| Abstract|| |
Most chronic kidney disease (CKD) biomarkers in the current clinical use are not sensitive enough and cannot be used to identify the early stage of the disease. Klotho is a transmembrane protein predominantly expressed in the renal tubules and implicated in managing phosphate homeostasis, together with fibroblast growth factor-23 (FGF-23); a bone-derived protein that increases urinary phosphate excretion. The present study was carried out on 50 patients CKD with different etiologies referred to the Internal Medicine Department and Out Patient Clinic of Tanta University Hospitals and 30 apparently healthy individuals of matched age and sex as a control group. They were subjected to the following assessment: detailed history taking, careful clinical examination, and laboratory investigations, including urea, creatinine, estimated glomerular filtration rate (eGFR), serum electrolytes, urinary albumin, urinary phosphorus (U-Ph), and specific laboratory tests for: Alpha Klotho (α-klotho) and FGF-23 by using ELISA technique. The present study shows that the mean value of serum creatinine, urea, phosphorus, urinary albumin, and FGF-23 were significantly increased, whereas there was a significant decrease in the mean value of eGFR, calcium, and U-Ph in the patients with CKD when compared with control group. Plasma level of serum α-klotho is significantly decreased in all patients with CKD when compared to the control group and there was a significant positive correlation between serum α-klotho level and eGFR, serum calcium level and U-Ph level. Plasma level of serum α-klotho is significantly decreased in all patients with CKD and serum α-klotho can be used as a good marker for early diagnosis and staging of CKD.
|How to cite this article:|
Khodeir SA, Okda HI, Abdalal HM. Clinical significance of fibroblast growth factor-23 and soluble alpha klotho in different stages of chronic kidney disease. Saudi J Kidney Dis Transpl 2019;30:108-18
|How to cite this URL:|
Khodeir SA, Okda HI, Abdalal HM. Clinical significance of fibroblast growth factor-23 and soluble alpha klotho in different stages of chronic kidney disease. Saudi J Kidney Dis Transpl [serial online] 2019 [cited 2021 Apr 22];30:108-18. Available from: https://www.sjkdt.org/text.asp?2019/30/1/108/252900
| Introduction|| |
Chronic kidney disease (CKD) is a universal health problem that is characterized bygradual irreversible renal damage. As renal function deteriorates, disturbances in mineral metabolism, such as hyperphosphatemia develop obviously. These metabolic disorders are firmly connected with poor prognosis and survival. Therefore, the early diagnosis and management of CKD are important to prevent the progression and extra renal complications. However, most current CKD biomarkers are not sensitive enough and cannot be used to recognize early-stage disease. The fibroblast growth factor-23 (FGF-23) and its co-receptor Klotho have attracted great interest and are presently recognized as playing a master role in the regulation of phosphate homeostasis. FGF-23, a bone-derived protein which increased urinary phosphate excretion in a Klotho-dependent manner to maintain phosphorus homeostasis and reducing serum level of Vitamin D3 (1–25 DH VD3). Klotho is a transmembrane protein which serves as a co-factor for FGF-23 to bind with its cognate receptor. Soluble Klotho (sKl) refers to the cleaved extracellular domain of membrane Klotho. Klotho is principally synthesized mainly in the kidney, brain, and other organs and play many biological functions, including modulation of ion transport, signal transduction, anti-renin angiotensin aldosterone, and anti-aging properties which mediated through multiple system effects. Klotho also suppresses 1α-hydroxylase in the kidney to regulate calcium metabolism and participates in the regulation of PTH synthesis in parathyroid gland by FGF-23.
The kidney has the highest levels of klotho expression and in addition is the major source of sKl. Previous studies have indicated that circulating soluble Alpha klotho (α-klotho) may declare a useful biomarker for the diagnosis of CKD. However, data on the relation between circulating α-klotho, FGF-23, and the other markers of mineral metabolism are limited. The aim of the present work was to study serum soluble α-klotho in different stages of CKD and its correlation with FGF-23 to focus more light on its clinical importance.
| Subjects and Methods|| |
This study was carried out on CKD patients with different etiologies includes diabetic nephropathy, chronic glomerulonephritis, obstructive nephropathy, and hypertensive nephropathy, who were admitted to the Internal Medicine Department and Out-patient Clinic of Tanta University Hospitals. The study involved 80 subjects classified into two groups as follows: Group 1 control group included 30 apparently healthy individuals acting as reference group with no history of kidney disease. Group 2 patients with CKD included 50 patients. Diagnosis was established on the basis of clinical, biochemical, and imaging assessment. They were further subdivided into five subgroups according to the level of estimated glomerular filtration rate (eGFR).
All patients were subjected to the following: detailed history taking, thorough clinical examination, laboratory investigations includes blood urea, serum creatinine, eGFR and urinary albumin, and phosphorus. Serum electrolytes include sodium, potassium, calcium, and phosphorus. Specific laboratory tests for serum soluble α-Klotho and FGF-23 by using ELISA technique.
A volume of 5 mL of venous blood were withdrawn with a sterile plastic syringe from each subject. Two milliliters were added to EDTA vacutainers for the assessment of human and human soluble α-klotho by ELISA. Blood was mixed then centrifuged for 20 min at speed of 2000–3000 rpm and the separated serum was kept frozen at -20°C, the remainning 3 mL blood were added to plain vacutainers, centrifuged as previous portion then separated. Serum was used for routine chemical analysis of urea, creatinine, and serum electrolytes. ELISA kit is based on the principal of double-antibody sandwich technique to detect Human soluble α-klotho and FGF-23.
| Statistical Analysis|| |
Statistical presentation and analysis of the present study was conducted, using the mean, standard deviation, Chi-square, linear correlation coefficient, receiver operating characteristic (ROC)-curve, and analysis of variance tests by Statistical Package for Social Science (SPSS) version 17.0 (SPSS Inc., Chicago, IL, USA). Chi-square hypothesis stated that the row and column variables are independent, without indicating the strength or direction of the relationship. The linear correlation coefficient was used for the detection of correlation between two quantitative variables in one group.
| Results|| |
The total studied participants were 80 patients with 48 males (60%) and 32 females (40%). In Stage I, there were two male (50%) and two female (50%). In Stage II, there were 11 male (68%) and five female (31%). In Stage III, there were nine male (60%) and six female (40%). In Stage IV, there were five male (55%) and four female (44%). In Stage 5, there were four males (66%) and two females (33%) and in the control group, there were 17 males (56%) and 13 females (43%). The patient's age ranged from 38–61 years with mean value of 50 ± 10.4 as shown in [Table 1] and [Table 2].
|Table 2: Comparison between patients and control groups regarding demographic data.|
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Serum calcium is ranged from 8.6–9.2 mg/dL with mean value of 8.9 ± 0.28 in the 1st stage. It ranged from 8.6–9.7 mg/dL with mean value of 9.1 ± 0.3 in the 2nd stage, from 8.6–9.6 mg/dL with mean value of 9.04 ± 0.31 in the 3rd stage, from 8.1–9.5 mg/dL with mean value of 8.6 ± 0.4 in the 4th stage. It ranged from 8.1–9.1 mg/dL with mean value of 8.6 ± 0.3 in the 5th stage and it ranged from 8.6–9.4 mg/dL with mean value of 9.11 ± 0.15 in the control group. There was a significant decrease in the mean value of Stages IV and V compared to Stages I, II, III, and control as shown in [Table 3].
|Table 3: Comparison between different stages of chronic kidney disease and controls regarding serum calcium, phosphorus, urinary phosphorus level, α klotho, and FGF-23 levels.|
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Serum level of phosphate is ranged from 2.7–3.7 mg/dL with mean value of 3.2 ± 0.4 in the 1st stage and from 2.7–3.9 mg/dL with mean value of 3.1 ± 0.3 in the 2nd stage. It ranged from 2.6–4 mg/dL with mean value of 3.3± 0.4 in the 3rd stage, from 2.9–4.1 mg/dL with mean value of 3.6 ± 0.38 in the 4th stage, from 3.4–5.4 mg/dL with mean value of 4.4 ± 0.6 in the 5th stage and from 2.7–3.7 mg/dL with mean value of 3.13 ± 0.29 in the control group. There was a significant increase in the mean value of Stages III, IV, and V compared to Stages I, II, and control as shown in [Table 3].
Urinary phosphorus (U-Ph) level is ranged from 750–1150 mg/24-h with mean value of 930 ± 172 in the 1st stage, from 820–990 mg/24-h with mean value of 896 ± 53 in the 2nd stage, from 700–800 mg/24-h with mean value of 750 ± 28 in the 3rd stage, from 500–720 mg/24-h with mean value of 632 ± 79 in the 4th stage and ranged from 400–750 with mean value of 593 ± 128 in the 5th stage. It ranged from 750–1150 mg/24-h with mean value of 904 ± 145 in the controls. There was significant decrease in the mean value of Stages III, IV, and V compared to Stages I, II, and control as shown in [Table 3].
Soluble serum α-klotho level is ranged from 10–10.8 ng/mL with mean value of 10.4 ± 0.3 in the 1st stage, from 7.2–9.9 ng/mL with mean value of 8.7 ± 0.9 in the 2nd stage, from 1.7–7 ng/mL with mean value of 4.5 ± 1.9 in the 3rd stage, from 1.1–1.7 ng/mL with mean value of 1.5 ± 0.18 in the 4th stage and from 0.7–1.07 ng/mL with mean value of 0.79 ± 0.18 in the 5' stage. It ranged from 10–10.9 ng/mL with mean value of 10.5 ± 0.2 in the control group. There was significant decrease starting from Stage II and more marked in Stages III, IV, and V compared to Stage I and control as shown in [Table 3].
Serum level of FGF-23 is ranged from 820–824 pg/mL with mean value of 822 ± 1.70 in the 1st stage, from 825–933 pg/mL with mean value of 885 ± 33 in the 2nd stage, from 932–1150 pg/mL with mean value of 1037 ± 75 in the 3rd stage, from 1168 pg/mL with mean value of 1506 ± 255 in the 4th stage and ranged from 1900–2340 pg/mL with mean value of 2091 ± 191 in the 5th stage. It ranged from 818–825 pg/mL with mean value of 821 ± 2.2 in the control group. There was a significant increase in the mean value of FGF-23 starting from Stage III and more marked in Stages IV, V compared to Stage I, II, and control group as shown in [Table 3].
Regarding the correlation between values of soluble serum α-klotho and different variables. There was significant positive correlation between soluble serum α-klotho level with eGFR, serum sodium (Na), serum calcium (Ca) and U-Ph. There were significant negative correlation between soluble serum α-Klotho (sKL) level with serum creatinine, serum phosphorus (Ph), serum potassium (K), blood urea, urinary albumin (U-albumin), albumin creatinine ratio, and FGF-23 as shown in [Table 4] and [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5].
|Figure 1: Correlation between α-Kloth and estimated glomerular filtration rate.|
eGFR: estimated glomerular filtration rate.
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|Figure 3: Correlation between α-Kloth and serum phosphorus level.|
Serum Ph: Serum phosphorus.
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|Figure 4: Correlation between α-Klotho and urinary phosphorus level.|
U-Ph: Urinary phosphorus.
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|Figure 5: Correlation between α- Klotho and FGF-23.|
FGF-23: Fibroblast growth factor-23.
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Regarding the correlation between values of FGF-23 level and different variables. There was significant positive correlation between (FGF-23) level with serum creatinine level, serum phosphorus level (Ph), serum potassium (K), blood urea, urinary albumin (U-albumin), albumin creatinine ratio, and FGF-23. There was significant negative correlation between serum FGF-23 level with eGFR, serum sodium (Na), serum calcium (Ca), and U-Ph as shown in [Figure 6].
|Figure 6: Correlation between FGF-23 and eGFR.|
FGF-23: Fibroblast growth factor-23, eGFR: estimated glomerular filtration rate.
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ROC of α-klotho at a cutoff value of ≤9.9, it showed a sensitivity of 92%, a specificity of 100%, with positive predictive value (PPV) 100%, negative predictive value (NPV) 88.2% and accuracy 97% to discriminate between normal and diseased persons with CKD. The area under the curve for α-klotho was 0.970 shown in [Figure 7].
ROC of FGF-23 at a cutoff value of >825 pg/mL it showed a diagnostic sensitivity of 90%, specificity of 100%, with PPV 100%, and NPV 85.7% and accuracy of 96.3% to discriminate between normal and diseased persons with CKD. The area under the curve for FGF-23 was 0.963 shown in [Figure 8].
|Figure 8: Area under the curve for FGF-23 = 0.963.|
FGF-23: Fibroblast growth factor-23.
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In a univarite analysis, there was significance in e-GFR, S-Klotho, FGF23, serum PH, U. albumin, Na, K, urea, age, weight, creatinine, Ca, U-PH, and alb/crea. In multivariate analysis, there is significance in e-GFR, S-Klotho, U. albumin and K. However, the FGF23, serum PH, Na, and Urea were nonsignificant as shown in [Table 5].
| Discussion|| |
Unfortunately, CKD usually occult and insidious at onset. Early diagnosis was difficult due to the lack of uniform application of simple tests for early detection and evaluation. α-klotho act as co-receptor that complexes with FGF receptor 1 (FGFR1) mediating signaling by the circulating hormone FGF-23 which is an important manager of mineral homeo-stasis.
The present study was designed to evaluate the usefulness of the serum soluble α-klotho value as well as FGF-23 in CKD patients and their relation to disease progression and extra renal complications.
In the present study, regarding the serum calcium level, there was a significant decrease in Stage III, IV, and V compared to Stage I and II. These findings agreed with Craver et al, who found that decreased serum calcium was involved in CKD progression but are more likely in advanced stages. Furthermore in this study, serum phosphate level showed a significant increase in the late stages of CKD (Stage III, IV, and V) and these findings agreed with Pavik et al, who found that at CKD stage IV and onward, serum levels of phosphate start rising.
In the present study, FGF-23 was found to be significantly increased in CKD patients compared to the control group starting from Group III and showed a positive relationship with CKD progression. These findings were in agreement with Pavik et, and Gutierrez et al al, who found that in CKD, circulating FGF-23 levels gradually increase with declining renal function and when patients reach end-stage renal disease, FGF-23 levels can rise to1000-fold up its normal range. By using ROC of FGF-23 at a cutoff value of >825 pg/mL it showed a diagnostic sensitivity of 90%, specificity of 100%, with PPV 100%, NPV 85.7%, and accuracy of 96.3% to discriminate between normal and diseased persons with CKD. The area under the curve for FGF-23 was 0.963. It was found also that serum soluble α-Klotho level was significantly decreased in Group I of CKD compared to control group and dropped gradually with progression of the disease. Serum soluble α-Klotho level was significantly decreased starting from Stage II CKD compared with Stage I and not only in the advanced stages of the disease and these findings were harmonized with Drew et al, whereas Seiler et al refused the hypothesis that CKD is characterized by reduced plasma level of secreted Klotho. This variation in hypothesis may be occurred because Seiler et al did not measure α-Klotho in healthy controls or Stage I CKD patients. ROC of α-klotho at a cutoff value of ≤9.9 showed a sensitivity of 92%, a specificity of 100%, with PPV 100%, NPV 88.2%, and accuracy 97% to discriminate between normal and diseased persons with CKD. The area under the curve for α-klotho was 0.970.
According to Hu et al, klotho and FGF-23 are key regulators of mineral metabolism in renal insufficiency. FGF-23 is a phosphaturic hormone released by osteocytes and binds to FGFRs with modest affinity. In vivo, Klotho is required for FGF-23-mediated receptor activation. Klotho forms a complex with the FGFR and thereby increasing its affinity for FGF-23. Thus, FGF-23 and Klotho adjust phosphate homeostasis by promoting renal phosphate excretion. Furthermore, Hu et al, reported that α-Klotho reduces tubular phosphate reabsorption.
The observed early decrease in soluble α-klotho levels can be explained by decrease shedding of membrane α-klotho expressed in renal distal tubules, which reflects a decrease in the amount of membrane α-klotho, and the subtle reduction in nephron number that may occur in early CKD stages. This explanation is consistent with Hu et al, and Rotondi et al,, who stated that since α-klotho is normally excreted in urine, its detectable reduction in serum levels along with progressive renal damage can be most probably referred to reduced renal synthesis.
In the present study, serum soluble α-Klotho and eGFR show a significant positive correlation while serum soluble α-Klotho and U ACR in all patients groups, including Stages I and II give significant negative correlation. Thus, these data demonstrated that serum soluble α-Klotho may represent a useful biomarker for detecting early stage CKD, However, FGF-23 level and the ordinary methods for the diagnosis of CKD as proteinuria and eGFR showed significant changes starting from Stage III. In agreement with our results Seibert et al concluded that in acute kidney injury serum soluble α-Klotho level are not associated with impaired kidney function, whereas in CKD, impaired soluble α-Klotho level observed and significantly correlated with eGFR.
The study also showed that soluble α-Klotho level was positively associated with serum calcium and U-Ph and negatively correlated with serum phosphorus and FGF-23 concentration in all CKD patients. These data matched with Hu et al, who stated that α-klotho and FGF-23 may play a key role in the pathogenesis of mineral and bone disorder in the early stages of CKD. In agreement with our results Tan et al, reported that soluble α-klotho demonstrate a significant association with phosphorus reabsorption independent of FGF-23 and this relationship deserve further exploration.
We can conclude that serum level of soluble α-klotho increased and FGF-23 levels decreased in early stages of CKD, and this associated with mineral metabolic changes in calcium and phosphorus. Hence, soluble α-klotho was early sensitive parameter for the early diagnosis of stages of CKD and extra renal bone complication.
| Limitation of the study|| |
Although this research was carefully prepared, and reached its aims, there were some unavoidable limitations such as uncontrolled dietary calcium intake, also this study was conducted on one of the Egyptian university hospitals, further larger prospective studies are needed to validate our findings.
Conflict of interest:
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Hanaa I Okda
Department of Internal Medicine and Nephrology, Faculty of Medicine, Tanta University, Tanta
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]