Scholarly article on topic 'Assessment the relationship between serum soluble klotho and carotid intima-media thickness and left ventricular dysfunction in hemodialysis patients'

Assessment the relationship between serum soluble klotho and carotid intima-media thickness and left ventricular dysfunction in hemodialysis patients Academic research paper on "Clinical medicine"

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{"Cardiovascular diseases" / "End-stage renal disease" / "Fibroblast growth factor-23" / "Soluble Klotho"}

Abstract of research paper on Clinical medicine, author of scientific article — Emad Abdallah, Osama Mosbah, Ghada Khalifa, Amna Metwaly, Omnia El-Bendary

Abstract Background The aim of our study was to assess the relationship between soluble Klotho (s-Klotho) and carotid intima–media thickness (CIMT) and left ventricular (LV) dysfunction in hemodialysis (HD) patients. Methods This is a cross-sectional study conducted on 88 patients with end-stage renal disease on regular HD. Serum levels of calcium, phosphorus, parathyroid hormone, and C-reactive protein were measured. The serum levels of s-Klotho and fibroblast growth factor-23 (FGF-23) were measured using an Enzyme linked immunosorbent assay (ELISA) kit. Echocardiography and measurement of CIMT were also conducted. The studied patients were divided according to the median s-Klotho level into 2 groups: patients with low s-Klotho (Group I) and patients with high s-Klotho (Group II). Results Mean value of s-Klotho was significantly low in HD patients compared to controls (P = 0.001), and mean value of FGF-23 was significantly high in HD patients compared to controls (P = 0.001). The mean values of parathyroid hormone, FGF-23, and phosphorus were significantly high in Group I compared to Group II, whereas the mean value of serum calcium was significantly low in Group I compared to Group II. The mean values of CIMT, LV mass (LVM), LVM index, and LV ejection fraction (LVEF) were high in Group I compared to Group II. Patients with low s-Klotho had significantly more coronary artery disease (CAD). In a regression analysis of s-Klotho with different markers of cardiovascular diseases, s-Klotho showed significant association with CIMT, LVEF, and CAD, but not with LVM and LVM index. Conclusion The present study showed that patients with a low s-Klotho were more often associated with increased CIMT, LV dysfunction, and CAD, and it seems that there was independent association between s-Klotho and CIMT, LVEF, and CAD.

Academic research paper on topic "Assessment the relationship between serum soluble klotho and carotid intima-media thickness and left ventricular dysfunction in hemodialysis patients"

Accepted Manuscript

Assessment the relationship between serum soluble klotho and carotid intima-media thickness and left ventricular dysfunction in hemodialysis patients

Emad Abdallah, Associate professor of nephrology, Osama Mosbah, Ghada Khalifa, Amna Metwaly, Omnia El-Bendary

KIDNEY RESEARCH

iND CLINICAL PRACTICE

PII: S2211-9132(15)30058-9

DOI: 10.1016/j.krcp.2015.12.006

Reference: KRCP 241

To appear in: Kidney Research and Clinical Practice

Received Date: 23 September 2015

Revised Date: 27 November 2015 Accepted Date: 16 December 2015

Please cite this article as: Abdallah E, Mosbah O, Khalifa G, Metwaly A, El-Bendary O, Assessment the relationship between serum soluble klotho and carotid intima-media thickness and left ventricular dysfunction in hemodialysis patients, Kidney Research and Clinical Practice (2016), doi: 10.1016/ j.krcp.2015.12.006.

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Manuscript title: Assessment the relationship between serum soluble klotho and carotid intima-media thickness and left ventricular dysfunction in hemodialysis patients

Short title: Soluble klotho in hemodialysis

1 112 Emad Abdallah* ; Osama Mosbah ; Ghada Khalifa ; Amna Metwaly ; Omnia El-

Bendary3

1Department of Nephrology, Theodor Bilharz Research Institute, Cairo, Egypt. 2

Department of Intensive Care Unit, Theodor Bilharz Research Institute, Cairo, Egypt. Department of Clinical Chemistry, Theodor Bilharz Research Institute, Cairo, Egypt.

Corresponding author: Emad Abdallah

Associate professor of nephrology, Theodor Bilharz Research Institute, Cairo, Egypt

Postal address: Theodor Bilharz Research Institute El-Nile St.Waraq El-Hadar

P.O.Box 30 Imbaba Giza, 12411 Egypt

Phone: 00201005767492

Fax: 20235409125

Email: drabdallah96@gmail.com

Background

The aim of our study was to assess the relationship between soluble klotho (s-klotho) and carotid intima-media thickness (CIMT) and left ventricular dysfunction in hemodialysis (HD) patients. Methods

This is a cross-sectional study conducted on 88 patients with end-stage renal disease on regular HD. Serum levels of calcium (Ca), phosphorus (Ps), parathyroid hormone (PTH) and C-reactive protein (C-RP) were measured. The serum levels of s-klotho and fibroblast growth factor-23 (FGF-23) were measured using ELISA kit. Echocardiography and measurement of CIMT were also conducted. The studied patients were divided according to median s-klotho level into two groups, patients with low s-klotho (group I) and patients with high s-klotho (group II) Results

Mean value of s-klotho was significantly low in HD patients compared to control (p = 0.0001) and mean value of FGF-23 was significantly high in HD patients compared to control (p = 0.0001). The mean values of PTH, FGF-23 and Ps were significantly high in group I compared to group II, while,the mean value of serum Ca was significantly low in group I compared to group II. The mean values of CIMT, left ventricular mass (LVM), LVM index (LVMI) and LV ejection fraction (LVEF) were high in group I compared to group II. patients with low s-klotho had significantly more coronary artery disease (CAD). Regression analysis of s-klotho with different markers of cardiovascular diseases, s-klotho showed significant association with CIMT, LVEF and CAD, but not with LVM and LVMI. Conclusion

The present study showed that, patients with a low s-klotho associated with increased CIMT, LV dysfunction and CAD more often and it seems that, there was independent association between s-klotho and CIMT,LVEF and CAD.

Keywords: Cardiovascular drseases; End-stage renal disease; Fibroblast growth factor-23; Soluble klotho.

Introduction

Chronic kidney disease (CKD) associated with increased levels of parathyroid

hormone (PTH) and fibroblast growth factor-23 (FGF-23) and hypocalcemia,

hyperphosphatemia, bone disease, vascular calcification and cardiovascular

morbidities collectively referred to as chronic kidney disease-mineral and bone

disorder (CKD-MBD) [1-3]. Recent reports suggested that increased levels of FGF-23

and decreased levels of soluble klotho (s-klotho) are a common manifestations of

CKD that develop earlier than increased levels of phosphate (Ps) or PTH [4,5].

A transmembrane (TM) protein known soluble a-klotho (s-klotho) is primarily

produced in the kidney distal tubular cells [6]. Soluble a-klotho acts as a coreceptor

for the bone derived protein FGF-23 [7,8]. Regulation of both renal handling of Ps

and renal synthesis of calcitriol need a cofunction of both FGF-23 and TM-klotho [9].

Soluble a-klotho is the circulating protein resulting from the shedding of the

extracellular domain of TM-klotho operated by two metalloproteinases of the a

disintegrin and metalloproteinase domain-containing protein (ADAM) family:

ADAM 10 and ADAM 17 [10]. In particular,s-klotho inhibits the sodium-phosphate

cotransporter NaPi2a expression in the proximal tubules thus generating a phosphaturic effect additive to and independent of FGF-23 [11-13] and activates the ion channel TRPV5 in the distal tubules, thus increasing tubular reabsorption of calcium (Ca) [14]. So, a-klotho, with its transmembrane and soluble forms, is deeply involved with the physiologic regulation of mineral metabolism [15]. CKD is a common risk factor for cardiovascular drseases (CVD) such as coronary artery disease (CAD), cerebrovascular stroke, peripheral vascular disease and heart failure [16]. Whereas part of CVD burden in patients with CKD is related to traditional risk factors, CKD associated disturbance in Ca-Ps homeostasis play a crucial role as well [2]. Recent studies showed that, FGF-23 and its co-receptor s-klotho have an important role in Ca-Ps homeostasis and they could be the missing link in the detrimental relationship between CKD and CVD [17].

Given the strong cardioprotective effects of klotho demonstrated in preclinical studies, the present study aimed to assess the association between s-klotho and carotid intima-media thickness (CIMT) and left ventricular (LV) dysfuntion in patients with ESRD on regular hemodialysis (HD). It was hypothesized that low levels of s-klotho are associated with a larger burden of CVD in these patients.

Methods Study population

This is a cross-sectional study conducted on 88 patients with end-stage renal disease

(ESRD) on regular HD of at least 6 months duration. All patients have been attending,

nephrology dialysis unit, Theodor Bilharz Research Institute, Cairo, Egypt. All

patients received three sessions of HD /week, each of 4 hours duration using

polysulphone dialyzer (Fresenius, St. Wendel, Germany) with surface area of 1.4 to

1.6. Patients with advanced congestive heart failure (defined clinically by elevated jugular venous pressure, ascitis, peripheral oedema and shortness of breath at rest and echocardiography with LVEF <30% requiring hospital-based support, a heart transplant or palliative care according to guidelines of the American College of Cardiology/American Heart Association) [18], sepsis or malignancy were excluded from the study. Informed written consent was obtained from all participants. The study protocol was approved by the Institute Ethics Committee and the study was performed in accordance with the Declaration of Helsinki.

The studied patients were divided according to median s-klotho level (cutpoint 476 pg/ml) into two groups, patients with low s-Klotho <476 pg/mL (group I, n=44) and patients with high s-Klotho >476 pg/ml (group II, n=44)

A control group consists of 28 normal population, were used to give our reference values for s-klotho, FGF-23, CIMT and echocardiographic findings. They were 17 males and 11 females with mean age 53.3±16.2 years, with normal renal function, and no evidence of acute or chronic underlying disease.

Each patient underwent a full through history and clinical examination. Demographic characteristics and coexisting conditions as atherosclerotic CAD diagnosed by electrocardiography and coronary angiography within 3 months of enrollment in the study were collected.

Fasting blood samples were collected from the patients before HD session, immediately centrifuged, aliquoted in vials and stored at -60°C till time of analysis. Thawing the test samples was carried on at a low temperature with mixing them completely before measurement. Routine examinations included complete blood picture, kidney function tests (serum urea, creatinine, sodium and potassium, and uric

acid), random blood sugar, serum electrolytes, lipids profile, serum albumin. Serum levels of Ca, Ps, PTH and C-reactive protein (C-RP) were measured. Serum levels of s-klotho and FGF-23 measurement

The serum levels of s-klotho were measured using an ELISA system (Immuno-Biological Laboratories, Gunma, Japan) [19], and this assay detects circulating soluble Klotho by using two monoclonal antibodies that specifically recognize the extracellular domain of Klotho, with a lower limit of detection of 6.15 pg/mL and he intra- and inter-assay coefficients of variation were <10%. The serum levels of FGF-23 were measured using a commercial sandwich ELISA kit (Kainos Laboratories, Inc., Tokyo, Japan) [20], that utilizes a 2-site ELISA for the full-length molecule. Two specific murine monoclonal antibodies recognize the biologically active FGF23, with a lower limit of detection of 3 pg/mL, and inter- and intraassay coefficients of variation of <5%.

Residual renal function (RRF) estimation

RRF was estimated by calculating glomerular filtration rate (GFR) expressed in mL/min/1.73 m2. GFR was estimated as the mean of urea and creatinine clearance using 24 hrs urine collections and the mean of the posthemodialysis and the prehemodialysis plasma urea and creatinine. RRF was considered zero in patients with a urinary output <100 mL/24 hours [21]. Kt/V was used to assess dialysis adequacy [22]. Echocardiography

After HD session, each patient underwent echocardiography to determine LV mass

(LVM), LV mass index (LVMI), and LV ejection fraction (LVEF). For determination

of LVM, the Devereux formula was used [23]. LV mass (gr): 1.04 [(LVID + PWT +

IVST)3 - LVID3 ] - 14. LVID = LV internal dimension. PWT = posterior wall

thickness. IVST = interventricular septal thickness. LVM was divided by body surface area to measure LVMI. According to the current guidelines, LV dysfunction was defined as a LVEF < 45%.

Measurement of carotid intima-media thickness (CIMT)

After HD session, each patient underwent ultrasonography of the carotid arteries using a high resolution real-time scanner with a 7.5 MHz transducer [24]. The examination was done with the patient in the supine position, and the common carotid artery and carotid bifurcation, were scanned on both sides. The carotid artery was scanned in the longitudinal and transverse directions. The site of the most advanced atherosclerotic lesion that showed the greatest distance between the lumen-intima interface and the media-adventitia interface was the maximum IMT value. When plaque was detected on ultrasonography, it was observed as localized thickening rather than a circumferential change in the vessel wall. The greatest thickness of the intima-media complex (including plaque) was used for the maximum IMT value. We identified patients having atherosclerosis based on atheromatous plaques of focal increases in IMT >1.1 mm in accordance with a prior study that showed the normal limit of IMT to be <1.0 mm [25].

Statistical analysis

Data are analysed as means ± standard deviations (SD) or as number (%). The 2-tailed

Student's i-test were used to compare the mean values of two groups (GraphPad

QuikCalcs). Categorical data compared using the Chi-square test. Spearman rank

correlation test was used to analyse the correlations between s-klotho levels and

markers of mineral metabolism. Multi-variate regression analysis (using MedCal

software) were used to analyse the association between s-klotho and markers of CVD,

with linear regression analysis model with unstandardized Beta's and a 95% confidence interval (CI) for CIMT, LVM, LVMI and LVEV and logistic regression analysis model for CAD. Linear regression analysis model was also used to determine the association between FGF-23 and CIMT, LVM, LVMI and LVEF and logistic regression analysis model for CAD. Statistical analyses of data were performed using SPSS for Windows, version 16.0 (SPSS Inc., Chicago, IL, USA).P value <0.05 considered to be significant and p value <0.01considered to be highly significant.

Results

Demographic characteristics of HD patients and controls included in this study are shown in table 1, where mean age of studied patients was 58.6±19.3 years. Fifty one (57.95%) of patients were males and 37 (42.05%) were females. The most common causes of ESRD were diabetes mellitus, hypertention, and glomerulonephritis [table 1].

Mean value of s-klotho was significantly low in HD patients compared to control group (477.9±76.2 versus 863.7±261.8, p = 0.0001) and mean value of FGF-23 was significantly high in HD patients compared to control group (60.5±17.6 versus 35.8±13.9, p = 0.0001) [table 2].

Patients with a low s-klotho (<476 pg/ml) had a high age (63.2±9.4 versus 53.8±16.2, p = 0.0013), FGF-23, PTH, Ps, and C-RP and low Ca and RRF [table 2] compared to group II. There was no significant differences in the mean values of serum creatinine, lipids profile, hemoglobin, serum glucose, serum albumin, serum uric acid and Kt/V between the two groups [table 2].

s-klotho and markers of mineral metabolism

The mean values of serum levels of PTH, FGF-23, and Ps were significantly high in patients of group I compared to patients of group II, while, the mean value of serum levels of Ca was significantly low in group I compared to group II [table 2]. Using spearman rank coefficient correlation, there was negative significant correlation between s-klotho and FGF-23 (r = -0.717, p = 0.0001, 95% confidence interval(CI) for r -0.806 to -0.598) [fig.1], PTH (r = - 0.484, p = 0.001, 95% CI for r -0.630 to -0.306) [fig.2], and Ps (r = -0.548, p = 0.0001, 95% CI -0.680 to -0.383) [fig.3], while there was significant positive correlation between s-klotho and Ca (r = 0.294, p = 0.0054, 95% CI 0.0903 to 0.474) [fig.4].

s-klotho and markers of CVD

The mean values of CIMT, LVM, LVMI, and LVEF were high in patients of group I compared to patients of group II. patients with low s-klotho had significantly more CAD 36 (81.1%) versus 22 (50.0%), p=0.0044} [table 3].

Using spearman rank coefficient correlation, there was positive significant correlation between s-klotho and LVEF ( r = 0.392, 95% CI 0.199 to 0.556, p = 0.0002) [fig.5]. Table 4 shows, the results of the multi-variate regression analysis of s.klotho with CIMT, LVM, LVMI, LVEF and CAD as different markers for CVD. After the adjustments with age, gender, mean blood pressure and duration of HD, s-klotho showed significant association with CIMT, LVEF and CAD, but not with LVM and LVMI.

FGF-23 and markers of mineral metabolism

Using spearman rank coefficient correlation, there was positive correlation between

FGF-23 and Ps (r = 0.497, p = 0.0001, 95% CI 0.321 to 0.640) and PTH (r = 0.405, p

= 0.0001, 95% CI 0.214 to 0.567), while there was negative correlation between FGF-23 and Ca (r = -0.237, p = 0.026, 95% CI -0.426 to -0.0293). FGF-23 and CVD markers

Table 4 shows the results of multi-variate regression models of FGF-23, total cholesterol, triglycerides, and blood glucose with CIMT, LVM, LVMI, LVEF and CAD as different markers for CVD. After the adjustments with age, gender, mean blood pressure and duration of HD, FGF-23, total cholesterol, triglycerides, and blood glucose were not independently associated with CIMT, LVM, LVMI, LVEF and CAD.

Discussion

In the present study, we measured the s-klotho levels and determined the association between s-klotho levels and markers of mineral metabolism ( FGF23, PTH, Ca, and Ps), and CVD (CIMT, CAD, LVM, LVMI, and LVEF) in patients with ESRD on regular HD. Our study showed that, there was negative significant correlation between s-klotho levels and serum levels of FGF-23, PTH, and Ps, and positive correlation between s-klotho and serum levels of Ca. Also the present study showed that lower levels of s-klotho are significantly associated with signs of CVD such as increased CIMT, more CAD and LV dysfunction.

Our study showed that serum levels of s-klotho were significantly decreased in patients

with ESRD on regular HD compared to the control group, as previously reported in

CKD patients [26] and patients on HD [27-29]. It has been reported that s-klotho levels are severely reduced in patients with CKD compared to control subjects [30]. However, it appears that s-klotho levels are not completely depleted, even in patients with ESRD on HD [27-29]. This finding suggests that a basal level of s-klotho may be produced from other organs than the kidneys, such as the brain and parathyroid glands, might exist in humans, as has been previously reported in mice [15,31,32]. Recent studies revealed that, s-klotho has been decreased in early stage of CKD, even before rising of Ps or PTH and emerged as a powerful player in Ca-Ps homeostasis that is thought to contribute to the high burden of CVD in CKD patients [5,33]. This study showed that, patients with a low s-klotho (<476 pg/ml) had a high age and low RRF compared to group II and there was no significant differences in the mean values of Kt/V between the two groups. Our results are in agreement with the results of Lindberg et al [28], who reported that patients with high s-klotho were more with good RRF and Sawires et al [29], who reported that there was no significant correlation between s.klotho and Kt/V and there

was negative significant correlation between s-klotho and age The present study showed that, patients with a low s-klotho associated with CIMT, LV dysfunction and CAD more often and it seems that, there was independent association between s-klotho and CIMT, LVEF and CAD using multi-variate regression analysis. Our results are in agreement with results of Kiagawa,et al [34], who reported that the serum klotho level was found to significantly correlate with markers of CKD-MBD and is an independent biomarker of arterial stiffness in patients with CKD. In contrary to our results reported by Buiten,et al [35], who showed that s-klotho was not independently associated with CVD in dialysis patients and did not support a direct cardioprotective effect of s-klotho.

Many clinical studies have suggested that s-klotho exerts strong cardioprotective effects. For instance, s-klotho has been shown to protect against vascular calcifications in rodent models of CKD while in humans without CKD, higher s-klotho levels have been related to a lower incidence of mortality and CVD [28,36-38]. Moreover, low s-klotho levels have been associated with increased arterial stiffness in CKD patients [34]. Further support for a direct role of klotho in vascular homeostasis comes from in vitro studies showing endogenous expression of s-klotho in human vascular smooth muscle cells (VSMCs) [39]. Interestingly, inhibition of s-klotho expression in aortic VSMCs resulted in accelerated calcification of these cells [39]. However, the exact role of s-klotho in the progression of CVD in dialysis patients remains to be elucidated. As long as CVD start to develop in the early stages of CKD [2,16] and there are many reports revealed that s-klotho start to decrease in the early stages of CKD [5,34]. Therefore, patients with ESRD on regular HD have been exposed to low s-klotho levels for a prolonged period of time predisposing to vascular calcifications and atheosclerosis. The role of s-klotho in the development of atherosclerotic disease in dialysis patients might be overshadowed by the large amount of other pathophysiological stimuli for CVD prevalent in these patients, such as obesity, diabetes, dyslipidemia and hypertension. This might explain the disparity between data found in rodents with only klotho deficiency and patients suffering from a wide variety of co-morbidities. However, our study showed that, there was no significant differences in the mean values of lipids profile, blood glucose, mean blood pressure, BMI and duration of dialysis between the two groups and by using multi-variate regression analysis, there was no independent association between FGF-23, blood glucose, total cholesterol and triglycerides and CVD markers. These finding may explain that, s.klotho seems to be independently associated with CIMT, LVEF and CAD as markers of CVD.

In our study, s-klotho are significantly correlated with markers of mineral metabolism, which are consistent with many studies in CKD patients [5,34], that reported that s-klotho correlated negatively with PTH and Ps and positively with Ca while no correlation was found between s-klotho and 1,25 dihydroxycholecalciferol (1,25D) and fractional excretion of calcium (FECa). Also consistent with studies in HD patients [35], that reported that serum s-klotho level was significantly associated with a lower plasma 25(OH)D and lower PTH [29].

Also in our study, FGF-23 was significantly associated with markers of mineral metabolism and FGF-23 was not independently associated with markers of CVD. Our results are consistent with studies in HD patients [35], that reported that FGF-23 showed a strong positive association with Ps and PTH and FGF-23 was not independently associated with CVD. Also our results comparable with results from studies in CKD patients, where FGF-23 levels correlated positively with PTH, Ps and negatively with 1,25D and no correlation existed with Ca and FECa [5].

This study has some limitations. Small samples of patients and data on dietary Ca, Ps and medications intake, which may affect the levels of serum Ps, Ca , s-klotho and FGF-23 not collected. Ps and PTH as a risk factors for CVD not statistically analysed. In conclusion, the present study showed that, patients with a low s-klotho associated with increased CIMT, LV dysfunction and CAD more often and it seems that, there was independent association between s-klotho and CIMT, LVEF and CAD. Conflict of interest

The authors declare that they have no conflict of interest.

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[27]Yokoyama K, Imura A, Ohkido I, Maruyama Y, Yamazaki Y, Hasegawa H, Urae J, Sekino H, Nabeshima Y, Hosoya T: Serum soluble alpha-klotho in hemodialysis patients. Clin Nephrol 77: 347-351, 2012

[28]Lindberg K, Amin R, Moe OW, Hu MC, Erben RG, Östman Wernerson A, Lanske B, Olauson H, Larsson TE: The kidney is the principal organ mediating klotho effects. J Am Soc Nephrol 25: 2169-2175, 2014

[29]Happy K, Sawires RM, Essam MF, Morgan RA, Mahmoud C: Serum Klotho: Relation to Fibroblast Growth Factor-23 and Other Regulators of Phosphate Metabolism in Children with Chronic Kidney Disease. Nephron 129: 293-299, 2015

[30]Koh N, Fujimori T, Nishiguchi S, Tamori A, Shiomi S, Nakatani T, Sugimura K, Kishimoto T, Kinoshita S, Kuroki T, Nabeshima Y: Severely Reduced Production of Klotho in Human Chronic Renal Failure Kidney. Biochem Biophys Res Commun 280: 1015-1020, 2001

[31]Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, Ohyama Y, Kurabayashi M, Kaname T, Kume E, Iwasaki H, Iida A, Shiraki-Iida T, Nishikawa S, Nagai R, Nabeshima YI: Mutation of the mouse Klotho gene leads to a syndrome resembling ageing. Nature 390: 45-51, 1997

[32]John GB, Cheng CY, Kuro-o M: Role of Klotho in aging, phosphate metabolism, and CKD. Am J Kidney Dis 58: 127-134, 2011

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[34]Kitagawa M, Sugiyama H, Morinaga H, Inoue T, Takiue K, Ogawa A, Yamanari T, Kikumoto Y, Uchida HA, Kitamura S, Maeshima Y, Nakamura K, Ito H, Makino H: A Decreased Level of Serum Soluble Klotho Is an Independent Biomarker Associated with Arterial Stiffness in Patients with Chronic Kidney Disease. PLoS One 8: e56695, 2013

[35]Buiten MS, de Bie MK, Bouma-de Krijger A, van Dam B, Dekker FW, Jukema JW, Rabelink TJ, Rotmans JI: Soluble Klotho is not independently associated with cardiovascular disease in a population of dialysis patients. BMC Nephrol 15: 197, 2014

[36]Hu MC, Shiizaki K, Kuro-o M, Moe OW: Fibroblast growth factor 23 and Klotho: physiology and pathophysiology of an endocrine network of mineral metabolism. Annu Rev Physiol 75: 503-533, 2013

[37]Semba RD, Cappola AR, Sun K, Bandinelli S, Dalal M, Crasto C, Guralnik JM, Ferrucci L: Plasma klotho and cardiovascular disease in adults. J Am Geriatr Soc 59: 1596-1601, 2011

[38]Navarro-Gonzalez JF, Donate-Correa J, Muros de FM, Perez-Hernandez H, Martinez-Sanz R, Mora-Fernandez C: Reduced Klotho is associated with the presence and severity of coronary artery disease. Heart 100: 34-40, 2014

[39]Lim K, Lu TS, Molostvov G, Lee C, Lam FT, Zehnder D, Hsiao LL: Vascular Klotho deficiency potentiates the development of human artery calcification and mediates resistance to fibroblast growth factor 23. Circulation 125: 2243-2255, 2012

Fig.1: Correlation coefficient between serum level of soluble klotho (s-klotho) and fibroblast growth factor-23 (FGF-23) {r = -0.717, 95% confidence interval -0.806 to-0.598, p = 0.0001}

Fig.2: Correlation coefficient between serum level of soluble klotho (s-klotho) and parathyroid hormone (PTH) {r = -0.484, 95% confidence interval -0.630 to -0.306, p = 0.0001}

Fig.3: Correlation coefficient between serum level of soluble klotho (s-klotho) and serum calcium (Ca) level {r = 0.294, confidence interval 0.0903 to 0.474, p = 0.0054} Fig.4: Correlation coefficient between serum level of soluble klotho (s-klotho) and serum phosphate (Ps) level {r = -0.548, 95% confidence interval -0.680 to -0.383, p = 0.0001}

Fig.5: Correlation coefficient between soluble klotho (s-klotho) and left ventricular ejection fraction (LVEF) {r = 0.392, 95% confidence interval 0.199 to 0.556, p = 0.0002}

Table 1: Demographic characteristics of the studied patients and control group.

Variable Studied 88 patients Control group P value

Age(years) 58.6±19.3 53.3±16.2 0.1920

Gender(M/F) 51/37(57.95%/4 2.05%) 17/11(60.7%/39. 3%) 0.9997

BMI(g/m2) 24.6±5.2 26.2±5.6 0.1666

SBP(mm/Hg) 124.6±15.5 119.2±11.3 0.0913

DBP(mm/Hg) 85.7±25.4 80.4±19.1 0.3121

Mean BP(mm/Hg) 98.7±28.8 93.4±22.6 0.3756

Duration of 59.5±35.4 - -

dialysis(months)

Anuria 30(34.1%) - -

Aetiology of ESRD

DM 33(37.5%)

HTN 27(30.7%)

GN 15(17%)

CIN 5(5.7%)

SLE 3(3.4%)

APKD 2(2.3%)

Unknown 3(3.4%)

BMI, body mass index; Mean BP, mean blood pressure; ESRD, end-stage renal

disease; DM, diabetes mellitus; HTN, hypertention; GN, glomeulonephritis; CIN,

chronic interstitial nephritis; SLE, systemic lupus erythematosus; APKD, adult polycystic kidney disease

Table 2: Laboratory parameters of the studied patients and control group

Variables All Control P value Group I(s- Group II(s- P value

patients( group(n=2 klotho<476 klotho>476

n=88) 8) pg/ml,n=44) pg/ml,n=44)

S.creatinine 7.9±1.8 0.82±0.3 0.0001 7.5±1.2 8.3±3.1 0.1141

(mg/dl)

S.Ca (mg/dl) 8.4±1.0 8.5±0.6 0.6178 8.12±0.8 8.7±1.3 0.0136

S.Ps (mg/dl) 1.9±1.1 0.93±0.2 0.0001 2.1±0.5 1.8±0.6 0.0126

S.PTH (pg/ml) 71.7±25. 31.23±23. 0.0001 76.9±21.3 67.1±24.5 0.0484

RRF(ml/min/1.73 2.1±1.5 2.4±1.8 3.1±1.3 00395

Kt/V 1.56±0.6 1.46±0.4 1.62±0.5 0.1011

T.cholesterol 154.4±16 122.3±14. 0.0001 151.4±17.4 148.3±17.3 0.4043

(mg/dl) .4 6 0.0052 124.4±36.8 122.2±36.6 0.7793

LDL-C (mg/dl) 123.3±37 101.5±27. 0.0001 117.7±22.3 115.3±19.2 0.5899

TGs (mg/dl) .3 8

116.5±20 .5 91.7±16.3

Hb (g/dl) 11.2±1.5 13.8±1.2 0.0001 10.8±1.5 11.2±1.4 0.1994

Bl.glucose 136.7±26 118.6±21. 0.0013 132.1±27.3 141.4±25.8 0.1024

(mg/dl) .4 3

C-RP (mg/l) 16.1±4.2 0.71±0.4 0.0001 20.9±5.1 17.3±6.2 0.0038

S.albumin (g/dl) 3.8±0.7 4.2±0.6 0.0075 3.7±0.4 3.9±0.9 0.1815

S.uric acid (mg/dl) 6.8±1.0 5.7±1.2 0.0001 6.7±0.7 7.1±1.4 0.0937

S-klotho (pg/ml) 477.9±76 .2 863.7±261 .8 0.0001 427.8±46.2 535.4±61.4 0.0001

S.FGF-23 (pg/ml) 60.5±17. 6 35.8±13.9 0.0001 67.8±24.2 50.3±11.2 0.0001

S.PTH, serum parathyroid hormone; S.Ca, serum calcium; S.Ps, serum phosphorus; RRF, residual renal function; T.cholesterol, total cholesterol; LDL-C, low density lipoprotein-cholesterol; TGs, triglycerides; C-RP, c-reactive protein; s-klotho, soluble klotho; S .FGF-23, serum fibroblast growth factor-23

Table 3: Echocardiographic findings ,CIMT and CAD of the studied patients and control group

Variable All ^ patients(n= 88) Control group(n=2 8) P value Group 1(s- klotho<474,n= 44) Group 11(s- klotho>474,n= 44) P value

LVM(g) 193.4±22.3 112.7±23. 6 0.000 1 198.6±26.3 188.2±18.3 0.0341

LVMI(g/m2) 110.4±21.5 66.4±21.3 0.000 1 120.7±23.2 110.2±18.6 0.0215

LVEF(%) 49.6±10.2 58.6±6.3 0.000 1 45.9±10.3 55.2±9.3 0.0001

CIMT(mm) 0.99±0.19 0.78±0.23 0.000 1 1.17±0.23 0.93±0.13 0.0001

CAD 58(65.9%) - 36(81.1%) 22(50.0%) 0.0044

LVM, left ventricular mass; LVMI, left ventricular mass index; LVEF, left ventricular ejection fraction; CIMT, carotid intima-media thickness; CAD, coronary artery disease

Table 4: Multi-variate linear regression analysis of the predictive factors for CIMT,LVM,LVMI,LVEF and CAD.

Variables

s-klotho

-0.213

-0.184

-0.123

0.251 0.04

0.342 0.05

FGF-23 0.132 0.114 0.134 -0.162 0.451

0.06 0.07 0.07 0.06 0.06

T.cholesterol 0.075 0.033 0.124 0.0038 0.312

0.13 0.16 0.08 0.31 0.09

Tiglycerides 0.046 0.113 0.031 0.0059 ^ 0.123

0.15 0.09 0.21 0.11 0.11

Bl.glucose 0.012 0.026 0.0168 0.0147 0.412

0.23 0.14 0,26 0.09 0.06

Adjusted for age,gender,mean blood pressure,and duration of dialysis. CIMT, carotid intima-media thickness; LVM, left ventricular mass; LVMI, left ventricular mass index; LVEF, left ventricular ejection fraction; CAD, coronary artery disease; s-klotho, soluble klotho; FGF-23, fibroblast growth factor-23; T.cholesterol, total cholesterol; Bl.glucose, blood glucose

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Fig.l: Correlation coefficient between serosa level of soluble Idotho (s-klotho) and

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Fibroblast growth factor-23 (FGF-23) {r = -0.717, 95% confidence interval -0.806 to-0.59S:p = 0.0001}

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Fig.2: Correlation coefficient between serum level of soluble klotho (s-ldotho) and

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Fig.3: Correlation coefficient between serum level of soluble klotho (s-klotho) and

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