The αKlotho gene, initially discovered in mice, exhibits a deficiency-induced phenotype akin to human aging.1 Highly expressed in the kidney, the Klotho protein functions as a receptor for fibroblast growth factor 23 (FGF23).2 Klotho forms the FGFR-Klotho complex by binding to the FGF receptor (FGFR) via the receptor binding arm, serving as a physiological receptor for FGF23.2 Known as a single-pass transmembrane protein, the Klotho protein undergoes cleavage by proteolytic enzymes, such as secretases, resulting in the secretion of its extracellular domain (ectodomain shedding).3 In patients with chronic kidney disease, renal Klotho transcript significantly decreases, and soluble αKlotho emerges as a promising early-stage kidney disease biomarker.4 Current methods enable the measurement of soluble αKlotho (sKlotho) levels. Although the origin of sKlotho was identified as αKlotho highly expressed in the kidney,5 measuring sKlotho in healthy humans through invasive tests like catheterization proves challenging. Consequently, few clinical studies have reported the renal origin of blood sKlotho in humans via catheter sampling. To address this, we opted to measure sKlotho in blood samples from patients undergoing adrenal venous sampling (AVS) for primary aldosteronism (PA) diagnosis. This approach aims to ascertain whether the kidneys secrete sKlotho and identify the clinical parameters associated with blood sKlotho.

Table 1 presents the baseline characteristics of the 12 patients. In summary, the median age was 44.5 [43.8–56.0] years, with 50% (n=6) being male. The mean eGFR was 78 (±22) (mL/min/1.73m2). For all 12 cases, the PAC/PRA ratio (ARR) exceeded 200(pg/mL/ng/mL/h). All patients were on calcium channel blockers and three of them on concomitant alpha blockers. The patients were not taking any medications (mineralocorticoid receptor antagonists, beta blockers and diuretics) that could significantly affect ARR. The sKlotho concentration at the suprarenal IVC (476±74.8) was significantly higher than that at the infrarenal IVC (434±68.2) (p=0.018) (Fig. 1A), with a change rate of 8.12±2.3% (Fig. 1B). Correlations were observed between serum sodium level and the change in sKlotho (r=−0.773, p=0.003) and between ARR and the change in sKlotho (r=−0.790, p=0.004) (Fig. 1C and D). Table 2 shows the association between other clinical parameters and the rate of change in sKlotho. Renal function indices, such as BUN, Cr, and eGFR, did not exhibit significant associations with the rate of change in soluble αKlotho.

Fig. 1.

Blood soluble αKlotho in the infrarenal and suprarenal vena cava (A). Change in αKlotho (%) from the infrarenal to suprarenal vena cava (B). Relationship between serum sodium (mmol/L) levels and change in αKlotho (%) (C). Relationship between PAC/PRA ratio (ARR) and change in Klotho (%) (D). Data are presented as mean with standard error of the mean (SEM). Black circles and bars indicate the mean and SEM of Klotho.

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In this study, we utilized ELISA to demonstrate in humans that the concentration of sKlotho at the suprarenal IVC surpasses that at the infrarenal IVC, affirming the kidney as the organ of origin for soluble αKlotho. Hu et al. conducted a study with blood from catheterized patients (n=9) evaluating heart failure and other conditions, revealing a step-up in sKlotho concentration from the infrarenal to suprarenal vena cava.8 They measured sKlotho concentrations as protein levels using Western blot.8 While catheterization is highly invasive and challenging in normal subjects, our study and theirs consistently depict a step-up in sKlotho concentration from the infrarenal to suprarenal vena cava, despite differing pathological conditions in the sampled patients.

Picciotto et al. extensively examined renal Klotho release parameters in patients undergoing cardiac catheterization (n=22), identifying oxygen uptake as a predictor of renal αKlotho release.9 Although our study did not assess oxygenation, we found a correlation between the change in sKlotho (%) and serum sodium and ARR. In PA, excessive aldosterone secretion induces sodium reabsorption, leading to increased fluid volume and hypertension.10 Klotho(+/−) mice with elevated aldosterone levels develop hypertension,11 suggesting a potential link between strong aldosterone production and impaired αKlotho secretion. However, no relationship was observed between baseline blood pressure and the extent of sKlotho change. A recent large-scale study reported no difference in serum sKlotho levels measured by ELISA between individuals with and without hypertension.12 On the other hand, partial correlation analysis, controlling for serum potassium levels, showed no correlation between changes in sKlotho and serum sodium (r=0.162, p=0.677) in this study. This could be a result of the pathophysiology of primary aldosteronism affecting serum potassium levels, but the small sample size precluded detailed statistical analysis. Further investigation is needed to explore the relationship between hyperaldosteronism severity and the extent of sKlotho change.

In our study, the change in sKlotho did not correlate with eGFR. Only two patients had an eGFR below 60mL/min/1.73m2, and none exhibited advanced renal impairment. Given aldosterone's renal damage effects13 and the potential decrease in blood sKlotho levels from the early stages of renal damage,4,14,15 it cannot be ruled out that subjects in this study might have lower blood sKlotho levels than healthy individuals. Blood sKlotho levels in our study were measured by ELISA rather than protein levels. Another study utilizing an immunoprecipitation immunoblot (IP-IB) assay showed a decrease in sKlotho in people with renal impairment.16 Additionally, it has been reported that whether measured by ELISA or IP-IB, when a blood sample undergoes two or more freeze-thaw cycles, the value significantly decreases compared to zero or one thaw cycle.16 Our study used samples frozen only once, and we believe that sample degradation did not have a significant effect. IB could measure distinct full-length proteins but is labor-intensive, while ELISA is a simple method, but the results are highly variable and less accurate than IB. In this study, the number of blood samples obtained was small and klotho could not be measured in IB.

However, this study has several limitations. First, as a small-scale study, it was not feasible to thoroughly investigate the patient background under which the sKlotho step-up disappears. Other factors relevant to the FGF23-Klotho endocrine system, such as FGF23, serum calcium, phosphate, and parathyroid hormone, were also not evaluated. Analyzing whether these parameters affect serum sKlotho levels or the change in sKlotho should be considered. Second, sKlotho was not measured using any method other than ELISA to verify if a similar αKlotho step-up exists.

In conclusion, Klotho was measured using ELISA, demonstrating the step-up of αKlotho concentration from infrarenal to suprarenal vena cava in humans. This supports the kidney's origin of circulating soluble αKlotho.

None.