Soluble Klotho is associated with mortality and cardiovascular events in hemodialysis

Patients with chronic kidney disease (CKD) are at increased cardiovascular risk due to the presence of atherosclerosis and arteriosclerosis in the vascular wall [1, 2]. The risk of cardiovascular events increases with advancing CKD stages [3], and more than 50% of patients with end-stage renal disease on renal replacement therapy die of cardiovascular causes [4]. Current knowledge suggests that, in parallel to the “classic” risk factors of cardiovascular morbidity and mortality (i.e. age, diabetes mellitus, hypertension and smoking), patients with CKD have several other factors that may pre-dispose to cardiovascular events, such as those related to the mineral and bone disorders (CKD – MBD), anemia and the chronic inflammatory state of “uremia” [5, 6]. In addition, intermediate outcomes, such as common carotid intima media thickness (ccIMT) and pulse wave velocity (PWV), which reflect the severity of atherosclerosis and arterial stiffness respectively, have been long known as independent determinants of cardiovascular events and mortality in hemodialysis patients [5‐7].

Klotho is a 130 kDa transmembrane protein, expressed predominantly in the distal convoluted tubules of the kidneys, but also in many other organs, which is involved in the regulation of human aging [8]. The extracellular domain of Klotho can undergo proteolytic cleavage by metalloproteinases and generate a 70-kDa soluble form, which is released in the circulation. Membrane Klotho binds to fibroblast growth factor receptor 1 (FGFR-1), converting it to a specific receptor for FGF-23 and thus exert its actions on mineral metabolism by regulating calcium, potassium and phosphorus excretion [9]. The biological functions of secreted Klotho may be independent of FGFR-1 and include modulation of endothelial nitric oxide synthesis, maintenance of endothelial integrity and inhibition of growth factor-1 signaling [10, 11]. Klotho knockout mice develop a syndrome similar to premature aging, with shortened life span, hyperphosphatemia, atherosclerosis and extensive vascular calcification [8]. Serum Klotho decreases from early CKD stages, partially because uremic toxins induce DNA methyltransferase protein expression, which downregulates Klotho through hypermethylation [12]. Therefore patients with CKD or on dialysis tend to generally have lower levels of Klotho than healthy individuals [13‐15].

Secreted Klotho has been associated with cardiovascular events and mortality in the general population and various sub-populations (i.e. the elderly or patients with diabetes) [16, 17], however relevant studies in CKD patients are inconsistent. Two analyses from a German cohort in CKD stages 2–4 showed that Klotho is not associated with cardiovascular morbidity or all-cause mortality [18, 19]. One pilot study also shows no such associations in hemodialysis patients [20]. However, a large cohort of French hemodialysis patients, showed that patients with serum Klotho above the first quartile had a significantly reduced occurrence of cardiovascular events and death [21]. The discrepancy between the above findings in hemodialysis, can be partially explained by differences of the aforementioned cohorts concerning factors known to affect cardiovascular outcomes, including age, dialysis vintage, comorbid conditions such as hypertension, diabetes mellitus and the presence of established cardiovascular complications. Furthermore, the mechanisms through which Klotho may affect cardiovascular events are unknown. A recent study suggested that decreased Klotho levels in CKD patients were associated with increased PWV, indicating arterial stiffness as a mediatory pathway, but causality could not be established [22].

In the aforementioned studies, no evaluations of intermediate outcomes such as the degree of atherosclerosis or arteriosclerosis were performed. Thus, this study aimed to examine the possible relationship between Klotho levels and cardiovascular outcomes in hemodialysis, with simultaneous exploration of possible confounding effects from common risk factors, FGF-23 and other CKD-MBD variables, PWV and ccIMT.

The association of secreted Klotho with cardiovascular morbidity and mortality was originally studied in non-CKD populations. In a cohort of 804 elderly (> 65 years old) individuals observed during 6 years, patients with Klotho level in the lowest tertile (< 575 pg/mL) had an increased risk of death compared with participants in the higher quartile (> 763 pg/mL) (HR:1.78, 95% CI 1.20–2.63) [16]. In a multicenter European study [25], Klotho levels were measured in 2948 patients referred for coronary angiography irrespective of kidney function, who were then observed for almost 10 years. The study showed that Klotho does not add predictive power to cardiovascular and mortality risk assessment in patients with normal renal function, since the HRs in the fourth quartile of Klotho levels compared to the first quartile were 1.14, 95%CI 0.94–1.38, for all-cause mortality and 1.03, 95%CI 0.80–1.31, for cardiovascular mortality. In contrast, a recent study in 168 patients with type 2 diabetes, followed for 7 years, showed that low Klotho is associated with macrovascular outcomes, as patients in the highest compared to the lowest quartile of Klotho levels had HR:0.471, 95%CI 0.307–0.725, p = 0.001 for the combined outcome of macrovascular complications (including coronary artery disease, cerebrovascular attacks and peripheral artery occlusive disease, with significant differences present also for the individual components [17].

With regards to patients with CKD, Seiler et al. have previously, shown in 312 patients with CKD stages 2 to 4, that levels of Klotho did not predict the occurrence of death or the initiation of renal replacement therapy during 2.2 years, (HR for logarithmic transformed Klotho 1.46, 95%CI 0.14–15.95, [18]. Another study from the same group, in a cohort of 444 patients with CKD stages 2 to 4, also showed that Klotho was not associated with cardiovascular outcomes since the HR for the third versus the first Klotho tertile, was 0.75, (95%CI 0.43–1.30) [19]. In a study in 239 hemodialysis patients observed for 2.5 years, Klotho levels were not associated with mortality (HR:1.22, 95%CI 0.66–2.28 for the third compared to the first Klotho tertile), but Klotho seemed to be protective against AF [20]. In contrast, Marcais et al., showed in 769 hemodialysis patients that, patients with serum Klotho above the first quartile (≥280 ng/L) had reduced occurrence of an endpoint that combined cardiovascular events and cardiovascular death (HR:0.39, 95%CI 0.19–0.78, p = 0.008), compared with patients with Klotho < 280 ng/L. This effect remained significant (HR:0.86, 95%CI 0.76–0.99, p = 0.03) after adjustment for a number of relevant cardiovascular risk factors [21]. Two smaller studies from Asia also suggest that low Klotho is associated with higher cardiovascular risk [26, 27].

FGF-23 is a 26-kDa protein secreted mainly by osteoblasts. It can be cleaved by convertase activity and therefore intact FGF-23 and C-terminal fragments of FGF-23, which are probably inactive, are detected in plasma [28]. As FGF-23 is primarily excreted in the urine, ESRD patients on dialysis have an enormous increase in plasma levels of both the intact molecule and the C-terminal fragments [29]. In CKD patients FGF-23 secretion rises well before serum parathyroid hormone (PTH) or phosphate concentrations, because of a deficiency of the necessary Klotho cofactor. Thus, as CKD progresses, FGF-23 levels rise and Klotho levels fall [9]. FGF-23 directly increases urinary fractional excretion of phosphate, impairs the synthesis and accelerates degradation of 1,25(OH)2D and is therefore implicated in the pathogenesis of secondary hyperparathyroidism. Moreover, elevated circulating FGF-23 are independently associated with vascular dysfunction, left ventricular hypertrophy, and death [29‐31].

Whether Klotho and FGF-23 act independently of each other on the cardiovascular system in CKD patients is not known. FGF-23 effects may be totally independent of Klotho, i.e. a direct action on the cardiomyocytes is described [32]. On the other hand Klotho has recently been shown to be associated with PWV, in a cross-sectional study in CKD, but causality could not be established [22]. The actions of Klotho have been extensively studied in animals, starting from the discovery of the Klotho gene in mice in 1997; Klotho-depleted animals exhibited a wide array of symptoms mimicking ageing, one of which was premature arteriosclerosis [8]. Klotho was found to have a cardioprotective effect in conditions of stress, by reducing the expression the transient receptor potential cation channels (TRPC channels) in the mouse heart [33]. Furthermore, Klotho was shown to maintain the integrity of the endothelium [11], inhibit the action of proinflammatory cytonkines such as TNF-alpha [34], and possibly have an anti-ageing effect on humans [35]. Most importantly, recent background studies have shown that Klotho protects against cardiac hypertrophy in mice independently of FGF-23 [36], whereas recombinant α-Klotho has been demonstrated to act therapeutically against uremic cardiomyopathy in mice [37].

Our study expands the aforementioned findings on the association of low-Klotho with increased cardiovascular risk, first by exhibiting that this association is independent from FGF-23 levels, or Ca x P product, both of which were associated with increased cardiovascular risk and mortality in hemodialysis [5, 31, 38]. Furthermore, in modelled analysis we observed that the association of low-Klotho with the primary outcome was additionally independent not only from classic cardiovascular risk factors but also from the intermediate endpoints of arterial stiffness, evaluated with the gold-standard method, i.e. carotid-femoral PWV, as well as the degree of atherosclerosis, evaluated by ccIMT. Of note, not only the association of low-Klotho with outcomes remained significant, but also the magnitude of the association was increasing with step-wise adjustment, going from 2.1 to 2.8-fold from univariate to the fully-adjusted model, in contrast to what commonly occurs in such analyses. Another strength of this study is the median follow-up of 5.5 years, which is the longest among the studies in hemodialysis. The main limitation is the small study sample. A detailed power estimation could not be performed as the baseline evaluation of the study took place before the publication of relevant studies in the field; to this end, this is a pilot study. This, however, did not affect our main findings, as the basic observations are very clear in terms of statistical significance, possibly due to the accumulation of events during the long follow-up. In our study, the baseline Klotho levels were on average somehow higher than in the few relevant studies in hemodialysis patients; this may due to differences in populations studied or the assays used. Lastly, we had a single evaluation of study variables (Klotho, PWV, ccIMT etc.) at baseline; as a result, they were not recorded overtime and at study-end, as commonly happens in cohort studies of this type.

This pilot study showed that low Klotho is associated with increased risk of cardiovascular events and cardiovascular death in hemodialysis patients. Moreover, this association between low Klotho and the occurrence of cardiovascular outcomes was found to be independent of several important parameters, that were previously shown to be associated with increased cardiovascular risk in dialysis, including cFGF-23, and Ca x P product, age, classical cardiovascular risk factors, a history of cardiovascular disease and also, cfPWV and ccIMT. These results suggest that low plasma Klotho may accelerate (or high Klotho may protect against) cardiovascular disease in individuals with CKD through mechanisms that are distinct from known cardiovascular risk factors. Future studies are expected to shed more light in the exact role of secreted Klotho in cardiovascular outcomes in these patients.