from a state of klotho deficiency. Thus, in addition to serving as a biomarker for CKD, klotho deficiency is also viewed as a pathogenetic indicator of renal and extra-renal complications in CKD [7].
Recent reports indicated klotho is associated with each component of MS. Arking et al. [8] showed that a functional variant of the Klotho gene was associated with increased systolic blood pressure and decreased HDL-cholesterol levels in 525 Jewish subjects. In experimental studies, klotho has been shown to ameliorate vascular endothelial dysfunction, to increase nitric oxide production, and to reduce elevated blood pressure in an animal models of MS [9,10]. Klotho polymorphism was significantly associated with glucose metabolism in apparently healthy Korean females [11]. In addition, genetic variants of Klotho have been associated with insulin resistance and hypertriglyceridemia [12]. Accordingly, it was suggested in all of these studies that klotho be considered a candidate molecule in MS. However, little is known about the association between klotho and MS per se in CKD patients. In the present study, we investigated the metabolic profile characteristics of Korean CKD patients and evaluated the association between serum klotho and MS using baseline cross-sectional data obtained from a large Korean CKD cohort.

Study population
We conducted a cross-sectional analysis of clinical profiles obtained at enrollment for the KoreaN Cohort Study for Outcome in Patients With Chronic Kidney Disease (KNOW-CKD) study. KNOW-CKD is a nationwide prospective cohort study, including predialysis subjects with CKD from stage 1 to 5, aged between 20 and 75 years and recruited from nine clinical centers of major university-affiliated Korean hospitals. Details of the rationale and study design of the KNOW-CKD have been described elsewhere [13]. Of 2238 CKD patients recruited between June 2011 and January 2016, 484 were excluded for the following reasons: 125 for missing serum klotho data; 9 for a serum klotho level lower than the detectable range; 3 for an extremely higher klotho level (> 6000 pg/ mL); and 347 for autosomal dominant polycystic kidney disease patients because large cystic masses in their kidneys and liver can cause an overestimation of abdominal obesity. Extreme klotho values were not influence value by Cook's distance analysis. Finally, 1754 CKD patients were included in the cross-sectional analysis. Klotho expression is known to decline in cases of acute kidney injury, regardless of whether it is caused by ischemia, infection, a toxin, or ureteral obstruction [14][15][16]. However, in the present study, we did not enroll patients with acute illness at the time of initial enrollment. Therefore, it is unlikely that initial klotho levels were influenced by acute illness. The study protocol was approved by the ethical committees of participating clinical centers, that is, by the Institutional Review Boards of Seoul National University Hospital (1104-089-359),   in 2011. All study subjects provided written informed consent. This study was performed in accordance with the principles of the Declaration of Helsinki.

Clinical data collection and laboratory analyses
Laboratory values and demographic characteristics at enrollment were extracted from an electronic casereporting form (http://www.phactax.org) developed by the assistance of the Division of Data Management at Seoul National University Medical Research Collaborating Center. Waist circumferences were measured midway between the lower part of the lowest rib and the upper iliac crest using a standardized protocol at clinics, as recommended by the World Health Organization (WHO) [17]. Brachial systolic blood pressure (SBP) and diastolic blood pressure (DBP) were obtained by averaging two measurements (separated by a minimum rest of 5 min in the sitting position) obtained using a calibrated oscillometric device (BP-203RV III; Omron Co., Kyoto, Japan). Serum creatinine levels were measured by an isotope dilution mass spectrometry (IDMS)-traceable method [18] at a central laboratory. Estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) creatinine equation [19]. Fasting blood glucose and triglyceride levels were measured in the hospital laboratories of participating center according to a standardized protocol. Overt proteinuria was defined as a 24-h urine protein result of > 500 mg/day. Serum α-klotho level was measured using an enzyme linked immunosorbent assay (ELISA) kit (Immuno-Biological Laboratories Co., Gunma, Japan) according to the manufacturer's instructions [20]. The intra-and inter-assay coefficients of variation of this kit were 2.7-3.5% (klotho levels 186.64-2968.78 pg/mL) and 2.9-11.4% (klotho levels 165.47-2903.01 pg/mL), respectively. The data for intra-and inter-assay coefficients of variations were validated in our central laboratory by measurements of a serum control in 20 repeats on each ELISA plate. The intra-and inter-assay coefficients of variations were 0.57-1.78% and 3.01-6.12% (klotho levels 120. .50 pg/mL), respectively. The standard curve was shown to be linear up to 4000 pg/mL. C-terminal FGF23 was measured using a second generation human FGF23 ELISA kit (Immutopics, San Clemente, California, USA) according to the manufacturer's instructions.

Results
Baseline clinical characteristics of subjects according to the presence of metabolic syndrome The clinical characteristics of patients at enrollment are shown in Table 1 Fig. 1. High blood pressure (95.8%) was the most prevalent component, followed by high fasting glucose (63.7%) and abdominal obesity (53.8%). Figure 2 presents the prevalence of MS across CKD stages. The prevalence of MS was > 50% even for early stage CKD. The prevalence of MS was higher in advanced stages of CKD (P < 0.001, P for linear trend < 0.001). As show in Fig. 3, the adjusted OR (adjusted for age, sex, eGFR, and overt proteinuria) of MS was significantly increased at eGFR levels of < 60 ml/min/1.73m 2 . Subjects were divided into two groups according to the presence of MS. Patients with MS were older (P < 0.001, Table 1) and had higher blood pressure (P < 0.001), body mass index (P < 0.001), and waist circumference (P < 0.001). At enrollment, diabetes mellitus (DM) (P < 0.001), hypertension (P < 0.001), and preexisting CV disease (P < 0.001) were more prevalent among subjects with MS. Mean eGFR (P < 0.001) was lower and uric acid (P = 0.025) level was higher in MS patients.  Fig. 4a). In addition, klotho levels tended to decrease as numbers of MS components increased ( Fig. 4b; P = 0.038).

Clinical characteristics of subjects stratified by serum klotho level
The clinical characteristics of patients dichotomized by serum klotho level (lower than median vs. equal to or higher than median) are shown in Table 2. Age age, sex, blood pressure, and underlying comorbidities were similar in these two groups. However, patients with a lower klotho level had a higher mean uric acid level (P < 0.001) and a higher C-reactive protein (CRP) level (P = 0.005). Mean eGFR (P < 0.001) and hemoglobin (P < 0.001) were lower in the low serum klotho level group. Serum klotho levels across CKD stages are shown in Fig. 5. Advanced CKD stages were associated with lower serum klotho levels (P < 0.001).  Table 3). As shown in Fig. 6, the adjusted OR of MS was significantly increased at serum klotho levels of < 518 pg/mL (the ROC cut-off value). Furthermore, adjusted for age, sex, eGFR, overt proteinuria, and drug information (ACEi or ARB, diuretics, statin), log klotho was still independently associated with the presence of MS (adjusted OR, 0.51; 95% CI, 0.27-0.98; P = 0.045).
ORs of relations between each MS component and serum klotho are shown in Table 4. Only high blood pressure (adjusted OR, 0.05; 95% CI, 0.01-0.29; P = 0.001) and hypertriglyceridemia (adjusted OR, 0.48; 95% CI, 0.27-0.87; P = 0.016) were found to be independently associated with log klotho. High fasting glucose, abdominal obesity, and low HDL cholesterol were not significantly associated with serum klotho.

Subgroup analysis
We further analyzed the relationship between klotho and MS in several groups. Significant interaction was found in age group (aged < 50 vs. ≥50 years), suggesting that the association between klotho and MS was particularly evident in subjects aged < 50 years (adjusted OR, 0.18; 95% CI, 0.06-0.56; P = 0.003 vs. adjusted OR, 0.72; 95% CI, 0.33-1.57; P = 0.412 in aged ≥50 years). Klotho levels tended to decrease as age increased (Pearson's correlation coefficient r = − 0.083, P < 0.001), but the value was not large. Klotho

Discussion
MS, a common clinical phenotype demonstrating as a combination of metabolic abnormalities including, hypertension, hyperglycemia, hypertriglyceridemia, decreased high-density lipoprotein cholesterol, and central obesity, is common in CKD [23]. In the present study, 63.7% of the 1754 CKD patients enrolled exhibited MS. The prevalence of MS exceeded 50% even in patients with early stage CKD and its prevalence was higher in advanced CKD stages. In the univariate analysis, serum klotho was significantly lower in patients with MS. After adjusting for various factors, such as, age, sex, eGFR, and overt proteinuria, serum klotho level remained an independent factor associated with MS in CKD.
In previous studies, genetic variants of Klotho increased the risk of MS which could be related to their susceptibility to high fasting glucose, high blood pressure, hypertriglyceridemia, and decreased HDL cholesterol [8,12]. Klotho has a role in the regulation of vascular tone through homeostatic interplay between the renin-angiotensin system and nitric oxide [24], which might explain the association between klotho and blood pressure. Furthermore, the Klotho has been shown to be a target gene for PPARγ, a key transcription factor controlling lipid metabolism and insulin sensitivity [25]. Thus, we speculated these factors might play major roles in the association of klotho with glucose and lipid metabolism.
MS is a cluster of risk factors of diabetes and CV disease. Subjects with CKD present higher incidence of CV disease and higher CV mortality than the general population [26,27]. Novel risk factors such as persistent inflammation, oxidative stress, and endothelial dysfunction can be linked to CV disease in CKD patients [28]. Fundamental manifestations of MS include insulin resistance and adipose tissue expansion, and the latter leads to oxidative stress and chronic inflammation, which aggravate insulin resistance [29][30][31]. Furthermore, these pro-inflammatory manifestations of MS might play roles in the pathogenesis of endothelial dysfunction and atherosclerosis [32]. Klotho is an anti-aging gene that extends life span when overexpressed and accelerates aging when disrupted [6]. Aging which reflects cellular senescence is closely associated with inflammatory reactions, oxidative damage, and endothelial dysfunction [33][34][35]. Klotho has also been shown to be an anti-inflammatory modulator [36,37], to be closely associated with oxidative stress [38,39]. Klotho plays a role in the protection against endothelial dysfunction [10]. The kidney is the principal organ responsible for the production of klotho, and CKD is known to be a klotho deficient state. Accordingly, the effects of klotho mentioned above suggest an association between klotho and MS beyond individual components of MS.
The present study is the first study, to our knowledge, to report the independent association between serum klotho level and MS per se especially in CKD patients. In previous studies, there were not many human studies, and most of studies were related to the association between genetic variants of Klotho and individual components of MS. In addition, few studies have been performed in patients with CKD. Furthermore, it was the strength of our study that we included a large-scale CKD cohort patients and obtained serum klotho level from a large number of our CKD patients. However, the study has several limitations that warrant consideration. First, because it was based on cross-sectional analysis, it could not demonstrate causality between klotho and MS. It could not provide detailed mechanistic links between klotho and MS. We just described the association between klotho and MS, not the causality. Second, serum klotho exhibits circadian variations [40]. They showed that midnight serum klotho level was an approximately 40% reduction with gradual return to near baseline values in the early morning hours. Serum klotho level decreased less than 20% compared with morning klotho level until 6 p.m. In the present study, blood samples were not obtained at any fixed time of the day. However, circadian variation of serum klotho may have been less in our study because blood sampling was done before 6 p.m.

Conclusions
Serum klotho levels were found to be independently and inversely associated with the presence of MS. Further studies are warranted to elucidate the nature of the mechanistic link between klotho and MS in CKD patients. Adjusted for covariates log klotho, age, sex, eGFR, and overt proteinuria OR Odds ratio, CI confidence interval, HDL high-density lipoprotein, eGFR estimated glomerular filtration rate by CKD-EPI creatinine equation