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ORIGINAL ARTICLE
Year : 2018  |  Volume : 31  |  Issue : 3  |  Page : 957-962

Evaluation of serum soluble klotho protein in patients with different degrees of chronic kidney disease


1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Al Minufiyah, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Menoufia University, Shebin Elkom, Egypt
3 National Blood Transfusion Services, Ministry of Health and Population, Shebein El Kom, Egypt

Date of Submission01-Mar-2017
Date of Acceptance18-Apr-2017
Date of Web Publication31-Dec-2018

Correspondence Address:
Marwa M Elwan Elshouny
National Blood Transfusion Services, Ministry of Health and Population, Shebein El Kom, Menoufia Governorate
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_136_17

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  Abstract 


Objectives
The aim of this study was to investigate serum α-klotho as a new biomarker for the diagnosis of chronic kidney disease (CKD), especially in its early stages.
Background
α-Klotho is a novel antiaging protein that was identified as a regulator of calcium and phosphate homeostasis, which is highly expressed in the kidney. The soluble form of klotho acts as an endocrine substance that exerts multiple actions including the modulation of renal solute transport and protection of the kidney.
Methods
A case–control study was conducted at the Clinical Pathology Department, Menoufia University Hospitals, Faculty of Medicine, during the period from January 2015 to November 2016. Sixty-eight patients suffering from CKD who were admitted to the hospital during the period from January 2015 to November 2016 were included in this study. In addition, 10 apparently healthy subjects with matching age and gender formed the control group. Routine laboratory investigations performed were: hemoglobin, urea, creatinine, uric acid, calcium (Ca2), phosphate (PO4), and albumin and the estimated glomerular filtration rate were measured and the α-klotho protein level was measured by ELISA.
Results
Serum soluble α-klotho protein levels were significantly lower in CKD patients compared with normal controls. Besides, α-klotho protein levels were positively correlated with estimated glomerular filtration rate, hemoglobin and were negatively correlated with renal function tests and phosphate.
Conclusion
Soluble α-klotho levels are significantly decreased in CKD not only in the advanced stages but also in the early stages.

Keywords: chronic kidney disease, estimated glomerular filtration rate, soluble α-klotho


How to cite this article:
El Saeed GK, Khodeer SA, Yassen YS, Elwan Elshouny MM. Evaluation of serum soluble klotho protein in patients with different degrees of chronic kidney disease. Menoufia Med J 2018;31:957-62

How to cite this URL:
El Saeed GK, Khodeer SA, Yassen YS, Elwan Elshouny MM. Evaluation of serum soluble klotho protein in patients with different degrees of chronic kidney disease. Menoufia Med J [serial online] 2018 [cited 2019 Jun 20];31:957-62. Available from: http://www.mmj.eg.net/text.asp?2018/31/3/957/248719




  Introduction Top


Chronic kidney disease (CKD) was prevalent worldwide in the middle-aged and old-aged population. It has been recognized as an important disease threatening human health[1]. Individuals with CKD are at risk of bone disorders, vascular abnormalities, and premature mortality due to changes in Ca2 and PO4 hemostasis[2].

The identification of klotho gene was a major discovery as the gene encodes multiple functions of protein regulation. Two forms of α-klotho protein have been reported: a membrane bound and a soluble one. The soluble form of klotho seems to function as a humoral factor and regulates glycoproteins on the cell surface including ion channels and growth factors[3].

α-Klotho protein expression in humans had only been described in the kidneys and parathyroid glands[4]. Recent evidence has suggested that α-klotho inhibits oxidative stress and regulates PO4, Ca2[5].

The aim of this work is to investigate serum α-klotho as a new biomarker for the diagnosis of CKD especially in the early stages.


  Patients and Methods Top


The case–control study included 68 patients of CKD (as a patient group), There were 40 men and 28 women; their mean age 62.52 ± 9.61 that ranged from 35 to 84 years.

The patient group was divided into five subgroups according to the GFR:

Subgroup 1 (stage 1 CKD): This group included 10 patients. There were seven men and three women with mean age 59.40 ± 6.86 that ranged from 50 to 70 years and their GFR ≥90 ml/min/1.73 m2.

Subgroup 2 (stage 2 CKD): This group included 20 patients. There were 12 men and eight women with mean age 61.65 ± 10.51 that ranged from 35 to 80 years and their GFR = 60–89 ml/min/1.73 m2.

Subgroup 3 (stage 3 CKD): This group included 14 patients. They included five men and nine women with mean age 67.92 ± 8.98 that ranged from 55 to 80 years and their GFR = 30–59 ml/min/1.73 m2.

Subgroup 4 (stage 4 CKD): This group included 10 patients. There were six men and four women, with mean age 65.50 ± 10.63 that ranged from 45 to 78 years and their GFR = 15–29 ml/min/1.73 m2.

Subgroup 5 (stage 5 CKD): This group included 14 patients, they included 10 men and four women with mean age 58.50 ± 7.68 that ranged from 45 to 70 years and their GFR = 15 ml/min/1.73 m2.

In addition, 10 apparently healthy, age-matched and gender-matched individuals were included as a control group. Their mean age was 61.10 ± 4.99 that ranged from 55 to 64 years and their mean GFR = 145.70 ± 40.01.

All individuals were subjected to clinical assessment including full history, general clinical examination and weight measurement.

Exclusion criteria

Patients with diabetes, history of autoimmune disease, neoplastic disease, patients on immunosuppressing drugs and pregnancy were excluded from the study.

The design was accepted by the local ethical committee. Informed consents were obtained from the shared patients.

Methods

Sampling

Samples were taken under complete aseptic conditions: 5 ml of venous blood were collected; 2 ml was added into the tube along with EDTA for assaying hemoglobin (Hb) and the rest 3 ml was added to plain vacutainer tubes, left to clot for 30 min and then centrifuged; the sera were separated into two aliquots, one for assaying routine laboratory parameters and the other kept at −20°C till the time of assaying of α-klotho protein.

Laboratory methods: Hb test was done on Sysmex xn-100 automated analyzer, renal function tests (urea, creatinine, and uric acid), Ca2, PO4, albumin were done by AU480 analyzer (Beckman Coulter (UK) Ltd, Oakley Court, Kingsmead Business Park, London Road, High Wycombe, United Kingdom) and estimated glomerular filtration rate (eGFR) by using Cockroft-Gault equation:



Measurement of serum α-klotho protein by ELISA: The microtiter plate provided in this kit has been precoated with an antibody specific to the target antigen. Standards or samples were added to the appropriate microtiter plate wells with a biotin-coated antibody preparation specific to the target antigen and then avidin conjugated to horseradish peroxidase was added to each microplate well and incubated. Then a tetramethylbenzidine substrate solution was added to each well. Only the wells that contain target antigen, biotin-conjugated and enzyme conjugated avidin will exhibit a change in colour. The enzyme substrate reaction was terminated by the addition of sulfuric acid solution and the color change was measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of the target antigen in the sample was determined by comparing the optical density of the sample with the standard curve.

Statistical analysis

Results were statistically analyzed by SPSS, version 20 (SPSS Inc., Chicago, Illinois, USA). Two types of statistics were done: Descriptive, for example, percentage (%), mean and SD and analytical. One-way analysis of variance (F test) was used to indicate the presence of any significant difference between several groups for normally distributed variables followed by post-hoc test to show any significant difference between the individual groups. Mann–Whitney test was used to indicate the presence of any significant difference between two groups not normally distributed, χ2-test was used to compare between two groups or more regarding one qualitative variable. P value less than 0.05 is considered significant. Receiver operating characteristic curve which is a graphical plot of the sensitivity versus false positive rate was done and Pearson's correlation (r) was used to show strength and direction of two quantitative variables.


  Results Top


As regards gender, age and weight there were no statistically significant differences between all the studied subgroups [Table 1].
Table 1: Comparison between subgroups regarding age, weight, and sex

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There were high statistically significant differences between controls and all other subgroups regarding serum creatinine (P < 0.001). Post-hoc test showed that there were high statistically significant differences between controls vs. subgroups 4 and 5, 1 vs. 4 and 5, 2 vs. 4 and 5, 3 vs. 4 and 5, 4 vs. 5 (P < 0.001) [Table 2].
Table 2: Comparison between controls and subgroups regarding laboratory parameters

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There were high statistically significant differences between controls and all other subgroups regarding eGFR (P < 0.001). Post-hoc test showed that there were high significant differences between controls versus subgroups (1, 2, 3, 4 and 5) (P < 0.001). Meanwhile, there were highly significant differences between subgroups (1 vs. 2, 3, 4, 5), (2 vs. 3, 4, 5) and (3 vs. 4, 5) (P < 0.001). There were significant differences between subgroups (4 vs. 5) (P = 0.048) [Table 3].
Table 3: Comparison between subgroups as regards glomerular filtration rate and α-klotho protein

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There were high statistically significant differences between controls and all studied subgroups regarding serum klotho protein (P < 0.001). Controls = 427.80 pg/ml, subgroup 1 = 323.60 pg/ml which was statistically higher than subgroup 2, 3, 4 and 5 (231.30, 199.93, 176.90, 134.21 pg/ml, respectively). Post-hoc test revealed that there were high significant differences between controls and subgroups (1, 2, 3, 4 and 5) (P < 0.001). Meanwhile, there were high significant differences between subgroups (1 vs. 2, 3, 4, 5), (2 vs. 3, 4, 5), (3 vs. 4, 5) and (4 vs. 5) (P < 0.001) [Table 3].

There were significant positive correlations between α-klotho protein and Hb, eGFR, while there were significant negative correlations between α-klotho protein and urea, creatinine and uric acid in total patients. Meanwhile, there were no significant correlations observed in the other studied subgroups and the other studied variables but the only significant positive correlations was observed between α-klotho protein and Ca, albumin in subgroup 2 [Table 4].
Table 4: Correlation between a-klotho protein and other variables among the subgroups

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There were highly statistical significant differences between all studied subgroups regarding serum α-klotho protein (P < 0.001). By using receiver operating characteristic curve between the control group and total patients, the cutoff value of α-klotho was 271 pg/ml, at this level, the area under the curve 0.85, sensitivity 100%, specificity 85.5%, accuracy 98%, positive predictive value (PPV) 98% and negative predictive value (NPV) 100%. Also, the cutoff level of α-klotho that differentiates between controls from subgroup 1 was 397 pg/ml, at this level, the area under the curve 0.99, sensitivity 100%, specificity 90%, accuracy 95%, PPV 91% and NPV 100%. The best cutoff level of α-klotho was 316 pg/ml that differentiate controls from subgroup 2, in which the area under the curve was 1, sensitivity 100%, specificity 100, accuracy 100%, PPV 100% and NPV 100% [Table 5] and [Figure 1],[Figure 2],[Figure 3].
Table 5: Receiver operating characteristic curve of cutoff value of a-klotho protein among controls both of total chronic kidney disease patients and subgroups

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Figure 1: Receiver operating characteristic curve of serum soluble α-klotho protein between total patients and controls.

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Figure 2: Receiver operating characteristic curve of serum soluble α-klotho protein between controls and subgroup 1.

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Figure 3: Receiver operating characteristic curve of serum soluble α-klotho protein between controls and subgroup 2.

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  Discussion Top


CKD is a worldwide public health problem[6]. Current recommendations by the Kidney Outcomes Quality Initiative and National Institute for Health Excellence are to use serum creatinine concentration to eGFR[7].

In the current study, the highest level of creatinine was recorded by the subgroup 5, followed by subgroups 4, 3, 2, 1 and lastly the control group. Also, there were greater statistically significant difference as regards to serum creatinine between all studied subgroups. These results are in accordance with that of Pandya et al.[8].

The current study has shown that there were high significant differences between all studied subgroups regarding eGFR. As showed by post-hoc test, these results are in accordance with Kim et al.[9].

The obtained results in the study revealed that the concentrations of serum α-klotho protein were significantly lower in CKD patients than controls. The mean level of α-klotho in subgroup 1 was statistically higher than subgroups 2, 3, 4 and 5. These results were in accordance with those revealed by Shimamura et al.[10]and Thorsen[11]. Also, Rotondi et al.[12] found that soluble α-klotho levels in CKD patients were significantly lower than its reference values, reduced levels in CKD stage 2, with a more significant reduction in CKD stages 3 and 4. Goldsmith and Cunningham[13] observed that α-klotho deficiency may initiate or aggravate dysregulated mineral metabolism which is involved in CKD progression and development of extra-renal complications. Milovanov et al.[14] reported that the serum level of the α-klotho is not only a marker for severity of CKD and its complications, but also a pathogenic factor of CKD progression. However, Isakova et al.[15] have suggested that initial fibroblast growth factor 23 increase in early CKD actually causes downregulation of klotho by reduced 1-25-dihydroxy cholecalciferol.

The current study addressed that there were significant positive correlation between α-klotho protein and hemoglobin, GFR in the total patient group. These results in accordance with Rotondi et al.[12] and Pavik et al.[16] found that there was positive correlation between eGFR and soluble α-klotho. Also, Milovanov et al.[14] found that there was a direct correlation between hemoglobin and α-klotho levels. However, Semba et al.[17]and Devaraj et al.[18] reported that circulating serum levels of soluble α-klotho are not always correlated with eGFR rate. Meanwhile, there were significant negative correlation between α-klotho and urea, creatinine in total patients. These results agreed with Shimomura et al.[10]. However, Seiler et al.[19]found stable soluble α-klotho levels in patients with CKD stages 2–4 and the plasma levels of secreted α-klotho were not associated with kidney function or the parameters of calcium–phosphate metabolism. Akimoto et al.[20]stated that there was no association between serum α-klotho levels and residual function. The only significant positive correlation was observed in subgroup 2 between α-klotho and Ca, albumin. Yamazaki et al.[21]found that serum soluble α-klotho correlated with age, correlated considerably with phosphate, creatinine but did not significantly correlate with Ca.

Although the reason for the discrepancies between those studies remains unclear, there were several possibilities; the number of evaluated patients was low. In fact, an increased number of cases would have reinforced the reliability of this study. In addition, it was difficult to differentiate secreted α-klotho from other short forms of α-klotho because of the highly conservative sequences between different klotho forms.


  Conclusion Top


Soluble α-klotho levels are found to be significantly decreased in CKD not only in the advanced stages but also in early stages.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Zeng J, Liu M, Wu L, Wang J, Yang S, Wang Y, et al. The association of hypertriglyceridemic waist phenotype with CKD and its sex difference: a cross-sectional study in Urban Chinese elderly population. Int J Environ Res Public Health 2016; 13:1233–1236.  Back to cited text no. 1
    
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Golembiewska E, Stpniewska J, Domanski M, Kedzierska K, Domanski M, Ciechenowski K et al. The role of klotho protein in CKD: studies in animals and humans. Curr Protein Pept Sci 2016; 17:821–826.  Back to cited text no. 3
    
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Ritter CS, Zhang S, delmez J, Finch JL, Slatopolsky E. Differential expression and regulation of klotho by paricalcitrol in kidney, parathyroid and aorta of uremic rat. Kidney Int 2015; 87:1141–1152.  Back to cited text no. 4
    
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Hill NR, Fatoba ST, Oke JL, Hirst JA, O'Callaghan CA, Lasserson DS, et al. Global prevalence of CKD: a systemic review and meta-analysis. Plos one 2016; 11:e0158765.  Back to cited text no. 7
    
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Pandya D, Nagrajappa AK. Ravi KS. Assessment and correlation of urea and creatinine levels in saliva and serum of patients with CKD, diabetes and hypertension: a research study. J Clin Diagn Res 2016; 10:zc58–zc62.  Back to cited text no. 8
    
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Kim HR, Nam BY, Kim DW, Kang MW, Han JH, Lee MJ, et al. Circulating α-klotho in CKD and relationship to progression. Am J Kidney Dis 2013;61:899–909.  Back to cited text no. 9
    
10.
Shimamura Y, Hamada K, Inoue K, Ogata K, Ishihara M, Kagwa T, et al. Serum level of soluble secreted α-klotho are decreased in early stages of CKD, making it a probable novel biomarker for early diagnosis. Clin Exp Nephrol 2012; 16:722–729.  Back to cited text no. 10
    
11.
Thorsen SI, Bleskestad HI, Jonsson G, Skadberg O, Goransson LG. Neutrophil gelatinase-associated lipocalin, FGF23 and soluble klotho in long-term kidney donors. Nophrol Extra 2016; 6:31–39.  Back to cited text no. 11
    
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Rotondi S, Pasquali M, Tartaglione L, Muci ML, Mandanici G, Sales S, et al. Soluble α-klotho serum level in CKD. Int J Endocrinol 2015; 2015:872193–887219.  Back to cited text no. 12
    
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Goldsmith DJ, Cunningham J. Mineral metabolism and vitamin D in CKD-more questions than answers. Nat Rev Nephrol 2011; 7:341–346.  Back to cited text no. 13
    
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Milovanov YS, Mukhin NA, Kozlovskaya LV, Milovanova SY, Markina MM. Impact of anemia correction on the production of circulating morphogenetic protein α-klotho in patients with stages 3B-4 CKD: a new direction of cardionephroproection. Ter Arkh 2016; 88:21–26.  Back to cited text no. 14
    
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Isakova T, Wahl P, Vargus GS, Gutierrez OM, Scialla J, Xie H, et al. FGF23 is elevated before parathyroid hormone and phosphate in CKD. Kidney Int 2011; 79:1370–1378.  Back to cited text no. 15
    
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Pavik I, Jaeger P, Ebner L, Wagner CA, Petzold K, Spichtig D, et al. Secreated klotho and FGF23 in CKD stage 1to5: a sequence suggested from a cross-sectional study. Nephrol Dial Transpl 2013; 28:352–359.  Back to cited text no. 16
    
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Semba RD, Cappola AR, Sun K, Bandinelli S, Dalal M, Crasto C, et al. Plasma klotho and cardiovascular disease in adults. J Am Geriatr Soc 2011; 59:1596–1601.  Back to cited text no. 17
    
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Devaraj S, Syed B, Chien A, Jialal I. Validation of an immunoassay for soluble klotho protein: decreased levels in diabetes and increased levels in CKD. Am J Clin Pathol 2012; 137:479–485.  Back to cited text no. 18
    
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Seiler S, Wen M, Roth HJ, Fehrenz M, Flugge F, Herath E, et al. Plasma klotho is not related kidney function and does not predict adverse outcome in patients with CKD. Kidney Int 2013; 83:121–128.  Back to cited text no. 19
    
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Akimoto T, Shiizaki K, Sugase T, Watanabe Y, Yoshizawa H, Otani N, et al. The relationship between the soluble klotho protein and the residual renal function among peritoneal dialysis patients. Clin Exp Nephrol 2012; 16:442–447.  Back to cited text no. 20
    
21.
Yamazaki Y, Imura A, Urakwa I, Shimada T, Murakawami J, Anono Y, et al. Establishment of sandwich ELISA for soluble α-klotho measurement: age dependent changes of soluble α-klotho levels in healthy subjects. Biochem Biophys Res Commun 2010; 398:513–518.  Back to cited text no. 21
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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