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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 33  |  Issue : 1  |  Page : 101-104

Evaluation of klotho protein level in chronic kidney disease patients


1 Department of aInternal Medicine, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of Internal Medicine, Shebin El-Kom Fever Hospital, Menoufia, Egypt

Date of Submission21-Jan-2019
Date of Decision04-Mar-2019
Date of Acceptance05-Mar-2019
Date of Web Publication25-Mar-2020

Correspondence Address:
Heba A.M. Anwar Dawoud
Qutor Tanta, El Gharbia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_11_19

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  Abstract 

Objective
The aim of this study is to assay the level of serum klotho protein in chronic kidney disease (CKDs) patients and the relation between its level and cardiovascular complications and mineral bone diseases.
Background
Klotho protein is a new diagnostic and prognostic biomarker in kidney diseases. Klotho protein deficiency plays an important role in the calcification of vascular and soft tissues. Cardiovascular diseases are the most important causes of morbidity and mortality in CKD patients.
Patients and methods
The study included 60 CKD patients on hemodialysis, 15 CKD patients without hemodialysis, and 14 apparent healthy persons; all are of different age and sex. All groups underwent the same examination and investigation: serum klotho levels assessment and other routine laboratory investigation, for example, blood urea, serum creatinine, serum phosphorous, serum calcium, complete blood count, and serum parathormone. Echocardiography and abdominal radiography are done to all the studied groups and the results are tabulated and analyzed.
Results
The serum klotho levels are markedly decreased in CKD patients. Serum klotho level is decreased with the increase in age; there was an inverse relationship between serum klotho, blood urea and serum creatinine (P = 0.001) and between serum klotho and phosphorous (P = 0.004); the relation between serum klotho and echo findings was significant (P = 0.025).
Conclusion
CKD is a statement of klotho deficiency, there is a marked decrease in serum klotho level in the CKD patients Cardiovascular complications especially aortic calcification were found with low-serum klotho level.

Keywords: cardiovascular diseases, chronic kidney diseases, echocardiography, hemodialysis, serum klotho


How to cite this article:
El-Arbagy AR, El-Barbary HS, Emara MM, Anwar Dawoud HA. Evaluation of klotho protein level in chronic kidney disease patients. Menoufia Med J 2020;33:101-4

How to cite this URL:
El-Arbagy AR, El-Barbary HS, Emara MM, Anwar Dawoud HA. Evaluation of klotho protein level in chronic kidney disease patients. Menoufia Med J [serial online] 2020 [cited 2020 Mar 30];33:101-4. Available from: http://www.mmj.eg.net/text.asp?2020/33/1/101/281261




  Introduction Top


Chronic kidney disease (CKD) is a public health challenge worldwide, associated with high rates of morbidity and mortality. It is also a strong risk factor for developing cardiovascular diseases[1].

There are a variety of uremia-specific nontraditional risk factors in CKD that may be amplified in the pathogenesis of cardiovascular complications including the presence of protein-bound uremic toxins such as indoxyl sulfate, low-serum klotho, albuminuria, hyperactive renin angiotensin aldosterone system, and abnormal bone mineral metabolism[2].

Klotho is a useful novel biomarker for renal diseases; it was originally identified as an anti-aging protein. Klotho present in transmembrane form acts as a coreceptor of FGF23 and a soluble form circulates in the body fluids including the blood, urine, and cerebrospinal fluid. The highest expression of klotho is in the kidney and brain[3].

Soluble klotho protein plays an important role in the modulation of ion transporter or channels, antioxidants, phosphaturic effect, renoprotection, and prevention of soft tissue calcification[4].

The current study aimed to evaluate the level of klotho protein in CKD stages and the relations between its level and cardiovascular complications.


  Patients and Methods Top


All participants gave written informed consent before inclusion into the study. After approval of Menoufia Ethics committee for the study proposal. Eighty-nine patients were divided into three groups, 60 CKD patients on dialysis, 15 CKD patients without dialysis, and 14 apparent healthy persons. All are of different age and sex from Kafr El Sheikh Hospitals.

The patients were excluded because of one or more of the following reasons: acute infection, connective tissue diseases, malignancy, history of thyroid gland dysfunction, recent myocardial infarction, recent major trauma less than 6 months, and oral anticoagulants. Full history is taken and physical examination was done to confirm or exclude symptoms and signs of uremia, hyperphosphatemia, hyperparathyroidism, and cardiovascular diseases. Blood samples were taken for routine laboratory investigation, for example, blood urea, serum creatinine, serum phosphorus, serum calcium, serum parathyroid hormone (PTH), complete blood count, and serum klotho. From each participant under complete aseptic technique, 5 ml venous blood samples were collected, samples were allowed to clot and then were centrifuged 20 min at the speed of 2000 rpm and the supernatant is removed. The kit assay human klotho level in the sample uses purified human klotho antibody to coat microtiter plate wells, makes solid-phase antibody, then add klotho to wells, combined with klotho antibody which is HRP labeled, becoming the antibody–antigen–enzyme antibody complex, after washing completely, add TMB substrate solution, and the TMB substrate becomes blue color. At HRP enzyme catalyzed reaction is determined by the addition of sulfuric acid solution and the color change is measured spectrophotometrically at a wave length of 450 nm. The concentration of human klotho in the samples is then determined by comparing the optical density of the samples to the standard curve. Radiography of the abdomen and echocardiography are done for all patients to exclude or prove aortic calcification and other cardiovascular changes.

The results were collected, tabulated, and statistically analyzed by an IBM-compatible personal computer with SPSS statistical package, version 20 on IBM compatible computer (Armonk, New York, United States). Two types of statistical analysis were done: descriptive statistics, for example n (%), mean, and SD. Analytic statistics for example, analysis of variance test (F) was used for comparison of quantitative variables between more than two groups of normally distributed data with least square difference test such as the post-hoc test. Kruskal–Wallis test (K) was used for comparison of quantitative variables between more than two groups of non-normally distributed data with Tamhane's test as the post-hoc test. χ2 was used to study the association between qualitative variables. A P value of less than 0.05 was considered statistically significant.


  Results Top


The klotho protein levels of 89 persons are analyzed. There was a marked decrease in its level in CKD patients and CKD patients on dialysis, but high level is seen in healthy persons.

There was highly significant increase in PTH level between the studied groups (P = 0.001) in the parathormone level in CKD patients on dialysis compared with healthy persons and significance of serum PO4 level which presents in high levels in the first and second groups of the study (P = 0.001). There is a highly significanct increase in serum klotho level (P = 0.001) in the third group. And there was a significant increase of blood urea (P = 0.041) in both first and second groups and significance of serum creatinine level in both first and second groups (P = 0.006). But there was no significance between the studied groups regarding hemoglobin (P = 0.398) and serum calcium (P = 0.113) [Table 1]. There was a significant inverse relationship between serum klotho and serum creatinine (P = 0.001) and an inverse relationship between serum klotho and serum phosphorous (P = 0.004) whereas there is high phosphorous and creatinine level in CKD patients with low-serum klotho level. There was statistical significant difference between serum klotho and hemoglobin level (P = 0.039) nonsignificance of PTH (P = 0.545). Also significant correlation is seen between the serum level of klotho protein and blood urea (P = 0.005) and there is a significant relation between serum klotho and age (P = 0.005); klotho level gradually decreases with the increase in age [Table 2].
Table 1: Comparison of studied groups regarding laboratory investigations

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Table 2: Correlation between serum level of klotho protein and other serological findings and age in all cases

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There was a significanct relation between serum klotho protein level and aortic calcification of the studied groups (P = 0.018) where aortic calcification is increased in CKD patients on dialysis whose klotho level is markedly decreased. A significant relation was observed between serum level of klotho protein and echocardiography groups (P = 0.025) where an abnormal echocardiograph is present more with low level of klotho protein in CKD patients [Table 3].
Table 3: Relation between serum level of klotho protein and cardiovascular changes by echo with focusing on aortic calcification

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  Discussion Top


Consistent with our findings, Litvin et al.[5] reported that CKDs describe abnormal kidney function and/or structure for more than 3 months. The risk of developing CKD increases with age. CKD amplifies the risk for cardiac morbidity and mortality. People with CKD also have high risk of acute kidney injury.

Consistent with our findings, Peralta et al.[6] reported that a patient is diagnosed to have CKD when the urinary albumin creatinine ratio is 30 mg/g or the estimated glomerular filtration rate (eGFR), as measured by the modification of diet in renal diseases study equation is 60 ml/min/1.73 m2 on at least two different occasions over 3 months.

Consistent with our findings, Luyckx et al.[7] reported that referral to a nephrologist is recommended once the patient is with an eGFR of 30 ml/min/1.73 m2, resistant hypertension, 30% loss of eGFR over 4 months, hyperkalemia 5.5 eq/l. Early referral to a nephrologist has been shown to improve the outcomes. Consistent with our findings, Hu et al.[8] reported that klotho was originally identified as an anti-aging protein, but has multiple biological actions the highest expression of which is in the kidney.

Consistent with our findings, Hu et al.[9] reported that klotho has extreme pleiotropic actions, including cytoprotection, anti-oxidation, anti-ptosis, and protection of vasculature. Consistent with our findings, Shimamura et al.[10] reported that klotho inhibits phosphate absorption and promotes phosphaturia that contributes to the prevention of vascular calcification.

Consistent with our findings, Hu et al.[11] reported that there are diagnostic, prognostic, and therapeutic roles of klotho in kidney diseases. Recombinant klotho administration is already used in animal models and maneuvers are available to enhance endogenous klotho.

Consistent with our findings, Lim et al.[12] reported that serum klotho is initially decreased in CKD patients. We also demonstrated a positive correlation between serum klotho and vascular calcification.

Consistent with our findings, Hu et al.[13] reported that serum klotho may be used as a simple, rapid, early biomarker of CKD and predictor of vascular calcification in CKD patients.

Consistent with our findings, Yamazaki et al.[14] reported that klotho deficiency appears not only as a biomarker for CKD but pathogenic for CKD development, progression, and complication. There are interesting similarity including hyperphosphatemia, ectopic soft tissue calcification, and decreased serum klotho suggests that CKD might be in a state of klotho deficiency.

Consistent with our findings, Huang and Moe[15] reported that serum phosphorus level is high in CKD patients with and without dialysis which is included in our study groups, where the kidney is the primary site of PO4 excretion and hydroxylation. vitamin D deficiency patients develop hypophosphatemia, which leads to a decrease in the calcium level by a negative feedback mechanism. The PTH level is increased which leads to secondary hyperparathyroidism. All these changes are combined with a decline in serum klotho in CKD patients.

Consistent with our findings, Navarro-Gonzalez et al.[16] reported that the relationship between serum klotho level and aortic calcification, when serum klotho protein is decreased in CKD patients leads to a reduction or inhibition of its phosphaturic effect that leads to vascular calcification and increase in cardiovascular disease and its complications with an increase in morbidity and incidence of mortality.


  Conclusion Top


Our study concluded that there is a significant decrease in serum klotho levels in hemodialysis patients compared with those apparently healthy persons. There is a significant inverse relationship between serum klotho and serum PO4, blood urea, serum creatinine, and age. However, there is nonsignificant relation between serum klotho and hemoglobin and PTH.

Cardiovascular complications, for example, left ventricular hypertrophy (LVH), ischemic heart disease (IHD), valvular disease, and pericardial effusion sclerotic aorta were present with low-serum klotho level.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Collistar D, Ferguson T, Komenda P, Tangri N. The patterns, risk factors and prediction of progression in chronic kidney disease:. a narrative review. Semin Nephrol 2016; 36:273–282.  Back to cited text no. 1
    
2.
Yieg J, Yoon J, SW An, Kuro O, HuangCL. Soluble klotho protects against uremic cardiomyopathy independetly of fibroblast growth factor 23 and phosphate. J Am Soc Nephrol 2015; 26:1150–1160.  Back to cited text no. 2
    
3.
Linderberg K, Amin R, Moe OW, Hu MC, Erben RJ. The kidney is the principale organ mediating klotho effects. J Am Soc Nephrol 2014; 25:2169–2175.  Back to cited text no. 3
    
4.
Grabner A, Faul C. The role of fibro blast growth factor23 and klotho in uremic cardiomyopathy. Curr Opin Nephrol Hypertens 2016; 25:314–324.  Back to cited text no. 4
    
5.
Litvin CB, Hyer JM, Ornstein SM. Use of clinical decision support to improve primary care identification and management of chronic kidney disease. J Am Board Fam Med 2016; 29:604–612.  Back to cited text no. 5
    
6.
Peralta CA, Shlipak MG, Judd S, Mcushman J. Detection of chronic kidney disease with creatinine, cystain C, and urine albumin to creatinine ratio and association with progression to end-stage renal disease and mortality. JAMA 2011; 305:1545–1552.  Back to cited text no. 6
    
7.
Luyckx VA, Naicker S, Mckee M. Equity and economics of kidney disease in sub Saharan Africa. Lancet 2013; 382:103–104.  Back to cited text no. 7
    
8.
Hu MC, Kuro-o M, Moe OW. Renal and extra renal actions of klotho. Semin Nephrol 2013; 33:118–129.  Back to cited text no. 8
    
9.
Hu MC, Shi M, Zhang J, Addo T, Cho HJ, BarkerSL, et al. Renal production, uptake and handling of circulating alpha klotho. J Am Soc Nephrol 2016; 27:79–90.  Back to cited text no. 9
    
10.
Shimamura Y, Hamada K, Inoue K, Ogata K, Shihara M, Kagawa T,et al. Serum levels of klotho are decreased in the early stages of chronic kidney disease, making it aprobable novel biomarker for early diagnosis. Clin Exp Nephrol 2012; 16:722–729.  Back to cited text no. 10
    
11.
Hu MC, Shi M, Zhang J, Quinones H, Kuro OM, Moe OW, et al. Klotho deficiency ia an early biomarker of renal ischemia reperfusion injury and its replacement is protective. Kidney Int 2010; 78:1240–1251.  Back to cited text no. 11
    
12.
Lim k, Lu TS, Molostvov G, Lee C, Lam FT. Vascular klotho deficiency potentiate the development of human artery calcification and mediates resistance to fibroblast growth factor 23. Circulation. 2012; 125:2243–2255.  Back to cited text no. 12
    
13.
Hu MC, Kuro-o M, Moe W. The emerging role of klotho in clinical nephrology. Nephrol Dial Transplant 2012; 27:2650–2657.  Back to cited text no. 13
    
14.
Yamazaki Y, Imura A, Urakawa I, Shimada T, Murakmi J, Aono Y,et al. Klotho and kidney function decline. Biochembiophys Res Commun 2011; 22:124–136.  Back to cited text no. 14
    
15.
Huang CL, Moe OW. Klotho anovel regulator of calcium and phosphorous homestasis. Pflugers Arch 2011; 462:185–193.  Back to cited text no. 15
    
16.
Navarro-Gonzalez JF, Donate-Correa J, Muros de Fuents M. Klotho is associated with the presence and severity of coronary artery disease. Heart 2014; 100:34–40.  Back to cited text no. 16
    



 
 
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  [Table 1], [Table 2], [Table 3]



 

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