Abstract
This study aims to determine fibroblast growth factor-23 and soluble α-Klotho levels in patients with autosomal dominant polycystic kidney disease. A total of 76 patients with autosomal dominant polycystic kidney disease and 32 healthy volunteers were included in the study. Serum fibroblast growth factor-23 and soluble α-Klotho levels were measured with ELISA kits. Parathyroid hormone, phosphate, calcium, creatinine, 25-hydroxyvitamin D3 levels, urinary protein to creatinine ratio and estimated glomerular filtration rate were also measured or calculated. Patients with autosomal dominant polycystic kidney disease had significantly higher serum parathyroid hormone (p<0.001), fibroblast growth factor-23 (p<0.001), soluble α-Klotho levels (p=0.001) and lower serum 25-hydroxyvitamin D3 levels (p<0.001) as compared with healthy volunteers. Serum fibroblast growth factor-23, soluble α-Klotho and 25-hydroxyvitamin D3 levels were similar in all five chronic kidney disease stages of autosomal dominant polycystic kidney disease (p>0.05). Fibroblast growth factor-23 (r=−0.251, p=0.034) and soluble α-Klotho levels (r=−0.251, p=0.034) were found to be negatively correlated with estimated glomerular filtration rate. This study shows increased fibroblast growth factor-23 levels in patients with autosomal dominant polycystic kidney disease which is in harmony with the general trend in patients with chronic kidney disease of other aetiologies, but, unlike them, also a significant increase in serum soluble α-Klotho levels in patients with autosomal dominant polycystic kidney disease suggesting an aberrant production or a decreased clearance of α-Klotho molecule. Considering the unique increases in erythropoietin levels due to erythropoietin production in renal cysts, we assume, patients with autosomal dominant polycystic kidney disease may potentially have different soluble α-Klotho production/clearance characteristics than the patients with other parenchymal renal diseases.
Significance of this study
What is already known about this subject?
Fibroblast growth factor-23 and its cofactor Klotho are molecules that regulate calcium and phosphate metabolism. Fibroblast growth factor-23 is markedly increased in patients with chronic kidney disease while Klotho levels are expected to be positively correlated with decreasing glomerular filtration rate.
What are the new findings?
Patients with autosomal dominant polycystic kidney disease had significantly higher fibroblast growth factor-23 (p<0.001) and soluble α-Klotho levels (p=0.001), and lower serum 25-hydroxyvitamin D3 levels (p<0.001) as compared with healthy volunteers. Serum fibroblast growth factor-23, soluble α-Klotho and 25-hydroxyvitamin D3 levels were similar in all 5 chronic kidney disease stages (p>0.05). Fibroblast growth factor-23 (r=−0.251, p=0.034) and soluble α-Klotho levels (r=−0.251, p=0.034) were found to be negatively correlated with estimated glomerular filtration rate.
How might these results change the focus of research or clinical practice?
Patients with autosomal dominant polycystic kidney disease may potentially have different soluble α-Klotho production/clearance characteristics than the patients with other parenchymal renal diseases.
Introduction
Autosomal dominant polycystic kidney disease (ADPKD) is one the most common genetic cause of end stage renal disease.1–4 ADPKD can easily be detected in early course of the disease, but unfortunately, its course generally cannot be effectively changed by treatment.5 Clinical presentation of ADPKD differs from the other chronic kidney disease (CKD) causes with less frequent occurrence of anaemia due to higher serum erythropoietin (EPO) levels.6 Recent studies indicate that in ADPKD patients, proximal tubules are the main site for EPO production and EPO is significantly enriched in cysts of proximal tubular origin.7
Fibroblast growth factor-23 (FGF-23) contributes in the regulation of calcium (Ca) and phosphate (P) metabolism FGF-23 and is produced by osteocytes, osteoblasts and directly increases urinary fractional excretion of P by reducing expression of sodium-phosphate cotransporter type II (NaPi-II). It also reduces intestinal P absorption by suppressing 1-α-hydroxylase activity.8–14 FGF-23, in order to exert its physiologic function requires its cofactor, Klotho protein. Klotho gene is reported to be expressed in the parathyroid glands, kidney and the choroid plexus. Klotho protein is translocated from the endosome to the cell membrane, extracellular domain of Klotho (soluble α-Klotho) may be disintegrated by the α-secretates ADAM 10 and 17 to generate large amounts of soluble Klotho into blood, urine and cerebrospinal fluid.15–19 Soluble α-Klotho increases Ca reabsorption in the distal tubule and decreases P reabsorption in the proximal tubules, independently from FGF-23.18 ,20–22 Serum soluble α-Klotho levels may be influenced by some factors such as age, calcium and phosphorus concentrations in healthy population.23
Recent studies have reported that FGF-23 is markedly increased in patients with CKD,24 ,25 while soluble α-Klotho levels are decreased in parallel with lower estimated glomerular filtration rates (eGFR).26 Although some studies in patients with ADPKD at CKD stages 1 and 2 suggest a decrease in plasma-soluble Klotho levels, there are insufficient data investigating the role and levels of FGF-23 and soluble α-Klotho and other main parameters of calcium-phosphate metabolism specifically in all stages of CKD in the ADPKD setting.5 Therefore, in this study, we aimed to describe the role of FGF-23 and soluble α-Klotho in bone and mineral abnormalities in patients with ADPKD.
Patients and methods
The study was approved by the ethical committee of Antalya Training and Research Hospital in accordance with ethical standards of the Declaration of Helsinki of 1975, as revised in 2000 and each patient has signed in with an informed consent. In total, 76 patients aged 22–84 years, with ADPKD at different stages of CKD, who did not require dialysis treatment or had previous kidney transplantation history, were included in the study. Patients with CKD were classified by eGFR according to the CKD Epidemiology Collaboration formula as CKD stage 1 (≥90 mL/min/1.73 m2), CKD stage 2 (60–89 mL/min/1.73 m2), CKD stage 3 (30–59 mL/min/1.73 m2), CKD stage 4 (15–29 mL/min/1.73 m2) and CKD stage 5 (<15 mL/min/1.73 m2 or dialysis). A total of 17, 19, 22, 12 and 6 patients were in CKD stages 1, 2, 3, 4 and 5, respectively.
A total of 31 healthy volunteers (HV), aged 40–68 years, which did not have medical history of any conditions, served as control group. Evaluation of patients and HV included past medical history, general clinical assessment, blood pressure measurement after 15 min rest, assessment of height, body weight, 8 hours fasting venous blood samples were collected in the morning. Sample sera were stored at −80°C. Samples were analysed for FGF-23, soluble α-Klotho, intact parathyroid hormone (iPTH), P, Ca, creatinine and 25-hydroxyvitamin D3 (25(OH)D3). Urinary protein to creatinine ratio was calculated in spot urine samples. Serum blood urea nitrogen, creatinine, Ca and P levels were also determined using commercially available assay kits (Beckman Coulter) and an autoanalyser (Beckman AU5800; Beckman Coulter Diagnostics, USA).
Measurement of serum 25(OH)D3 levels was performed using direct competitive chemiluminescence immunoassay method (DiaSorin, Stillwater, Minnesota, USA). The limit of detection was 3.5 ng/mL, and the coefficient of variation ranged between 4.8% and 11.1% 25(OH)D3 for this assay. Serum iPTH levels were determined using commercially available assay kits (Beckman Coulter) and an autoanalyser (Access DxI800; Beckman Coulter Diagnostics, USA). The iPTH assay is a two-site immunoenzymatic (‘sandwich’) assay. iPTH assay is linear up to 3500 pg/mL. The limit of detection is 1 pg/mL, and the coefficient of variation (CV) ranges between 3.5% and 6.4% for this assay. Serum soluble α-Klotho levels and FGF-23 levels were measured by using a commercially available ELISA kit (YH Biosearch, Shanghai, China) (%CV: <10 for both parameters, serum soluble α-Klotho levels assay range: 0.05–20 ng/mL, FGF-23 assay range: 5–1500 pg/mL). Those assays also employed the quantitative sandwich enzyme immunoassay technique. In order to avoid intratest variations, measurements were made in duplicate, simultaneously using the same ELISA kit.
Statistical analysis
The results were statistically evaluated, using Statistical Package for the Social Sciences (SPSS; V.16.0, Chicago, Illinois, USA). Normal distribution was evaluated by Shapiro-Wilk's test. Continuous variables with normal distribution were expressed as mean±SD and continuous variables with abnormal distribution were expressed as median (minimum-maximum). Categorical variables were expressed as frequency and percentage. According to the presence of normal distribution of the parameters, for the analysis of continuous variables the Mann-Whitney U test and t-test (independent samples t-test) were used. Univariate analysis of variance was used to detect the factors that affected the serum FGF-23 and Klotho levels. Pearson and Spearman's rho tests were used for correlation analysis. With 95% CI, a p value <0.05 was considered to be statistically significant.
Results
Patients with ADPKD had significantly higher serum iPTH (p<0.001), FGF-23 (p<0.001), soluble α-Klotho levels (p=0.001) and lower serum 25(OH)D3 levels compared with HV group (p<0.001) (table 1). When patients are classified into CKD stages, serum FGF-23, soluble α-Klotho and 25(OH)D3 levels were comparable in different stages of CKD (p>0.05). While 25(OH)D3, FGF-23 levels were similar in patients with CKD stage 1 and HV, FGF-23 levels were higher in CKD stages 2, 3, 4, 5 compared with HV. Soluble α-Klotho levels were higher in CKD stages 2, 3, 4 compared with HV. Soluble α-Klotho levels were similar in patients with CKD stages 1 and 5, and HV (table 2).
Soluble α-Klotho levels were in positive correlation with 25(OH)D3 (r=0.27; p=0.025;) and FGF-23 (r=0.818; p<0.001), and in negative correlation with eGFR (r=−0.251; p=0.034). There was no significant correlation between serum soluble α-Klotho and age (r=0.90; p=0.446) (table 3).
Analysis of correlation between serum FGF-23 levels and various renal function parameters showed that serum FGF-23 levels were in negative correlation with eGFR (r=−0.251; p=0.034) (table 3).
For determination of influence of Ca, P, 25(OH)D3, age and eGFR on serum soluble α-Klotho levels in patients with ADPKD, regression analysis was made. Serum soluble α-Klotho levels were related with only 25(OH)D3 (F=8.986; p=0.004) (table 4).
Discussion
In this study, we reported increased serum soluble α-Klotho and FGF-23 levels in patients with CKD with ADPKD. In patients with CKD; FGF-23 levels increase with declining renal function.27 ,28 Hyperphosphataemia associated with CKD, triggers FGF-23 production, and serum FGF-23 levels increase in the early stages in CKD conditions.27 Increase of serum FGF-23 levels is recognised before the elevation of serum P and iPTH levels in patients with CKD29 and relationship between serum FGF-23 levels and GFR is not found in earlier stages of CKD, where patients are normophosphataemic, suggesting that there may be an end-organ resistance to FGF-23 due to decreased production of its cofactor Klotho.30 ,31 Pavik et al5 found a significant decrease in soluble Klotho levels in ADPKD at CKD stages 1 and 2, suggesting a plasma Klotho reduction in early stages of ADPKD. Unlike Pavik's study, our patient group included patients with ADPKD from all stages of kidney insufficiency including stages 3, 4 and 5. Our results were also different with suggesting a statistically significant increase in serum soluble α-Klotho levels in patients with ADPKD. Although low Klotho levels are expected in CKD, some authors have suggested that, Klotho levels may be significantly increased in patients with CKD with higher creatinine levels (>2 mg/dL) compared with the patients with CKD with relatively lower creatinine levels (<1.2 mg/dL).32 In our patients, we have found that serum soluble α-Klotho levels were similar in patients with CKD stages 1 and 5 with healthy control participants, and were even higher in CKD stages 2, 3, 4 compared with healthy group. In addition to unexpectedly high levels of serum soluble α-Klotho levels we have found, in our patients with ADPKD, serum soluble α-Klotho levels were not affected by neither eGFR, nor serum inorganic phosphorus levels suggesting an autonomous production of α-Klotho. Some other authors have not found any correlation between soluble α-Klotho levels and declining renal function.33 We should remember that the possibility of autonomous production of some biologic substances is not unfamiliar in ADPKD pathophysiology. The renal cysts of ADPKD develop by overproliferation of tubular cells. While the cysts grow, cystic tubular epithelial secretion accumulates within cytes. Patients with ADPKD are known to suffer with less severe CKD-related anaemia, even in the advanced stages of their disease, due to autonomous production of EPO. As, Ito et al34 have shown positive EPO staining in the cytoplasm of the cystic epithelial cells and also in cystic fluid, therefore, in case of EPO, phenomenon of tubular secretion accumulation has been demonstrated in ADPKD. Eckardt et al7 have found that single interstitial cell juxtaposed to proximal tubular cysts may exert autonomous production of EPO inside the cysts, which ameliorates the anaemia during end-stage polycystic kidney disease. With respect to sodium concentrations, cysts in patients with ADPKD are mostly of proximal tubule origin, and cysts originated from proximal tubules contain the highest concentrations of EPO.7
On the other hand, α-Klotho mRNA is expressed strongly in the kidney, especially in renal tubules.18 Proximal and distal convoluted tubules express Klotho protein, therefore, kidney tubules are suggested to be the main production site for soluble α-Klotho molecule. Thus, Klotho protein and EPO molecules are produced by proximal tubular epithelium, and at least in case of EPO, molecules secreted by tubular epithelial cells may built up within cystic fluid and later systemic circulation in ADPKD. We believe, same mechanism may be valid for Klotho and aberrant tubular production of Klotho in ADPKD may be an explanation for our finding of unexpectedly high plasma α-Klotho levels in patients with ADPKD. The presence of exogenously given soluble α-Klotho in proximal lumen, suggests that α-Klotho traffics across renal tubules from basolateral membrane to luminal side, and tubules may also be the clearing site for α-Klotho molecule.18 The other explanations of high levels α-Klotho may be impaired renal clearance of soluble α-Klotho in patients with ADPKD and maybe some other forms of CKD.33 Therefore, renal cysts of ADPKD may be an aberrant source for α-Klotho and, additionally, decreased clearance of soluble α-Klotho due to cyst-induced tubular damage may contribute in unexpectedly increased levels of circulating α-Klotho we have found in our patients.
But unfortunately, our study was designed to be a clinical one and has not aimed to perform advanced procedures such as measuring α-Klotho contents in aspirated cyst fluids. Therefore, at this point, our speculations about the pathophysiologic explanations for the cause/causes of our extraordinary findings suggesting increased circulating α-Klotho levels in ADPKD, needs to be supported by further experimental and clinical studies.
On the other hand, besides measuring α-Klotho levels in cystic fluids, urine and membrane-bound Klotho were beyond the scope of our study; we had some other limitations, such as relatively small size of the study population, potential effects of treatments, for example, RAS inhibitors, active vitamin D analogues, have all been disregarded. But we believe, if it is true, possibility of high α-Klotho levels in ADPKD deserves special attention and may have some clinical and pathophysiological impacts.
In conclusion, our data show that elevated FGF-23 and soluble α-Klotho levels were present in patients with ADPKD. Further research is needed to identify and explain the cause of paradoxical outcomes related to FGF-23 and soluble α-Klotho levels in patients with ADPKD.
Acknowledgments
Prior presentation: Parts of this study was presented in abstract format in the 32nd National Congress of Nephrology, Hypertension, Dialysis and Transplantation, Antalya, Turkey, 21–25, October, 2015. Consequently, an abstract version of this study was published in Turkish Nephrology, Dialysis and Transplantation Journal, 2015; Volume 24, number 3, supplement 1.
Footnotes
Contributors FS and AI were involved in research design and writing. SD and HYE were involved in data collection. RC and FFE were involved in editing.
Competing interests None declared.
Patient consent Obtained.
Ethics approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Provenance and peer review Not commissioned; externally peer reviewed.