1. Background
The Klotho gene and its related protein were identified as a putative aging factors in 1997, when the aging process was aggravated in a group of Klotho knockout mice (1). Klotho is expressed mainly in the kidneys, parathyroid glands, brain choroid plexus, and testes (2-4). Studies have confirmed Klotho expression in other tissues, including the aorta, colon, thyroid gland, and pancreas, but the kidney remains the strongest Klotho-producing organ (5).
There are two types of Klotho: circulating and membrane-bound. The latter functions as a co-receptor for fibroblast growth factor-23 (FGF23). The membrane-bound form, after losing its membrane domain, enters into the circulation as soluble Klotho (sKl), acting as a hormone with anti-aging and anti-oxidative stress properties; sKl can also be directly generated by alterative splicing of the Klotho transcript (2, 5). Klotho deficiency is an early biomarker for chronic kidney disease, and its upregulation could protect the kidney from fibrosis progression (6). The beneficial effect of physical activity in preventing premature mortality has been established by epidemiological studies showing that exercise may delay aging through various mechanisms. Exercise-induced Klotho upregulation could be one explanation. Klotho upregulates nitrous oxide (NO) production and inhibits angiotensin II-induced reactive oxygen species production within endothelial cells (7). In an epidemiological study, handgrip strength, an indicator of total body muscle strength, was correlated with plasma Klotho concentration (8).
2. Objectives
The purpose of this study was to determine whether plasma Klotho levels are influenced by aerobic exercise. For this purpose, plasma Klotho levels were measured in a group of trained athletes.
3. Materials and Methods
In this study, 30 healthy football players (males aged 18 – 22 years) participated. All participants were performing daily morning and evening exercise training. The controls were 28 healthy young males (age range 18 – 27 years). All subjects were nonsmokers and free of cardiovascular disease, as indicated by their medical history. None of the subjects took cardiovascular medications or hormone replacement therapy, and they all maintained routine eating habits. In the experimental group, Klotho concentration was measured the morning after a session of afternoon training, with blood samples collected from the antecubital vein. All participants had abstained from caffeine and fasted for at least 8 h before sampling. The measurements were performed at constant room temperature (22°C). We did not measure the level of physical activity in the control group; they were healthy young males with normal daily physical activity, but none were trained athletes. Serum concentrations of cholesterol and triglycerides were also measured in both groups. Plasma Klotho concentrations were measured with the ELISA technique, using a soluble Klotho ELISA assay kit based on the manufacturer’s instructions (Human Klotho ELISA Kit; Hangzhou Eastbiopharm Co., Ltd., Hangzhou, China).
4. Results
The demographic characteristics and measurements of the athlete group and the controls, respectively, were as follows: age, 18 – 22 versus 18 – 27 years; body mass index, 22.3 ± 1.4 versus 24.9 ± 1.3 kg/mg; total cholesterol, 5.3 ± 0.4 versus 5.7 ± 0.3 mmol/L; triglycerides, 1.5 ± 0.1 versus 1.7 ± 0.15 mmol/L; serum calcium, 9.8 ± 0.8 versus 9.7 ± 0.6; serum phosphorus, 4.4 ± 0.3 versus 4.93 ± 0.34; systolic blood pressure, 117 ± 5 versus 119 ± 6 mmHg; diastolic blood pressure, 70 ± 4 versus 71 ± 3 mmHg; and plasma Klotho, ng/mL 3.375 ± 1.48 ng/mL versus 1.39 ± 0.43 ng/mL (P < 0.05).
We found no significant differences between the groups for total cholesterol, triglycerides, and blood pressure. The control subjects were within close range of the previously proposed Klotho concentrations for normal individuals, while the athlete group had significantly higher plasma Klotho concentrations.
5. Discussion
The results of this study showed that aerobic exercise training induces an increase in plasma Klotho levels. Plasma Klotho levels were only measured one time, the day after exercise in the athlete group; therefore, it is not known whether this elevation continues over time. Our study population and the controls were healthy young adult males, and their serum calcium and phosphate levels were within the normal range. In the study group, we collected the blood samples the morning after the last evening exercise, so we cannot rule out the acute effect of exercise on plasma Klotho levels. It has been shown that aerobic exercise training induces increased plasma Klotho concentrations and decreased arterial stiffness in postmenopausal women (9). Exercise training might increase circulating Klotho due to increases in peroxisome proliferator-activated receptors (PPAR) and decreases in angiotensin II type I receptor (AT1R) signaling (10). Aerobic exercise-induced increases in plasma Klotho concentrations could be responsible for exercise-induced decreases in arterial stiffness (11), enhancing vascular protection and ameliorating endothelin-induced arterial stiffness. Secreted Klotho protects endothelial cells and smooth muscle cells through NO production (12) and suppression of oxidative stress (13-15). Klotho-induced endothelial NO production regulates endothelial cell calcium influx (9). Transforming growth factor beta-1 (TGF-β1) and endothelin-1 (ET-1) receptor activation negatively affect arterial stiffness, and their levels are decreased by exercise training (16). Interestingly, in a cross-sectional study, low plasma Klotho concentrations were independently associated with disability among the elderly (17). Exercise-induced increment of serum Klotho could be due to increased Klotho secretion or increased splicing of membrane-bound Klotho (9).
The kidney is the major source of sKl production (18), and membrane-bound Klotho is also a co-activator of FGF23, which is prominently expressed in distal convoluted tubule (DCT) and proximal convoluted tubule (PCT) cells; these locations are essential for its function as a phosphaturic substance (11). Klotho deficiency is an early biomarker for chronic kidney disease (CKD), and a progressive decline in urine Klotho occurs with CKD progression (6, 11, 19). Endogenous Klotho may influence the processes of inflammation, oxidative stress, and vascular calcification and remodeling (20). Secreted Klotho directly blocks phosphate-induced dedifferentiation of vascular smooth muscle cells into osteoblast-like cells. Secreted Klotho also prevents the transformation of endothelial cells to osteoblast-like cells (21, 22).
Klotho production is affected by many physiological and non-physiological conditions. Angiotensin II downregulates renal Klotho protein expression (23), and AT1R blockade increases circulating Klotho. Conversely, oxidative stress downregulates Klotho production (23).
Further studies are needed in order to clarify the dynamics of Klotho production and secretion, and to understand the mechanisms of exercise-induced Klotho secretion or shedding.
