Republic of Iraq/Kurdistan Regional Government Ministry of Higher Education & Scientific Research University of Sulaimani/College of Medicine Department of Physiology
The Role of Fibroblast Growth Factor-23 as a Clinical Biomarker in End Stage Renal Disease Patients on Hemodialysis in Sulaimani City A THESIS SUBMITTED TO THE COLLEGE OF MEDICINE/UNIVERSITY OF SULAIMANI IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN PHYSIOLOGY
Prepared by
Dr. Shahram Hama Ali Aziz M.B.Ch.B
Supervised by
Dr. Kawa Hussein Amin M.B.Ch.B C.A.B.M
January 2017
Supervisor’s Certification
I certify that this thesis was carried out under my supervision at the University of Sulaimani/College of Medicine in partial fulfillment of the requirements for the degree of Master of Science in Physiology.
Supervisor Dr. Kawa Hussein Amin Lecturer, M.B.ch.B, C.A.B.M Faculty of Medical Sciences/School of Medicine University of Sulaimani
Quotation
الر ِح ِيم من َّ ِب ْس ِم هللاِ َّ الر ْح ِ اء أَ ِخي ِه َكذَ ِل َك ِك ْدنَا ع ِ ع ِ اء أَ ِخي ِه ث ُ َّم ا ْستَ ْخ َر َج َها ِم ْن ِو َ فَ َبدَأَ ِبأ َ ْو ِع َيتِ ِه ْم قَ ْب َل ِو َ ِين ْال َم ِل ِك ِإ ََّّل أَ ْن يَشَا َء َّ َّللاُ ن َْرفَ ُع دَ َر َجا ٍ ت ِليُو ُ س َ ف َما َكانَ ِليَأ ْ ُخذَ أَخَاهُ فِي د ِ ع ِليم َم ْن نَشَا ُء َوفَ ْوقَ ُك ِِّل ذِي ِع ْل ٍم َ صدَقَ هللاُ العَظيم َ
Dedication
This work is dedicated to:
My dear parents My beloved wife My adored son My dear sisters
Acknowledgement
I would like to express my deepest gratitude to the (University of Sulaimani), (Ministry of Higher Education & Scientific Research), and (Consultative Council of Kurdistan Regional Government) for giving me the opportunity and confirming my rights in starting my post-graduate study. I also owe a debt of gratitude to all the lecturers and personals in the departments
of
(physiology,
Biochemistry,
Community
Medicine,
Microbiology, Higher Education, Scientific Research and ethics) in University of Sulaimani for helping me obtain the necessary skills and requirements for conducting a research, and to the (Sulaimani Dialysis Center) for giving me the permission to conduct my research there. I must also thank my thesis supervisor (Dr. Kawa Hussein Amin) and all my family members for supporting me during the entire length of this work. A special thank you goes to (Prof. Dr. Ari Sami), (Prof. Dr. Kamal Ahmed Said), (Dr. Kosar Ali Murad), (Ms. Aveen), (The Honorable Chief Justice Shwan Muhialdin Ali), (The Honorable Justice Dr. Sardar Yasin Hamad Amin), (The Honorable Justice Dr. Osman Yasin Ali), (The Honorable Justice Star Sofi Hameed), (Mr. Counsel Sami Kamal Saeed), (Ms. Rukhosh), (Mr. Dana), (Dr. Nasreen abdulrahim wafi) ,(Dr. Shirwan Hama Salah Omer), (Dr. Shirwan), (Mr. Anwar), (Ms. Rukhosh), (Mr. Bahzad Omar Ali), (Ms. Mriam), (Dr. Sarhang), (Dr. Shaho A. Ezzaddin) and (Mr. Najmadeen).
Abstract Background: Fibroblast Growth Factor 23 (FGF23) is a bone derived circulating hormone that markedly increases in end stage renal disease (ESRD) patients where high concentration of this hormone has been found to be associated with increased risks for having a poor Phosphate (PO4) homeostasis, more severe anemia and lower dialysis adequacy. Objectives: This study aims to determine the association between FGF23 with Phosphate homeostasis, hemoglobin, dialysis vintage and adequacy (Kt/V) in the patients with ESRD. Subjects and Methods: In this Case-Control study, 52 ESRD patients on regular hemodialysis for at least 6 months in Sulaimani dialysis center and 26 healthy subjects were randomly enrolled. For both groups; blood pressure, hemoglobin, serum urea, creatinine, Phosphorus, Calcium, Sodium, intact FGF23, parathyroid hormone (PTH) and 25-hydroxy vitamin D were measured. Kt/V values of the patients were also calculated. Results: FGF23 was markedly elevated in the patients compared to the controls by 87 folds. It was positively correlated with Phosphorus (P: <.001), PTH (P: <.001) and dialysis vintage (P: .001) but was negatively correlated with 25-hydroxy vitamin D (P: .01), Calcium (P: .05) and Kt/V (P: .02) in the patients. FGF23 was not correlated to hemoglobin level in the patients. Conclusions: FGF23 is a useful clinical biomarker in end stage renal disease patients where high level of this hormone is associated with poor Phosphate homeostasis, lower dialysis adequacy (Kt/V) and longer dialysis vintage however its association with severity of anemia is vague. ii
CONTENTS Page no. Abstract ........................................................................................................... ii List of Tables ........................................................................................................................ viii List of Figures.......................................................................................................................... ix List of Abbreviations ......................................................................................xi
CHAPTERS I. Introduction 1.1. Overview ........................................................................................ 1 1.2. Objectives ....................................................................................... 3
II. Literature Review 2.1. Fibroblast growth factor 23 (FGF23) ............................................. 4 2.1.1. Structure and biochemical properties ............................... 4 2.1.2. Mechanism of action ......................................................... 5 2.1.3. Physiological Function...................................................... 6 2.1.3.A. FGF23 functions in the kidneys.......................... 7 2.1.3.B. FGF23 functions in the intestine......................... 8 2.1.3.C. FGF23 functions in the parathyroid gland .......... 8
iii
CONTENTS (continued) Page no. 2.1.3.D. FGF23 functions in other organs ........................ 8 2.1.4. Regulation of FGF23 level ............................................... 9 2.1.4.A. Systemic factors .................................................. 9 2.1.4.B. Local factors......................................................11 2.2. Klotho ...........................................................................................11 2.3. Diseases associated with dysregulation of FGF23 .......................12 2.4. Chronic kidney disease (CKD) ....................................................12 2.4.1. Definition and stages.......................................................12 2.4.2. Epidemiology ..................................................................13 2.5. Complications of chronic kidney disease .....................................14 2.5.1. Anemia ............................................................................14 2.5.2. Chronic kidney disease–mineral and bone disorder .......15 2.5.2. A. Increased FGF23 level .....................................17 2.5.2.B. Hyperphosphatemia ..........................................20 2.5.2.C. Hypocalcemia ...................................................22 2.5.2.D. Vitamin D Deficiency .......................................22 2.5.2.E. Secondary hyperparathyroidism (SHPT) ..........23 2.6. Treatment of chronic kidney disease............................................25 2.7. Dialysis .........................................................................................27 iv
CONTENTS (continued) Page no. 2.8. Dialysis Adequacy........................................................................28
III. Subjects and Methods 3.1 Study design ..................................................................................30 3.2. The case group..............................................................................30 3.2.1. Inclusion criteria .............................................................30 3.2.2. Exclusion criteria ............................................................30 3.3. The control group .........................................................................31 3.4. Study settings ...............................................................................31 3.5. Ethical considerations ..................................................................31 3.6. Instruments and procedures ..........................................................32 3.7. Statistical analysis ........................................................................35
IV. Results 4.1. Participants’ demographics characteristics ..................................37 4.2. Participants’ clinical characteristics .............................................37 4.3. Participants’ laboratory characteristics ........................................39 4.4. Medical treatment in the patients .................................................41
v
CONTENTS (continued) Page no. 4.5. Comparison between the participants according to their clinical and laboratory characteristics (Cases Vs. Controls) ...........................41 4.6. Serum FGF23 level in the hemodialysis patients.........................43 4.7. Factors associated with serum FGF23 level in the patients .........45 4.8. FGF23 and demographic features of the patients ........................46 4.9. FGF23 and etiology of chronic kidney disease ............................47 4.10. FGF23 and Phosphate homeostasis in the patients ....................49 4.10.1. FGF23 and serum inorganic Phosphorus in the patients ......................................................................................49 4.10.2. FGF23 and secondary hyperparathyroidism in the patients ......................................................................................51 4.10.3. FGF23 and Vitamin D statues in the patients ...............53 4.10.4. FGF23 and serum total Calcium in the patients ...........55 4.10.5. FGF23 level as a predictor for poor Phosphate homeostasis in hemodialysis patients .......................................56 4.11. FGF23 and anemia in the patients ..............................................58 4.12. FGF23 and dialysis adequacy (Kt/V) .........................................60 4.13. FGF23 and dialysis vintage ........................................................62
vi
CONTENTS (continued) Page no.
V. Discussion 5.1. Discussion ....................................................................................64 5.2. Limitations of the study................................................................72 5.3. Strength of the study ....................................................................72
VI. Conclusion and Recommendations 6.1. Conclusions ..................................................................................73 6.2. Recommendations ........................................................................73
REFERENCES.........................................................................................74
APPENDIX ................................................................................................96
vii
LIST OF TABLES
Table
Title
Page no.
4.1
Distribution of the participants according to their general demographic characteristics
37
4.2
Distribution of the participants according to their clinical characteristics
38
4.3
Distribution of the participants according to their measured laboratory characteristics
39
4.4
Comparison of clinical and laboratory characteristic of the cases and the controls with the statistical difference between them Clinical and laboratory characteristics of the hemodialysis patients stratified according to their serum intact FGF23 concentration and the statistical difference between them Correlations of FGF23 with other measured parameters in the patients according to Spearman’s correlation test
42
Evaluation of factors that are associated with FGF23 Log10 value in the patients according to multivariate regression analysis Difference in FGF23 concentration according to the demographic features of the hemodialysis patients and the statistical difference between them Difference in FGF23Log10 values in the hemodialysis patients according to the three main etiologies of CKD and the statistical difference between them using ANOVA test Characteristics of the area under the curve according to receiver operating characteristic curve analysis
46
4.5
4.6 4.7
4.8
4.9
4.10
viii
44
45
47
48
57
LIST OF FIGURES
Figure
Title
Page no.
3.1
Changes in the color of the samples inside the 96-Well plate of the human parathyroid ELISA Kit during running the assay Changes in the color of the samples inside the 96-Well plates of the human intact FGF23 and 25hydroxycholecalciferol ELISA kits during running the assays Distribution of the hemodialysis patients according on their CKD etiology Difference in FGF23 concentration between the hemodialysis patients according to their serum Phosphorus concentration Correlation between serum inorganic Phosphorus and serum intact FGF23 levels in the hemodialysis patients according to Spearman’s correlation test Difference in FGF23 concentration between the hemodialysis patients with controlled secondary hyperparathyroidism and hemodialysis patients with uncontrolled secondary hyperparathyroidism Correlation between serum intact parathyroid hormone and serum intact FGF23 levels in the hemodialysis patients according to Spearman’s correlation test Difference in FGF23 concentration between the hemodialysis patients according to their Vitamin D statues
34
3.2
4.1 4.2
4.3
4.4
4.5
4.6
ix
34
48 49
50
51
52
53
LIST OF FIGURES (continued) Figure
Title
Page no.
4.7
Correlation between serum 25-hydroxy vitamin D and serum intact FGF23 levels in the hemodialysis patients according to Spearman’s correlation test Difference in FGF23 concentration between the hemodialysis patients according to their Calcium concentration Correlation between serum total Calcium and serum intact FGF23 levels in the hemodialysis patients according to Spearman’s correlation test Receiver operating characteristic (ROC) curve of intact FGF23 with poor Phosphate homeostasis as status variable Difference in FGF23 concentration between the hemodialysis patients according to the severity of their anemia Correlation between hemoglobin and serum intact FGF23 levels in the hemodialysis patients according to Spearman’s correlation test Difference in FGF23 concentration between the hemodialysis patients according to their dialysis adequacy (Kt/V) Correlation between dialysis adequacy (Kt/V) and serum intact FGF23 levels in the hemodialysis patients according to Spearman’s correlation test Difference in FGF23 concentration between the hemodialysis patients according to their dialysis vintage Correlation between dialysis vintage and serum intact FGF23 levels in the hemodialysis patients according to Spearman’s correlation test
54
4.8
4.9
4.10
4.11
4.12
4.13
4.14
4.15
4.16
x
55 56 57 58 59 60 61 62 63
List of Abbreviations
Abbreviations
Meanings
1,25-dihydroxyvitamin D
1,25-dihydroxycholecalciferol or Calcitriol
1α-hydroxylase
25-hydroxyvitamin D3 1-alpha hydroxylase
25-hydroxyvitamin D
25-hydroxycholecalciferol or Calcifediol
24,25-dihydroxyvitamin D ANOVA test
24,25-Dihydroxycholecalciferol or (24R)-hydroxycalcidiol Analysis of variance test
Ca
Calcium
CaSR
Calcium-sensing receptor
CKD
Chronic kidney disease
CKD-MBD
Chronic kidney disease - Mineral and bone disorder Cardiovascular system
CVS CYP24A1 gene
DNA
Cytochrome P450 family 24 subfamily A member 1 gene Cytochrome P450 family 27 subfamily B member 1 gene Duration of time from the first day of starting dialysis Deoxyribonucleic acid
EPO
Erythropoietin
ESRD
End stage renal disease
CYP27B1 gene Dialysis vintage
xi
List of Abbreviations (continued)
Abbreviations
Meanings
FGF15
Fibroblast growth factor 15
FGF19
Fibroblast growth factor 19
FGF21
Fibroblast growth factor 21
FGF23
Fibroblast growth factor 23
FGFR
Fibroblast growth factor receptor
FGFRs
Fibroblast growth factor receptors
g/dl
Gram per deciliter
GFR
Glomerular filtration rate
HD
Hemodialysis
IQR
Interquartile range
kg
Kilogram
Kt/V
Pseudo-dimensionless number used to quantify the dialysis adequacy A small peptide which is a member of the cathelicidin family of antimicrobial peptides with amino acid sequence:
LL37
(LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES)
mEq/L
Milliequivalent per liter
mg/dl
Milligram per deciliter
xii
List of Abbreviations (continued)
Abbreviations ml/min/1.73 m2
Meanings
mmHg
Milliliter per minute per average adult body surface area Millimeter of mercury
mRNA
Messenger Ribonucleic acid
n
Sample size
ng/ml
Nano gram per milliliter
NPT2a
Pct.
Sodium-Dependent Phosphate Transport Protein 2A Sodium-Dependent Phosphate Transport Protein 2B Sodium-Dependent Phosphate Transport Protein 2C Percentage
pg/ml
Pico gram per milliliter
Pi PO4
Inorganic Phosphate or Inorganic Phosphorus Phosphate
PTH
Parathyroid hormone
SD
Standard deviation
SHPT
Secondary hyperparathyroidism
TRPV5
Transient receptor potential cation channel subfamily V member 5
NPT2b NPT2c
xiii
List of Abbreviations (continued)
Abbreviations
Meanings
VDR
Vitamin D receptor
Wnt pathway
Wnt: A term composed of fusion between two words, first is "wg" derived from the Drosophila gene “wingless” and second is "int" derived from its mammalian homolog which is “proto-oncogene integration-1”. The Wnt pathways are a group of signal transduction pathways made of proteins that pass signals into a cell through cell surface receptors
xiv
CHAPTER I INTRODUCTION
INTRODUCTION
1.1. Introduction Fibroblast growth factor 23 (FGF23) is a bone-derived circulating hormone that is considered as one of the most important regulators of serum inorganic Phosphorus (Pi) and Vitamin D concentrations in the body and it has various direct and/or indirect effects on bone and mineral homeostasis (1,2)
. FGF23 regulates the Phosphate homeostasis by its direct inhibitory
action on certain types of Sodium dependent Phosphate co-transporters in the renal proximal tubules and it reduces vitamin D concentration directly through suppressing renal CYP27B1 and stimulating CYP24A1 gene activities (3-5). Changes in FGF23 concentration have been observed in several genetic and acquired diseases, the most common one of which is chronic Kidney disease (CKD) especially in its end stages where high concentration of FGF23 is found in the patient (6-8). In these patients, failure of the kidneys to excrete Phosphate gradually leads to a related series of events that could result in multiplication of FGF23 concentration for hundreds to thousands folds and eventually development of Chronic kidney disease-mineral and bone disorder (CKD-MBD), a disorder that has been linked with a very high risks of morbidity and mortality
(5)
. FGF23 is thought to be the earliest
biomarker for mineral and bone disease in chronic kidney disease patients 9)
(5,
and previous studies in dialysis patients have linked poorer Phosphate
homeostasis with higher FGF23 concentration
1
(10)
; they also indicated their
associations with lower vitamin D level and secondary hyperparathyroidism (SHPT) level
(12)
(11)
. High FGF23 level has also been linked to lower hemoglobin
, lower residual renal function
lower dialysis adequacy
(13)
(10)
, longer dialysis vintage
(10)
and
. These studies clearly suggest FGF23 to be a
potentially valuable diagnostic and/or prognostic biomarker in chronic kidney disease patients. Other studies and clinical researches have raised the likelihood of targeting FGF23 with therapeutic agents in order to reduce the high morbidity and mortality rates that is currently seen in chronic kidney disease patients (14, 15). All these available data have motivated us to study the role of FGF23 as a clinical biomarker in our end stage renal disease (ESRD) patients in order to have the basic information about the situation when FGF23 enters the clinical practice as it is expected in the future whether it was as a diagnostic, a prognostic or a therapeutic tool (16,17).
2
1.2. Objectives The aims of the study are: 1. To evaluate the relationship between FGF23 and Phosphate homeostasis in end stage renal disease patients on hemodialysis. 2. To assess any correlation between FGF23 and hemoglobin level in end stage renal disease patients on hemodialysis. 3. To determine any correlation between FGF23 and dialysis adequacy (Kt/V) in end stage renal disease patients on hemodialysis. 4. To demonstrate any correlation between dialysis vintage and FGF23 concentration in end stage renal disease patients on hemodialysis.
3
CHAPTER II LITERATURE REVIEW
LITERATURE REVIEW
2.1. Fibroblast growth factor 23 (FGF23) 2.1.1. Structure and biochemical properties Fibroblast growth factor 23 is a bone-derived circulating peptide that is encoded by the FGF23 gene which is located on chromosome 12 in humans
(1)
. It is the most important regulator of serum Phosphate and
possibly calcitriol levels and it is required for maintenance of normal bone and mineral homeostasis (1,2). Fibroblast growth factors (FGFs) comprise a family of 22 members (there is no FGF15 in humans) grouped into 7 subfamilies
(2)
. They have numerous effects on development, repair,
metabolism and various endocrine signaling pathways (2,5). FGF23 belong to the FGF19 subfamily alongside FGF19 and FGF21 (5)
. This subfamily has a unique structure that enables them to avoid
capturing in the extracellular matrices and this allows them to function as endocrine factors (5). On the other hand this characteristic will reduce their affinity for FGF receptors (FGFRs) which has been shown to be weak (5). Even though the FGFRs are widespread in different organs, FGF23 activity was detected only in certain tissues including kidney, parathyroid gland, and pituitary gland, suggesting the existence of an essential cofactor to induce downstream signaling
(2,5)
. After binding proteins to FGF23 in renal
homogenate was analyzed, the major protein was found to be Klotho
(8)
.
Klotho alone was not sufficient for FGF23 signaling, it was shown to work as a co-receptor for FGF23 together with certain subtypes of FGFRs 4
(2,5)
.
Because FGF23 is produced by the bone, circulates in serum and acts on the kidney to regulate serum Phosphate and calcitriol levels, therefore it is considered to be a hormone rather than a local cytokine like other classical members of FGF family (8). Structurally, FGF23 was the 23rd member of the FGF family to be discovered (1). DNA sequencing indicates that FGF23 is a protein with 251 amino acids (8) and a molecular mass of near 30 Kilo-Daltons (5). FGF23 is proteolytically processed between arginine 179 and serine 180 by proprotein convertase (furin) to generate smaller amino and Carboxyl-terminal fragments
(8)
. The biological significance of these fragments is not fully
understood and they are considered to be inactive which means that the fulllength functional protein is deactivated via cleavage into amino and Carboxyl-terminal chains
(5,8)
. In bone, FGF23 is predominantly expressed
by osteocytes, and to a lesser extent by osteoprogenitor cells, osteoblasts, cementoblasts, odontoblasts, and chondrocytes (18). FGF23 is also expressed in the bone marrow, ventrolateral thalamic nucleus, thymus and lymph nodes however their exact contribution to the circulating FGF23 levels is not clear (19). Apart from its secretion, bones are also responsible for many gene products and local factors that regulate the expression and elimination of FGF23 such as sclerostin, metalloendopeptidase homolog PEX, dentin matrix protein 1 and matrix extracellular phosphoglycoprotein, Therefore this hormone is regarded as a bone hormone (19). 2.1.2. Mechanism of action Klotho binds to multiple FGFR to increases their affinity to FGF23 and to define the tissue specificity of FGF23 effects (19). It interacts with one
5
of the four FGF receptors with heparin sulfate proteoglycans as a co-factor to form a ternary FGF:FGFR: Heparin Sulfate Proteoglycans-signaling complex that is required for receptor activation and mediation of FGF23 actions
(19)
. After this, they will stimulate the downstream signaling
molecules including early growth response element-1 and induce phosphorylation of FGF receptor substrate-2a, extracellular signal-regulated kinase (ERK), P38 mitogen-activated protein kinases, Jun N-terminal kinase, and Protein kinase B (Akt) (4). The expression of Klotho is limited to certain tissues such the distal convoluted tubule and to a lower extent the parathyroid and pituitary glands, heart and few others which might explain the tissue restricted physiologic actions of FGF23
(5)
. High FGF23 concentration under pathological
conditions such as in CKD exerts its non-specific effects on various organs independent of Klotho through other pathways such as (Calcineurin- nuclear factor of activated T-cells pathway), FGF23 can bind to FGFRs with low affinity in the absence of Klotho in these situations (20-22). In mice, it was found that FGF23 has a half-life of around 20 minutes and this was prolonged in certain disease such as CKD (18). Further studies indicated a longer half-life of circulating FGF23 in human (around 1 hour) (18)
. After cloning of FGF23, several enzyme-linked immunosorbent assays
for FGF23 have been established (18). 2.1.3. Physiological Function Identification of FGF23 has reformed our understanding of mineral metabolism in general and Phosphate homeostasis specifically. FGF23 was initially discovered in 2000 by Yamashita and his colleagues through attempts to identify the predicted existence of phosphate-regulating 6
hormones ‘phosphatonins’ (1). Our understanding of the functions of FGF23 is obtained from our knowledge of the distribution of the Klotho-FGFR complexes in the body and various studies both in vivo and vitro demonstrating the cell type and organ-specific actions of FGF23 (19). These studies indicate that FGF23 mainly targets the kidney, parathyroid gland, choroid plexus, and the pituitary gland (19). Among these, the kidney is the principal target for FGF23 where FGF23 is considered as one of the main regulators of Phosphate reabsorption and production of vitamin D (19,23). 2.1.3.A. FGF23 functions in the kidneys: In kidneys, molecular studies indicated that FGF23 decreases serum Phosphate level through reduction and internalization of the Sodium dependent Phosphate co-transporters (NPT2a and NPT2c) in the renal proximal tubules
(23,24)
. Other studies in
normal humans have demonstrated the presence of circulating FGF23 in plasma, which is consistent with the physiological role of FGF23 in Phosphate regulation (25). It should be mentioned that the highest expression of Klotho-FGFR complexes is in the distal tubule whereas the major biologic effects of FGF23 occurs in the proximal tubule. It is thought that either the small amount of Klotho-FGFR in proximal tubules is sufficient to mediate the biological activity of FGF23 or FGF23 may stimulate distal tubules to release certain paracrine factors that affect proximal tubular function (distal to proximal feedback loop) (19, 26). Studies have shown that FGF23 affects vitamin D level prior to the reduction of serum Phosphate
(8)
. FGF23 has two effects to reduce serum
vitamin D level: first it directly suppresses renal 1 α-hydroxylase, leading to decreased conversion of 25-hydroxyvitamin D to its active metabolite Calcitriol (1,25-dihydroxyvitamin D). Second is to directly enhance vitamin D degradation through stimulation of 25-hydroxyvitamin D-24-hydroxylase 7
expression which converts 1,25-dihydroxyvitamin D into more hydrophilic metabolites with less activity which is 24,25-dihydroxyvitamin D (8). FGF23 is also involved in Sodium homeostasis through directly regulating the membrane abundance of the sodium chloride cotransporters in distal tubules and hence increasing Sodium reabsorption
(27)
. In addition,
high FGF23 has been linked with increased renin-angiotensin system activity therefore FGF23 may have a role in increasing blood volume and pressure and so kidney damage
(27)
. FGF23 also influences Calcium (Ca)
homeostasis by increasing the distal tubular Calcium absorption via increasing the expression of epithelial Calcium channels (TRPV5) in a Klotho dependent fashion (28). 2.1.3.B. FGF23 functions in the intestine: In the intestine, FGF23 reduces Phosphate absorption indirectly through vitamin D dependent decrease in the Sodium
dependent
Phosphate
co-transporters
(NPT2b)
expression.
Subsequently, this effect will also decrease Calcium absorption (29). 2.1.3.C. FGF23 functions in the parathyroid gland: In parathyroid gland, FGF23 directly reduces Parathyroid hormone (PTH) transcription and secretion (30). This is suggested by the presence of Klotho-FGFR complexes in the gland and the effects of FGF23 injection on it
(30)
. In vitro studies
indicated that FGF23 acts directly on the parathyroid gland to activate the Mitogen-activated protein kinase pathway and decrease PTH secretion (31). 2.1.3.D. FGF23 functions in other organs: In brain, the function of FGF23 in choroids plexus is not completely explored but there are evidences for FGF23 to play a role in PO4 regulation in cerebrospinal fluid (32). In Pituitary gland, there are no Klotho-FGFR complexes and the role of FGF23 is vague yet there are evidences of severe growth retardation in FGF23 null mice and 8
upregulation of early response gene expression in response to acute FGF23 administration to mice
(19)
. In bones, there is no expression of Klotho and
there are no strong evidences for direct effect of FGF23 (5). Changes in bone mineralization have been observed in patients with abnormal FGF23 level which could be indirect through its effect to reduce serum vitamin D (33). 2.1.4. Regulation of FGF23 level Both systemic and local bone-derived factors regulate FGF23 expression in osteocytes via both transcriptional and posttranslational mechanisms (2). 2.1.4.A. Systemic factors: Major systemic factors identified to regulate serum FGF23 concentration are Calcitriol and serum Phosphate (2). The most rapid stimulus for FGF23 expression both in vitro and in vivo is Calcitriol producing a response in serum FGF23 level within 3–4 h after intravenous injection (24). This effect mediated through the vitamin D receptor (VDR) because studies in VDR-null mice did not show an increase in FGF23 levels after vitamin D injection
(32)
. They also identified vitamin D-responsive
element on the FGF23 promoter which is required for calcitriol stimulation of FGF23 production
(32)
. In addition to its direct effect, studies have
indicated that Vitamin D regulate FGF23 indirectly through extracellular signaling pathways, which are mediated by leptin and Interleukin-6
(31)
.
Phosphate loading in mice increases FGF23 levels even though changes in serum Phosphate level do not appear to have an immediate effect on FGF23 production and the response to alterations in dietary Phosphate intake usually appears after a lag time of up to 1 week
(26).
Nevertheless, in vivo
studies have shown that both Phosphate and calcitriol independently increase FGF23 level (34). 9
Parathyroid hormone also stimulates FGF23 expression and secretion by bones. Studies in patients with primary hyperparathyroidism, patients with secondary hyperparathyroidism during CKD and patients with Jansen’s metaphyseal chondrodysplasia have demonstrated such association between PTH and FGF23
(31,35,36)
. FGF23 is also dependent on Klotho to induce
FGFR signaling and mediate its biological activity (55). This is evidenced by Klotho knockout mice who exhibit nearly an identical biochemical phenotype similar to FGF23-knockout mice even with the high circulatory FGF23 levels (37). In addition to the transmembrane Klotho mediating the effect of FGF23, the circulating form can increase FGF23 production and thereby suppress Phosphate reabsorption (38). Acid-base statue may also affect FGF23 production where increased FGF23 mRNA expression has been shown in animal tissues exposed to an acid medium (39). Another factor is Iron statues where small increase in FGF23 level after intravenous iron administration in patients on hemodialysis has been observed
(40)
. Hypoxia may also increase FGF23
production independently from serum iron level; this has been observed in cells grown under low oxygen tension and in rats exposed to hypoxia (41). Estrogen also upregulates FGF23 expression both in vivo studies in animals and in vitro studies in human (42). Calcium also affects FGF23 regulation. In mice, Calcium deficiency decreases serum FGF23 level, which is thought to be a part of a negative feedback mechanism to stimulate vitamin D expression
(43)
. Various other factors have been shown to have effects on
FGF23 expression such as glucocorticoids (5), insulin (44), aldosterone (45) and serum Magnesium (46).
10
2.1.4.B. Local factors: In addition to systemic factors, recent studies have suggested a role for local bone-derived factors, bone remodeling and mutations in genes that regulate bone mineralization in direct or indirect regulation of FGF23 production
(2,5)
. Some of these factors include
Phosphate-regulating neutral endopeptidase X-linked, Dentin matrix acidic phosphoprotein
1
(2)
,
matrix
extracellular
phosphoglycoprotein,
Ectonucleotide pyrophosphatase/ phosphodiesterase family member 1, High molecular weight-Fibroblast growth factor-2
(5)
, secreted frizzled related
protein-4 (34), ratio of inorganic phosphate to pyrophosphate in bone (31). Further studies are required to identify new factors and explain the exact mechanisms by which different systemic and local factors affect FGF23 secretion and function.
2.2. Klotho Klotho is an enzyme that was originally discovered as an anti-aging factor (20). Klotho exists as a membrane-bound (type 1 membrane protein) that is required for FGF23-mediated receptor activation and also has two circulating forms (5). The trans-membrane Klotho is mainly expressed in the kidney and in the choroid plexus (4). It is also found in the parathyroid gland, sinoatrial cells of the heart and several other organs
(5)
while the soluble
forms are presenting in both plasma and urine (20). Circulating Klotho has been noticed to increase FGF23 level
(38)
.
Oppositely high FGF23 reduces Klotho gene transcription in the kidney and decreases both membrane and soluble proteins (47, 48). This has been observed in CKD patients (48).
11
2.3. Diseases associated with dysregulation of FGF23 Abnormal regulation of serum FGF23 is found in a number of genetic and acquired disorders (7). High FGF23 level has been found in disorders such as in autosomal dominant/recessive/X-linked hypophosphatemic rickets, tumor-induced osteomalacia and chronic kidney disease while low FGF23 has been noticed in others such as familial tumoral calcinosis (7,8). Generally, excessive FGF23 production results in hypophosphatemia, decreased Calcitriol, elevated PTH and rickets/osteomalacia while FGF23 deficiency results in hyperphosphatemia, elevated calcitriol, suppressed PTH and soft tissue calcification
(8,19)
. In addition, abnormalities in glucose
homeostasis, growth, thymic function (19), erythropoiesis (49), dyslipidemia (18) and age-related changes
(19)
have been linked to abnormal FGF23
concentration in various in vivo and in vitro studies. Numerous studies have confirmed an association between higher FGF23 concentration and increased morbidity and mortality (18,50). Further studies in these disorders will reinforce previous findings and recognize new elements concerned with FGF23 metabolism in the hope of finding superior diagnostic and therapeutic strategies in management of these disorders. Among these disorders, chronic kidney disease is the most common one that is associated with secondary increased FGF23 level (8,18).
2.4. Chronic kidney disease (CKD) 2.4.1. Definition and stages Chronic kidney disease is a progressive loss in kidney function over a period of months or years. It is present when a patient’s glomerular filtration rate (GFR) remains below 60 ml/min/1.73 m2 for more than 3 months which 12
represents loss of half or more of the adult level of normal kidney function, irrespective of the presence or absence of kidney damage
(51,52)
. CKD is
classified into five stages based on severity (51): Stage 1: there is slightly decrease renal function with GFR remain normal or relatively high, typically (≥90 ml/min/1.73 m2) Stage 2: GFR is (60–89 ml/min/1.73 m2). Stage 3: GFR is (30–59 ml/min/1.73 m2). Stage 4: GFR is (15–29 ml/min/1.73 m2). Stage 5: GFR is (<15 ml/min/1.73 m2) The 5th stage is often called end-stage renal disease (ESRD) where there is a total and permanent kidney failure. Here the diseased kidneys cannot get the body rid of the accumulated fluid and waste products any longer thus the patient will be needing renal replacement therapy which typically is renal transplant or dialysis as a holding measure until transplant (51). 2.4.2. Epidemiology CKD is a growing epidemic public health problem that affects millions of people worldwide, estimated to be 5–15% of the world population
(51,53)
. CKD patients have an increased risk of morbidity and
mortality compared to the general population (53). According to the United States renal data system (USRDS), the number of incidence and prevalence of CKD, ESRD, dialysis and kidney transplant patients is continuously growing with each year (54). CKD and especially ESRD has a huge burden on both community and healthcare system, according to the same data, in 2012 the total cost of ESRD patients in United States was $28.6 billion, accounting for 5.6% of the Medicare budget costs (54). All these alerting data 13
regarding CKD requires a special attention and calls for new studies in order to improve the currently available diagnostic and therapeutic measures.
2.5. Complications of chronic kidney disease With decrease in renal function, the patients suffer from numerous complications such as fluid-electrolyte disturbance, acid-base imbalance, anemia, skeletal and cardiovascular disorders, and others
(51,53)
. In the
following sections we will explain how abnormalities in serum Phosphate and FGF23 levels play roles in the development of some of these disorders. 2.5.1. Anemia There is a high prevalence of anemia among CKD patients with its prevalence and severity being increased with deterioration in renal function, reaching its peak in dialysis patients where it is presents in approximately three quarters of the patients
(53)
. The cause of anemia in CKD is
multifactorial but mostly it is due to reduction in erythropoietin (EPO) production by the diseased kidneys (53). Other factors include malnutrition, bleeding tendencies, abnormal renin-angiotensin-aldosterone activities, increased inflammation, low serum Calcitriol levels, bone marrow suppression etc. (53,55). Anemia is one of the risk factors for cardiovascular disease and congestive heart failure thereby death in CKD patients (53,56,57). Abnormalities in FGF23 and Phosphate metabolism have been linked to development of anemia in CKD patients. In previous studies, high serum FGF23 level was correlated with low hemoglobin level in patients with CKD stages 3, 4 (56) and in patients on dialysis (12). It was shown that FGF23 level above 200 pg/ml is a major risk factor for having a low hemoglobin level (56). In an animal study, it has been shown that FGF23 has a direct inhibitory 14
effect on pre and postnatal erythropoiesis which is through its effect on generation and differentiation of hematopoietic stem cells
(49)
. In mice,
inactivation of FGF23 gene has been shown to result in severe increase in EPO and Hypoxia-inducible factor mRNA synthesis in kidney, bone marrow and liver while injection of FGF23 resulted in rapid decrease in serum EPO level (49). The exact action of FGF23 on erythropoiesis and development of anemia in CKD is not clear
(49)
. FGF23 may indirectly promote anemia
development by inhibition of vitamin D
(58)
. Calcitriol has been found to
have an effect in up-regulation of EPO receptor expression and stimulation of the erythroid precursor cells (59). Studies in normal population (58), early CKD (60) and ESRD (61) patients have shown such correlations. In addition to FGF23, circulating Klotho has been shown to regulate prenatal and postnatal hematopoiesis and play a role in hematopoietic development which is thought to be due to its direct effects on hematopoietic stem cell differentiation and erythroid cell generation and maturation
(62)
.
High FGF23 level may further stimulate anemia progression by suppressing Klotho expression thereby inhibiting its hematopoietic actions (48). 2.5.2. Chronic kidney disease–mineral and bone disorder Chronic kidney disease–mineral and bone disorder (CKD-MBD) is one of the complications of CKD which is identified as a systemic disorder of mineral and bone metabolism
(63)
. CKD-MBD manifested by
three forms of abnormalities and the patients may exhibit one, two or all the three forms. The first one is disorders in mineral metabolism where there is a derangement of Ca, PO4 and various hormones associated with their homeostasis such as FGF23, PTH and Vitamin D. Second one is skeletal remodeling disorders where there is a derangement in mineralization, bone 15
volume, bone growth and bone turnover. The third one is calcification of cardiovascular and soft tissues
(63,64)
. CKD-MBD links kidney and bone
diseases with cardiovascular complications and contributes to the high morbidity and mortality rate seen in CKD patients (21,63). From the second stage of CKD, there is a reduction in renal excretion of Phosphate. This results in an increased secretion of FGF23 in an attempt to maintain normal serum Phosphate level
(64)
. At first, FGF23 has an
adaptive role where it stimulates phosphaturia and helps in preventing the accumulation of Phosphate. However, with more decline in renal function and mass, the increased FGF23 cannot maintain normal serum Phosphate level (64). Human and animal studies have demonstrated that the increased FGF23 level is the earliest detectable alteration in CKD-MBD patients (5,9). Increased FGF23 level inhibits vitamin D activation. Low Calcitriol level directly acts on the parathyroid gland via VDR and indirectly through reduction in serum Calcium to promote PTH secretion. At the same time, high serum Phosphate level stimulates parathyroid gland to increase PTH secretion directly and indirectly through lowering serum Calcium and Calcitriol levels. Patients with ESRD usually suffer from high FGF23 and PO4 levels, low Ca and Calcitriol levels, high PTH level which eventually leads to SHPT and parathyroid hyperplasia with increased resistance to the action of PTH on bone. These derangements in mineral and hormonal balance with changes in bone remodeling over a period of months or years will lead to a chain of events that promote the development of various skeletal and cardiovascular abnormalities and ultimately death (21,24,53,63-65). It should be mentioned that there are new hypotheses for the development of CKD-MBD such as the direct effect of kidney injury and the 16
role of molecules that inhibit the canonical Wnt pathway (21). However, these concepts require further evidences to be recognized and get under the scope of large scale researches. Various mineral, hormonal, skeletal and vascular changes occur in patients with CKD-MBD, some of these changes include: 2.5.2.A. Increased FGF23 level: According to recent studies, increased FGF23 precedes changes in Ca, PO4 and PTH as one of the earliest detectable biomarkers of the CKD-MBD (5, 9). FGF23 concentration increases with early decline in GFR, usually when it is around (70-90 ml/min/1.73 m2) (9)
. FGF23 increases in response to two main events: First, decreased GFR
and accumulation of Phosphate in the blood. Second, decreased expression of Klotho and type 1 FGFR in the kidney and the parathyroid gland which results in a decreased responsiveness to FGF23 in these organs. In addition, a reduction in kidney mass and the number of intact functioning nephrons decrease the ability to excrete the accumulated FGF23 (24,47,66). On the other hand, some researchers have proposed other hypotheses to explain the high FGF23 level seen in CKD patients such as Iron deficiency anemia (67), direct kidney injury and activation of Wnt pathway
(21)
, inflammation
(68)
and
effects of drugs (69). However the first two mentioned events remain the most common and acceptable explanations (21,24,52,53). In early stages of CKD, increased FGF23 represents an adaptive response which exerts a protective effect to maintain normal serum Phosphate level (66). With progression of CKD, FGF23 level is markedly elevated reaching 1000s times above the upper limit of the normal range (17,66)
. The PTH further stimulates FGF23 release from bone directly and
indirectly through alterations in extracellular matrix factors in order to increase renal Phosphate excretion (31). However, due to reduction in renal 17
mass and function with decreased expression of Klotho, elevated FGF23 levels cannot enhance urinary Phosphate excretion any longer and FGF23 level continues to build up in the blood
(17)
. Thus in late stages of CKD,
FGF23 cannot maintain its protective effect to prevent hyperphosphatemia while various adverse off-target effects of FGF23 appearing in other organs (18,66)
. High FGF23 level results in decreased serum Calcitriol, low serum
Calcium and increased PTH secretion thereby SHPT
(31,64)
. The role of
FGF23 in development of these abnormalities has been observed in rats with experimental CKD which have shown improvement in these derangements following administration of FGF23 antibodies (70). FGF23 has been shown to suppress PTH mRNA expression in vitro and decreases serum PTH in vivo under normal physiological conditions (5,30)
. In fact, it is thought that high FGF23 detected before PTH in early
CKD because of the inhibitory effect of FGF23 on parathyroid gland
(24)
.
Therefore, it is logical to think that the high level of FGF23 in ESRD should be a protective measure against the development of SHPT. However, studies have found a strong correlation between elevated FGF23 levels and the severity of SHPT in CKD patients which is thought to be caused by down regulation of Klotho and FGFR in parathyroid gland which leads to unresponsiveness to the high FGF23 level (5,31). Furthermore, studies have found that in late stages of CKD, PTH and bone remodeling are the main regulators of FGF23 and the driving force behind amplification of FGF23 level (26). It should be noted that one of the effects of FGF23 is to suppress the Klotho gene transcription in the kidney therefore; high FGF23 may lead to a vicious cycle increasing its own level (24,47). This decline in Klotho is
18
detected early in CKD patients usually from stage 2 and it includes both membrane and circulating forms (47,48). Due to various effects on serum Phosphate, Calcium, vitamin D, PTH and Klotho as well as its off-target effects on various other organs in CKD, FGF23 contributes to most of the complication of CKD-MBD and death (53,70)
. FGF23 represent a sensitive prognostic marker in CKD patients
especially those in early stages
(71)
. FGF23 independently predicts the
progression of CKD and its predictive value has been surpassed only by proteinuria (72). In a study in elderly subjects with normal or mildly impaired renal function, it was found that FGF23 is a predictor of fracture risk
(73)
.
Another study has shown FGF23 to be associated with infection and sepsis by inhibiting the synthesis of the antimicrobial molecule LL37 in peripheral blood monocytes (18) and by direct action on polymorphonuclear leukocytes and macrophages (74) which might explain the increased risk for infection in CKD patients, especially those on dialysis between FGF23 and inflammation
(75)
(18)
. Moreover, an association
, fat mass and dyslipidemia
(76)
, and
insulin resistance (77) in CKD has been found. Some studies even predicted a role for FGF23 in increased risk for stroke and intracranial hemorrhage (78), and impairment of learning and memory in CKD patients due to its direct action on hippocampal neurons (79). In renal transplant patients, high FGF23 has been linked to increased risk for transplant rejection (50). FGF23 have been found to provide prognostic information regarding treatment programs for CKD patients such as efficiency of Vitamin D therapy and prevention of refractory hyperparathyroidism (80). A large number of studies have established a link between FGF23 level and cardiovascular disorders and a stronger association was detected in 19
CKD patients (81). FGF23 has been associated with increased distal sodium reabsorption, effective circulating, volume expansion, hypertension, and cardiac hypertrophy through its action to regulate sodium-chloride transporter in the distal convoluted tubule (27). FGF23 has a direct effect in development of left ventricular hypertrophy by activation of the Calcineurinnuclear factor of activated T-cells pathway in cardiac muscle cells (21,22). It also has been shown to predict the risk for aortic calcification independently (82)
. Other studies have linked high FGF23 level to atrial fibrillation (83), heart
failure
(84)
, endothelial dysfunction
(85)
and progression of vascular
calcification (86). High FGF23 causes a decrease in soluble Klotho level in addition to its transmembrane form
(47,48)
. Many studies have indicated a link between
Klotho deficiency and vascular calcification, vascular stiffness and mortality (87)
. Moreover, circulating Klotho has a wide range of systemic functions (5),
it is safe to assume that FGF23-induced Klotho deficiency in CKD patients will promote the development of some of these adverse systemic complications. FGF23 may also stimulate the development of vascular complications indirectly through stimulating vitamin D deficiency or SHPT (88,89) (90)
. Finally, FGF23 is an independent risk factor for mortality in normal
, CKD (91), ESRD (91) and kidney transplant (50) patients.
2.5.2.B. Hyperphosphatemia: Hyperphosphatemia (Pi > 4.5 mg/dL) is an electrolyte disturbance in which there is an abnormally high level of Phosphate in the blood. It is one of the manifestations of CKD-MBD (92). From the second stage of CKD, difficulties in renal Phosphate excretion appears as the amount of functioning renal tissue cannot excrete the extra Phosphate reaching blood from intestinal absorption and serum Phosphate 20
begin to build up
(53,64,92)
. However, Severe hyperphosphatemia does not
appear until the (GFR is < 25 ml/min/1.73 m2) (92). Hyperphosphatemia through its effects on Calcium, vitamin D, PTH and FGF23 over a period of time will affects bone mineralization and remodeling leading to development of bone abnormalities and fracture at the same time it promotes calcification in soft tissue and cardiovascular system (CVS) which results in various disorders in heart, vasculature and different other organs (53,64,92). In addition to these effects, researchers have found that hyperphosphatemia can be toxic to some cells such as chondrocytes and osteoblasts, leading to apoptosis in these cells
(93)
. This toxic effect may
contribute further to the bone damage seen In CKD patients. Studies have shown that hyperphosphatemia is an independent risk factor for further deterioration in residual renal unit anatomy and function (94), SHPT (64), bone disorder (92), homodynamic disturbance (95), cardiovascular disorders such as vascular calcification
(96)
, calciphylaxis
(97)
, myocardial infarction
(98)
and
mortality (99). A high PO4 level above 5 mg/dl is associated with higher rate of mortality compared to CKD patients with near-normal serum PO4 levels (100)
. However, recent studies suggest that Serum PO4 can be misleading in
risk assessment of CKD patients especially in early stages when there is normophosphatemia. They indicate that serum FGF23 level might be a better biomarker than serum PO4 level for risk assessment in these patients (4,101). Phosphate can be maintained in near normal level in patients with CKD and most of its complications can be delayed if the treatment which consists of dietary restriction of Phosphate and using Phosphate binders started early in the course of the disease in addition to later dialysis modifications in ESRD patients
(92)
. However, neglecting high serum
21
Phosphate in CKD patients can result in changes in skeletal, soft and (64)
cardiovascular tissues that can be difficult if not impossible to treat
.
Despite all the measures to control serum Phosphate level, many ESRD patients on dialysis continue to have a persistent hyperphosphatemia (92). 2.5.2.C. Hypocalcemia: Hypocalcemia (serum Ca < 8.5 mg/dL) is an electrolyte disturbance in which there is an abnormally low level of Calcium in the blood. It is one of manifestations of CKD-MBD
(102)
.
Serum Calcium is low because of several factors such as the precipitation of Calcium ions with extra Phosphate ions found in the blood, low Calcitriol level and the resistance of PTH action on bones
(21,53,64)
. Low serums
Calcium alongside other factors contribute to the increased PTH secretion and SHPT
(64,66)
. After a period of months or years these effects will
contribute to various bone, soft tissue and vascular abnormalities (21,53,64). If uncontrolled, Sever Hypocalcemia may cause life-threatening dysrhythmias and cardiac dysfunction, neuromuscular irritability such ass tetany or paresthesia, abdominal cramps and others (103). Hypocalcemia is a risk factor for SHPT thereby fracture and metabolic bone disorders (53) and has been linked with rapid progression of CKD
(104)
, cardiovascular
abnormalities such as left ventricular dysfunction (105), lower mean arterial pressure, higher left ventricular mass index (104), congestive heart failure (106) and death (107). CKD patients and even ESRD patients may have a normal or high serum Ca level due to usage of Calcium or vitamin D supplementations. However, these agents may increase the risk for vascular calcification (108). 2.5.2.D. Vitamin D Deficiency: Calcitriol level decreases in patients with CKD due to decrease in megalin expression in the proximal tubule which
22
decreases the uptake of 25-hydroxyvitamin D in the kidney, also megalin function declines due to damages from low molecular weight proteinuria (109)
. In addition, high FGF23 level inhibits vitamin D activation and promote
its catabolism (26). Beside Calcitriol, 25-hydroxyvitamin D is also decreased in CKD patients via different mechanisms such as leakage of Vitamin D binding protein (DBP) bound to 25-hydroxyvitamin D with proteinuria (109). Calcitriol deficiency leads to a reduced intestinal PO4 and Ca absorption which will stimulate PTH secretion (109,110). It also reduces the expression of VDR in various tissues including in parathyroid gland which will lead to the development of resistance to calcitriol-mediated regulation of PTH secretion and thereby increased PTH secretion (111). Furthermore, it stimulates skeletal resistance to the calcemic actions of PTH and these effects all together contribute to the development of SHPT (112). Vitamin D deficiency in early stages is an adaptive response against hyperphosphatemia rather than a true vitamin D deficiency state (26). However, in late stages after disruption of parathyroid-kidney-bone axis, there is a severe vitamin D deficiency which promotes abnormal skeletal development, and vascular complications (26). Studies in CKD patients have found that the risk for progression into ESRD is higher in those with lower vitamin D level (113) and low vitamin D status has been linked with poor outcomes including development of skeletal (114)
and cardiovascular disorders and mortality (115). Furthermore, vitamin D
has many non-classical roles in various organs in the body and its decline in CKD will disrupt the normal metabolism in most of these organs (116). 2.5.2.E.
Secondary
hyperparathyroidism
(SHPT):
Secondary
hyperparathyroidism refers to the excessive secretion of parathyroid hormone by the parathyroid glands and the associated hyperplasia of the 23
glands. It is a one of the manifestations of CKD-MBD
(117)
. The most
common cause of SHPT is chronic kidney disease where it represents a maladaptive process developing in response to the decreased renal function and the associated mineral and hormonal abnormalities (117). PTH secretion increase through several mechanisms. First, there is an increased Phosphate and FGF23 concentration which will lead to a reduced serum Calcium and vitamin D, these effects stimulate PTH secretion. Additionally, Phosphate is thought to be directly stimulating the parathyroid gland to increase PTH secretion. Secondly, along with these abnormalities, there is also a downregulation of VDR and Calcium-sensing receptor (CaSR) on the parathyroid gland even in early stages which produces an inappropriate response from the gland to Ca and Calcitriol abnormalities. Third, high PTH causes a downregulation of the PTH receptors on the bone which leads to an increased resistance to the calcemic action of PTH on bone (64,117,118). All these changes will lead to a vicious cycle that continuously increasing PTH release and promoting parathyroid hyperplasia (64,117,118). Among factors that contribute to SHPT, hypocalcemia is thought to be primary risk factor (119). PTH has been shown to stimulate FGF23 expression via both cyclic adenosine monophosphate protein kinase A and Wnt pathways (31). In fact, SHPT and bone remodeling is thought to be the main driving force behind high FGF23 level seen in patients with ESRD (26). Thus, high PTH multiplies the magnitude of abnormalities found in mineral metabolism, skeleton, soft tissue and CVS (53,64). Continuous stimulation of the gland by various factors stimulates it to undergo diffuse polyclonal hyperplasia followed by monoclonal nodular hyperplasia
(120)
. With this hyperplasia, expression of
both VDR and CaSR are decreased which will result in an inappropriate 24
response of the gland to mineral and hormonal imbalances in addition to reduced capacity of the gland to respond to treatment (120). Various studies have linked SHPT to abnormalities in skeleton including bone mineralization, excess bone resorption and high fracture risk (117,118)
. Apart from being associated with high FGF23, hyperphosphatemia,
hypocalcemia and low vitamin D levels
(64,117)
, SHPT alone has been
correlated to calcification in soft tissue and CVS
(121)
, Calciphylaxis
(122)
,
cardiovascular diseases in CKD patients such as left ventricular hypertrophy (123)
and increased mortality rate in CKD patients (124). If neglected, SHPT
progresses to tertiary hyperparathyroidism with development of autonomous PTH secretion in the gland which is usually out of the control of Calcium and vitamin D regulation. Here, despite vigorous treatment including kidney transplantation, the parathyroid gland will continue to secret excessive amounts of PTH which will produce a hypercalcemia rather than hypocalcemia in the patients but at a heavy price of severe damage to skeleton and soft tissue and vascular calcification. Surgical intervention is usually mandatory to correct the problem (117).
2.6. Treatment of chronic kidney disease The aim is to treat the original disease, relief symptoms and improve quality of life, prevent complications and halt its progressions into ESRD. Care should be given to the nutritional status, hypertension, anemia, hyperlipidemia, fluid & electrolyte balance, acid-base status, bone health and all the risk factors for cardiovascular disease (51,125) which is considered to be the main cause of mortality in CKD patients (126). After progression to ESRD, the only treatment is dialysis until renal transplant (if feasible) (125).
25
Despite the successful use of lifestyle changes, medications and dialysis strategies in treating CKD patients, the morbidity and mortality in patients with CKD remain very high (53,126,127). Average life expectancy in dialysis patients is 5-10 years (128). Even though renal replacement therapy can prolong life, the quality of life will be severely affected. Various drugs are in production that may offer additional protection and potentially reduce the high morbidity and mortality rates
(129)
. Because FGF23 is a strong
predictor of CKD progression and is linked with numerous cardiovascular complications, considering its diagnostic and/or prognostic potentials during clinical assessment of CKD patients and targeting it with FGF23 lowering agents could have a significant role in reducing the extent of the CKD complications and in prolonging the lives of the patients in the future (14, 15). Several approaches have been shown to reduce FGF23 level indirectly such as dietary changes, Phosphate restriction, Phosphate binders, Cinacalcet, parathyroidectomy and new therapeutic agents such as Etelcalcetide
(18)
. In addition, there are new drugs that are currently under
clinical trials to target FGF23 or its receptors (14, 15). However, to date limited data is available for their effects and their potential side effects. On the other hand there are some draw backs that keep FGF23 from entering clinical practice including the absence of a solid cut-off point and the need for better standardization of the current assays (16). The only way to overcome these obstacles is through further researches and clinical trials with the goal of reducing the CKD burden on the community and providing a better quality of live to the patients through utilizing FGF23 and other agents as new tools in their management.
26
2.7. Dialysis Dialysis is a process for removing waste and excess water from the blood of patients with ESRD and sometimes patients with acute kidney injury (128,130). In ESRD, it is considered a holding measure until kidney transplantation but sometimes it is the only supportive measure that is available when transplantation is not feasible
(130)
. Dialysis machines are
considered artificial kidneys that can perform most of the kidney functions however, they are not perfect since they cannot perform the endocrine functions of the kidney and they have their own complications (131). There are two main types of dialysis which are hemodialysis (HD) and peritoneal dialysis (PD)
(131)
. In HD, the machine removes waste
products and excessive water from the blood outside of the body on the basis of diffusion of solutes and ultrafiltration of fluid between the blood and dialyzer fluid across a semi-permeable membrane (130). In peritoneal dialysis, the patient’s peritoneum is used as a membrane across which the fluid and solutes can be exchanged with the blood (130). It is a less common form of dialysis and has its own advantages and disadvantages compared to HD (131). Dialysis is indicated usually when the patients lose around 85-90% of kidney function with GFR <15 ml/min/1.73m2
(128)
. However, it may be
indicated in some patients before reaching this GFR based on the clinical signs and symptoms (125,130). Usually the dialysis session lasts for about 4 hours, three times per week but in practice it depends on the kidney function and the extent of the built-up waste products in the body, also depends on the size of the patients, type of the dialysis, weight gain, patient’s signs and symptoms, and many other factors (128). 27
2.8. Dialysis Adequacy Dialysis Adequacy is a measure of how well the dialysis is working to determine the number and duration of dialysis that is required to keep the patient healthy and functional
(132)
. There are several methods for
determining dialysis adequacy which are depending on measuring the clearance of solutes that accumulate in the body. Unfortunately none of them are perfect (133). The most common forms of dialysis adequacy assessment depend on urea clearance and are the Kt/V and the Urea reduction ratio (URR) (133). Urea clearance is used because it is easy and cheap to measure, urea concentrations correlate with symptoms and its clearance rate correlates with improvements in the patient’s morbidity and mortality and it is retained in parallel with other significant toxins (133). Kt/V was developed by Frank Gotch and John Sargent and it is based on urea kinetic assuming that urea is generated at a constant rate by protein metabolism and is removed at a constant rate by residual renal function
(133)
. Kt/V is derived from the Urea
reduction ratio, however; it is more accurate than it in measuring the amount of urea removed during dialysis because it also considers the amount of urea removed with excess fluid
(134)
. K in Kt/v represents the dialyzer’s urea
clearance rate; t is duration of dialysis while V is volume of urea distribution which is roughly equal to patient's total body water (134). In Patient on regular HD, dialysis adequacy should be assessed monthly (134). The targeted Kt/V value for patients on three sessions a week is 1.4 with minimum accepted value of 1.2 (133). It should be noted that the values are different for patients receiving more or less than three sessions a week or on peritoneal dialysis
(135)
. Along the dialysis adequacy, the
healthcare personals should take into account other factors such as volume 28
and blood pressure control, mineral metabolism, clinical symptoms, anemia, nutritional state, quality of life and others in assessing patient’s wellbeing and establishing therapeutic measures
(133)
. There are several factors that
affect dialysis adequacy such as dialyzer surface area, length of treatment, blood flow rate, dialysate flow rate, type of vascular access, nutritional status,
correct
blood
sampling
procedures,
missed
treatments,
cardiopulmonary recirculation, cooperation from patients, residual renal function (133). Also there are several clues for inadequate dialysis that are seen in dialysis patients such as continuous tiredness, weakness, gastrointestinal disturbance, loss of weight, severe decrease in blood count and general uremic signs
(136)
. When dialysis has been shown to be
inadequate in a patient, several modifications can be done in an attempt to improve it such as changes in blood flow rate, dialyzer mass transfer-area coefficient, dialysis time and frequency, dialysate flow, needle size, ensuring adequate anticoagulation, dialytic modality and others (137). Assessment of dialysis adequacy can help in recognizing those patients that are at higher risk for developing poor outcomes (135). In some studies, plasma FGF23 level was observed to be correlated with lower dialysis adequacy (13) which means that effective dialysis is one of the factors that determine the concentration of FGF23 in ESRD patients. On the other hand, this possible correlation between the two can be a clue for health-care personals in identifying the patients that are in need for dialysis modification in order to improve their dialysis adequacy after measuring their FGF23 concentrations. Confirmation of these findings and utilizing it in clinical practice will boost the clinical significance of FGF23 even further.
29
CHAPTER III SUBJECTS AND METHODS
SUBJECTS AND METHODS
3.1. Study design This study was a Case-Control observational study.
3.2. The case group Fifty two randomly selected patients with ESRD who were on regular three sessions/week hemodialysis therapy for 3-4 hours on “Gambro dialysis machines” in the Sulaimani dialysis center in the period between 1st of April and 1st of August 2016 were enrolled in the study. 3.2.1. Inclusion criteria 1. End stage renal disease patients on hemodialysis therapy from Sulaimani dialysis center. 2. Hemodialysis duration of three to four hours per session. 3. Hemodialysis frequency of three sessions per week. 4. Dialysis vintage more than 6 months. 5. Age above 18 years old. 3.2.2. Exclusion criteria 1. History of parathyroidectomy. 2. History of any chronic mineral and/or musculoskeletal disorders unrelated to CKD. 30
3. History of malignancy. 4. History of Chronic viral infection.
3.3. The control group Twenty six healthy adult subjects with no history of chronic kidney disease nor abnormal renal function (high urea or creatinine) who were matched for age, sex and race with the patients were also enrolled in the study during the same time period (1st of April and 1st of August 2016).
3.4. Study settings This study was conducted in Sulaimani Dialysis Center in Sulaimani city in the period between 1st of April and 1st of August 2016. The center offers dialysis therapy for over 100 ESRD patients monthly residing in Sulaimani city and the surrounding areas. The laboratory investigations (except FGF23, 25-hydroxyvitamin D and PTH assays) were done in the same center’s laboratory department. The serum samples were kept frozen in -40 °C inside aliquots in Sulaimani Blood Bank center in Sulaimani city. The remaining assays (FGF23, 25-hydroxyvitamin D and PTH) were performed in Shahid Hadi Center’s laboratory department in Sulaimani city.
3.5. Ethical considerations This study was approved by (University of Sulaimani/ College of Medicine ethical committee) and all the procedures were conducted in accordance with the ethical principles of Helsinki declaration (6th revision in 2013). Verbal Informed consent was obtained from all the participants prior to their participation in the study. 31
3.6. Instruments and procedures A pre-tested questionnaire was assigned to obtain the necessary information from the participants through interviewing method. In the patient group, after explaining the nature and purpose of the study and obtaining their approval, pre-dialysis weight and mean blood pressure (using conventional Riester mercury sphygmomanometer in both of their arms in 45 degree position) were recorded prior to the hemodialysis sessions. Blood samples were collected via their arteriovenous fistula before starting their hemodialysis session in EDTA-tubes and in serum tubes. The patients were interviewed and necessary information was collected while they were undergoing their hemodialysis sessions. Ultrafiltration volume, blood flow rate were also recorded. Hemoglobin level was measured from the EDTAtubes using (Swelab Alpha hematology analyzer). The blood in serum tubes were centrifuged for 5 minutes at 2,822 relative centrifugal force in (Human Humax 4K centrifuge). Following centrifuging, the urea, creatinine, Calcium and Phosphorus levels were assessed using standard manual procedures for measuring these parameters and their resultant values were read using (Human Humalyzer 2000 spectrophotometer). Serum Sodium is also measured using (i-smart i-sens electrolyte analyzer). In the control group, after explaining the nature and purpose of the study and obtaining their approval, blood samples were taken via their median cubital vein and similar to the patients had their level of blood Urea, serum creatinine, serum Calcium, serum Phosphorus, serum Sodium and hemoglobin assessed with the same procedures in addition to recording weight, necessary medical history and their blood pressures using the same
32
type of sphygmomanometer. All the procedures mentioned above were done in the Sulaimani dialysis center. The remaining serums (around 2-3 ml) were transferred into aliquots, transported immediately into Sulaimani blood bank Center and kept frozen in -40 °C until the time for the remaining assays. Dialysis duration and post dialysis body weight for the patients were recorded and blood samples were again collected after the sessions to determine patient’s Kt/V values for dialysis adequacy using Medindia dialysis efficiency (Kt/V) calculator tool which calculates the Kt/V value according to the “Daugirdas” equation (138). For
determining
the
levels
of
FGF23,
vitamin
D
(25-
hydroxycholecalciferol) and PTH, we used human intact FGF23 ELISA Kit (Cat.No: MBS035508), human 25-hydroxycholecalciferol ELISA Kit (Cat.No: MBS019728) and human parathyroid ELISA Kit (Cat.No: MBS2505074),
respectively.
(MyBioSource,Inc.)
company.
They
were
Mybiosource
all
purchased
human
(FGF23,
from 25-
hydroxycholecalciferol and PTH) ELISA Kits employ the quantitative sandwich enzyme immunoassay technique with their Intra-assay and interassay coefficients of variation being <15%. FGF23 and PTH levels were expressed in pg/mL while 25-hydroxycholecalciferol level was expressed in ng/ml. The kits were run in Shahid Hadi Center’s laboratory and the manufacturer's instructions were followed to measure FGF23, 25hydroxycholecalciferol and PTH levels in the samples. The absorbance values of the samples in the plate wells were read using (Teco diagnostics TC84 automated spectrophotometer). Microsoft excel was used for determining the standard curves and necessary mathematical calculations
33
were performed in order to obtain the concentration of FGF23, PTH and vitamin D from their absorbance values. Figure 3.1:
Figure (3.1) shows changes in the color of the samples inside the 96-Well plate of the human parathyroid ELISA Kit during running the assay. Figure 3.2:
Figure (3.2) shows changes in the color of the samples inside the 96-Well plates of the human intact FGF23 and 25-hydroxycholecalciferol ELISA kits during running the assays. 34
3.7. Statistical analysis After data collection and prior to data entry and analysis, the variables of study were coded. Data entry performed via using an excel spreadsheet then the statistical analysis was performed by SPSS program, version 21 (IBM SPSS statistics package software program for statistical analysis). Normality of the data was tested using Shapiro-Wilk test. The data was demonstrated as mean ± Standard deviation (SD) for normally distributed variables or median ± Interquartile range (IQR) for non-normally distributed variables. The data was presented in tabular forms to show the distribution of the participants according to their general demographic and measured clinical and laboratory characteristics. Tabular forms was also used to demonstrate the comparison between the participants according to their clinical and laboratory characteristics (Cases Vs. Controls), the clinical and laboratory characteristics of the patients stratified according to their serum intact FGF23 concentration with the statistical difference between them, the correlations of FGF23 with other measured parameters in the patients according to Spearman’s correlation test, evaluation of factors that are associated with FGF23Log10 value in the patients according to multivariate regression analysis, the difference in FGF23 concentration according to the demographic features of the hemodialysis patients and the statistical difference between them, the difference in FGF23 Log10 values in the hemodialysis patients according to the three main etiologies of CKD and the statistical significant difference between them using (Analysis of variance) ANOVA test and the characteristics of the area under the curve according to receiver operating characteristic (ROC) curve analysis. 35
“Pie in 3-D” chart was used to shows the distribution of the hemodialysis patients according on their CKD etiology. “Clustered bars in 3D” was used to shows the difference in FGF23 concentration between the hemodialysis patients according to their Phosphate homeostasis, severity of their anemia and dialysis adequacy (Kt/V). “3-D Clustered Column” chart was used to shows the difference in FGF23 concentration between the hemodialysis patients according to their dialysis vintage. “Scatter with only markers” charts were used to shows the Correlation between serum intact iFGF23 with inorganic Phosphorus, serum intact parathyroid hormone, serum 25-hydroxy vitamin D, serum total Calcium, hemoglobin, dialysis adequacy (Kt/V) and dialysis vintage in the hemodialysis patients. Independent t test, Mann-Whitney U test and Analysis of variance (ANOVA) test were used to test the statistical difference between different groups regarding their FGF23 concentration or their FGF23 Log10 values. Spearman’s rho test was used to assess the correlation coefficient between FGF23 and other measured parameters and to evaluate the statistical significance of their correlation. Multivariate regression analysis was performed for FGF23 to determine the factors that independently predict its concentration. Because the FGF23 data contained extreme skewness, it is first transformed to logarithmic scale then the linear regression analysis was performed. Receiver operating characteristic (ROC) curve was generated for determination of predictive value of FGF23 concentration for Phosphate homeostasis in the hemodialysis patients. All the tests were two tailed and P value ≤ 0.05 was considered to be statistically significant.
36
CHAPTER IV RESULTS
RESULTS
4.1. Participants’ demographics characteristics The distribution of the 78 participants according to their general demographic characteristics is shown in table 4.1. The patients’ median age was 55 years ± 17 IQR (range: 26-87 years) while the control’s median age was 51.5 years ± 17.5 IQR (range: 29-67 years). Table 4.1: Table (4.1) shows the distribution of the participants according to their general demographic characteristics: Variables
Cases n: 52
Percentage
Controls n: 26
Percentage
26 26
50% 50%
13 13
50% 50%
16 10 51.5 ± 17.5 4 16 6
61.5% 38.5%
Sex Male Female Race Kurds 32 61.5% Arabs 20 38.5% 55 ± 17 Age (years ± IQR) Young 8 15.4% Middle 32 61.5% Old 12 23.1% *Abbreviations: n: Sample size. IQR: Interquartile range.
15.4% 61.5% 23.1%
4.2. Participants’ clinical characteristics The distribution of the participants according to their measured clinical characteristics is shown in table 4.2. 76% of the patients had high blood pressure compared to the controls. 83% of the patients had pre37
dialysis blood pressure above the pre-dialysis target range (130/80 mmHg). 38% of the patients reported having poor blood sugar control. Table 4.2: Table (4.2) shows the distribution of the participants according to their clinical characteristics: Variables
Cases n: 52
Pct.
Control n: 26
Pct.
Systolic blood Pressure ( <130 mmHg) (139) Within reference range Above reference range
11 41
21.1% 78.9%
14 12
53.8% 46.2%
Diastolic blood pressure ( <85 mmHg) (139) Within reference range Above reference range
12 40
23.1% 76.9%
16 10
61.5% 38.5%
Target blood pressure (140-142) < 130/80 mmHg < 140/90 mmHg
9 14
17.3% 26.9%
Dialysis adequacy (Kt/V) (1.4) (135)
1.53 ± 0.15 SD
-
Below target range Above target range Weight (kg) Pre-dialysis weight Post-dialysis weight Dialysis vintage (months)
10 42
Dialysis blood flow rate (ml/min)
260 ± 30 IQR
19.2% 80.8%
65 ± 13.5 IQR 62.5 ± 14.5 IQR 29 ± (28 IQR)
67.5 ± 16 IQR -
*Abbreviations: n: Sample size. Pct.: percentage. mmHg: millimeter mercury. SD: Standard deviation. IQR: Interquartile range. kg: kilogram. ml/min: milliliter per minute.
38
4.3. Participants’ laboratory characteristics The distribution of the participants according to their measured laboratory characteristics is shown in table 4.3. In the patients, median FGF23 concentration was 3146 pg/ml (Range: 493 to 14561 pg/ml) while in the control it was 36 pg/ml (range: 14 to 96 pg/ml). Table 4.3: Table (4.3) shows the distribution of the participants according to their measured laboratory characteristics: Variables Intact FGF23 (20-60 pg/ml) (17) Below reference range Within reference range Above reference range Inorganic Phosphorus (2.5-4.5 mg/dl) Below reference range Within reference range Above reference range Calcium (8.5-10.2 mg/dl) Below reference range Within reference range Above reference range Intact PTH (15-65 pg/ml) Below reference range Within reference range Above reference range Target intact PTH (150-450 pg/ml) (140,143) Below target range Within target range Above target range
Cases n: 52
Pct.
Control Pct. n: 26
0 0 52
0 0 100%
2 20 4
7.7% 76.9% 15.4%
0 18 34
0 34.7% 65.3%
0 22 4
0 84.6% 15.4%
22 28 2
42.3% 53.8% 3.9%
4 19 3
15.3% 73.1% 11.6%
0 0 52
0 0 100%
1 20 5
3.9% 76.9% 19.2%
5 33 14
9.6% 63.4% 26.9%
-
Abbreviations: n: Sample size. PTH: Parathyroid hormone. Pct.: percentage. mg/dl: milligram per deciliter. pg/ml: picogram per milliliter. ng/ml: nanograms per milliliter. mEq/L: milliequivalents per liter. g/dl: gram per deciliter.
39
Table 4.3 (continued): Table (4.3) shows the distribution of the participants according to their measured laboratory characteristics: Variables 25-hydroxy vitamin D (30-60 ng/ml) Below reference range Within reference range Above reference range Vitamin D statues (144) Normal concentration (>30 ng/ml) Insufficiency (16-29 ng/ml) Mild deficiency (5-15 ng/ml) Severe deficiency (<5 ng/ml) Serum Sodium (135-145 mEq/L) Below reference range Within reference range Above reference range Hemoglobin (Male 14-17.5 g/dl) (Female 12-15 g/dl) Below reference range Within reference range Above reference range Target hemoglobin (9-11.5 g/dl) (145) Below target range Within target range Above target range
Cases n: 52
Pct.
Control Pct. n: 26
46 6 0
88.5% 11.5% 0
17 9 0
6 25 21 0
11.5% 48.1% 40.4%
9 38 5
17.3% 73.1% 9.6%
3 22 1
11.6% 84.5% 1.9%
52 0 0
100% 0 0
10 14 2
38.5% 53.8% 7.7%
21 26 5
40.3% 50% 9.6%
65.4% 34.6% 0
-
-
Abbreviations: n: Sample size. PTH: Parathyroid hormone. Pct.: percentage. mg/dl: milligram per deciliter. pg/ml: picogram per milliliter. ng/ml: nanograms per milliliter. mEq/L: milliequivalents per liter. g/dl: gram per deciliter.
40
4.4. Medical treatment in the patients The antihypertensive medications were one or a combination of (Angiotensin Converting Enzyme Inhibitors and Calcium Channel Blockers). 65% of the diabetic patients reported using insulin ± their antidiabetic medications. All the patients reported following the recommended dietary restriction. All of them were receiving (synthetic Erythropoietin injections, Calcium-containing Phosphate binder tablets and vitamin D “αhydroxy vitamin D3” capsules). 63% of the patients reported receiving oral or parenteral Iron supplementations. There was no history of blood transfusion in the last 3 weeks.
4.5. Comparison between the participants according to their clinical and laboratory characteristics (Cases Vs. Controls) There were statistically significant differences between the cases and the control group regarding all their measured clinical and laboratory characteristics except for their serum Sodium concentration (P: .91). The average value for all the parameters in the patients were above or below the reference range except for serum Sodium concentration which was maintained within the normal range (135-145 mEq/L). Serum FGF23 concentration was significantly elevated in the patients compared to the control group by 87 folds. All the measured clinical and laboratory characteristic of the participants with the statistical differences between them using Independent t and Mann-Whitney U tests are shown in tables 4.4.
41
Table 4.4: Table (4.4) shows the comparison of clinical and laboratory characteristic of the cases and the controls with the statistical difference between them: Variable
Cases (Mean or median) n: 52
S.D or IQR
Controls (Mean or median) n: 26
S.D or
P value
IQR
Urea (mg/dl)
135.73
41.94 SD
12.04
4.02 SD
< 0.001
Calcium (mg/dl)
8.44
0.81 SD
9.15
0.79 SD
0.001
Phosphorus (mg/dl)
4.97
1.26 SD
3.48
0.78 SD
< 0.001
Sodium (mEq/L)
138.54
4.29 SD
138.77
3.65 SD
0.91
Creatinine (mg/dl)
5.25
3.15 IQR
0.93
0.38 IQR
< 0.001
Hemoglobin (g/dl)
9.5
2.7 IQR
13.25
3.08 IQR
< 0.001
FGF23 (pg/ml)
3146.0
2075 IQR
36.0
15.5 IQR
< 0.001
25-hydroxy vitamin D (ng/ml)
17.7
13.22 IQR
28.42
15.07 IQR
0.001
Parathyroid hormone (pg/ml)
361.5
240.8 IQR
43.68
35.51 IQR
< 0.001
Systolic blood Pressure (mmHg)
145.0
30.0 IQR
127.5
12.5 IQR
0.001
Diastolic blood pressure (mmHg)
95.0
15.0 IQR
87.5
10.0 IQR
< 0.001
*P value of ≤ 0.05 is considered to be statistically significant (Independent t and MannWhitney U test). Abbreviations: SD: Standard deviation. IQR: Interquartile range. n: Sample size. mg/dl: milligram per deciliter. pg/ml: picogram per milliliter. ng/ml: nanograms per milliliter. mEq/L: milliequivalents per liter. g/dl: gram per deciliter. mmHg: millimeter of mercury.
42
4.6. Serum FGF23 level in the hemodialysis patients The median FGF23 concentration was 3146 pg/ml. All the patients had high serum intact FGF23 concentration (range 493 to 14561 pg/ml) above the reference range (20 to 60 pg/ml) (17) and above the control group range (14 to 96 pg/ml). Only one patient had serum intact FGF23 level below the expected range for ESRD patients (500 to 50000 pg/ml) (17), the rest of the patients were within that expected range. The clinical and laboratory characteristic of the patients stratified according to their FGF23 concentration into patients with FGF23 concentration < the median values (3146 pg/ml) vs. patients with FGF23 concentration ≥ median value (3146 pg/ml) with the statistical differences between them are shown in table 4.5. There were statistically significant differences between the two groups in their Phosphorus (P: <.001), parathyroid hormone (P: <.001), creatinine (P: .02), dialysis vintage (P: .02) and dialysis adequacy (P: .04) according to Independent t and Mann-Whitney U tests.
43
Table 4.5: Table (4.5) shows the clinical and laboratory characteristics of the hemodialysis patients stratified according to their serum intact FGF23 concentration and the statistical difference between them: Variable
Mean or Median ± SD or IQR
P value
Group 1 (n:26)
Group 2 (n:26)
Urea (mg/dl)
128.92 ± 40.33 SD
142.54 ± 43.20 SD
0.25
Calcium (mg/dl)
8.62 ± 0.79 SD
8.26 ± 0.80 SD
0.11
Phosphorus (mg/dl)
4.10 ± 1.15 SD
5.84 ± 0.61 SD
< 0.001
Sodium (mEq/L)
138.81 ± 4.31 SD
138.27 ± 4.34 SD
0.66
Dialysis adequacy Kt/V
1.59 ± 0.16 SD
1.47 ± 0.11 SD
0.04
Creatinine (mg/dl)
4.5 ± 2.1 IQR
6.4 ± 2.7 IQR
0.02
Hemoglobin (g/dl)
9.9 ± 2.4 IQR
9 ± 2.3 IQR
0.12
25-hydroxyvitamin D (ng/ml) Parathyroid hormone (pg/ml) Systolic B.P mm. Hg
19.86 ± 13.01 IQR
17.6 ± 10.94 IQR
0.66
249.0 ± 154.3 IQR
445.8 ± 246.1 IQR
< 0.001
147.5 ± 31.3 IQR
145.0 ± 27.5 IQR
0.82
Diastolic B.P mm. Hg
97.5 ± 13.8 IQR
95.0 ± 13.8 IQR
0.78
Vintage (month)
18.0 ± 14.3 IQR
36.0 ± 16.5 IQR
0.02
*Group 1: Patients with FGF23 level < the median value (3146 pg/ml). Group 2: patients with FGF23 level ≥ the median value (3146 pg/ml). *P value of ≤ 0.05 is considered to be statistically significant (Independent t and MannWhitney tests). *Abbreviations: SD: Standard deviation. IQR: Interquartile range. n: Sample size. mg/dl: milligram per deciliter. pg/ml: picogram per milliliter. ng/ml: nanograms per milliliter. mEq/L: milliequivalents per liter. g/dl: gram per deciliter. mmHg: millimeter of mercury. 44
4.7. Factors associated with FGF23 level in the patients According to Spearman’s correlation test in the patients, FGF23 was positively correlated with Phosphorus (P: < .001), PTH (P: <.001) and dialysis vintage (P: .001) but it was negatively correlated to Calcium (P: .05), 25-hydroxyl vitamin D (P: .01) and dialysis adequacy Kt/V (P: .02). However no correlation was found between FGF23 and hemoglobin (P: 0.39). Correlations of FGF23 with other parameters in the patients are shown in table 4.6. Table 4.6: Table (4.6) shows the correlations of FGF23 with other measured parameters in the patients according to Spearman’s correlation test: Variable
Correlation coefficient (r)
P value
Urea
0.218
0.12
Creatinine
0.202
0.15
Calcium
- 0.265
0.05
Phosphorus
0.62
< 0.001
Sodium
0.035
0.81
Hemoglobin
- 0.121
0.39
25-hydroxyvitamin D
- 0.32
0.01
Parathyroid hormone
0.68
< 0.001
Dialysis adequacy Kt/V
- 0.41
0.02
Systolic blood pressure
0.028
0.84
Diastolic blood pressure
0.027
0.85
Dialysis vintage
0.5
0.001
*P value of ≤ 0.05 is considered to be statistically significant.
45
Multivariate regression analysis with serum Phosphorus, PTH, 25hydroxy vitamin D, Calcium, dialysis vintage and adequacy as independent variables revealed serum Phosphorus (P: <.001), PTH (P: .002) and Calcium (P: .05) as the factors that independently predict FGF23 (Log10) value in the patients. Evaluation of factors that are associated with FGF23 (Log10) value in the patients according to multivariate regression analysis is shown in table 4.7. Table 4.7: Table (4.7) shows evaluation of factors that are associated with FGF23 Log10 value in the patients according to multivariate regression analysis: Variable
Standardized regression coefficient (beta) P value
Phosphorus
0.093
< 0.001
Parathyroid hormone
0.001
0.002
25-hydroxyvitamin D
-0.005
0.08
Calcium
-0.055
0.05
Dialysis adequacy Kt/V
- 0.058
0.71
Dialysis vintage
0.002
0.14
*P value of ≤ 0.05 is considered to be statistically significant.
4.8. FGF23 and demographic features of the patients No statistically significant difference was found in FGF23 concentration based on the sex (P: .87) or race (P: .63) of the participants according to Mann-Whitney U test. No statistically significant difference was found in FGF23Log10 values based on the different age groups of the patients according to ANOVA test (P: .78). No statistically significant
46
correlation was found between FGF23 concentration and the age of the patients according to spearman’s correlation test (P: .44). Table 4.8: Table (4.8) shows the difference in FGF23 concentration according to the demographic features of the hemodialysis patients and the statistical difference between them: Variables
Median FGF23 (pg/ml) or Mean FGF23Log10
SD or IQR
P value
Sex Male Female
2925 3256
2873 IQR 1584 IQR
0.87
Race Kurds 3220 2546 IQR 0.63 Arabs 3071 1841 IQR Age (years ± IQR) Young (FGF23Log10) 3.38 0.27 SD 0.78 Middle (FGF23Log10) 3.4 0.31 SD Old (FGF23Log10) 3.46 0.3 SD *P value of ≤ 0.05 is considered to be statistically significant (Mann-Whitney U and ANOVA tests). Abbreviations: SD: Standard deviation. IQR: Interquartile range.
4.9. FGF23 and etiology of chronic kidney disease The etiology of CKD in the patients is shown in figure 4.1. Diabetic nephropathy was the most common cause while hypertensive nephropathy and glomerulonephritis were second and third respectively. This order was unaffected neither by race nor sex. No statistically significant differences were found regarding FGF23Log10 values among the three main etiology groups of CKD patients according to ANOVA test (P: .38) as shown in table 4.9. 47
Figure 4.1
Figure (4.1) shows the distribution of the hemodialysis patients according to their CKD etiology. Table 4:9: Table (4.9) shows the difference in FGF23Log10 values in the hemodialysis patients according to the three main etiologies of CKD and the statistical difference between them using ANOVA test: Etiology of CKD
FGF23Log10 value ± (SD)
Diabetics
Hypertensive
Glomerulo-
P
Nephropathy
Nephropathy
nephritis
value
3.28 ± (0.58)
3.2 ± (0.46)
3.45 ± (0.23)
0.23
*P value of ≤ 0.05 is considered to be statistically significant (ANOVA test). Abbreviations: CKD: Chronic kidney disease. SD: Standard deviation. 48
4.10. FGF23 and Phosphate homeostasis in the patients 4.10.1. FGF23 and serum inorganic Phosphorus in the patients 18 hemodialysis patients had their serum inorganic Phosphorus level within the normal range (Pi: 2.5 to 4.5 mg/dl) while 34 of them had hyperphosphatemia (Pi > 4.5 mg/dl). Patients with hyperphosphatemia had higher
FGF23
concentration
compared
to
the
patients
with
normophosphatemia as it is shown in figure 4.2 and there was a statistically significant difference between the two groups regarding their FGF23 concentration (P: <.001) according to Mann-Whitney U test. Figure 4.2:
Figure (4.2) shows the difference in FGF23 concentration between the hemodialysis patients according to their serum Phosphorus concentration. 49
Figure 4.3:
Figure (4.3) shows the Correlation between serum inorganic Phosphorus and serum intact FGF23 levels in the hemodialysis patients according to Spearman’s correlation test.
50
4.10.2. FGF23 and secondary hyperparathyroidism in the patients 38
hemodialysis
patients
had
controlled
secondary
hyperparathyroidism (PTH ≤ 150-450 pg/ml) (143) while 14 had uncontrolled secondary hyperparathyroidism (PTH > 450 pg/ml) uncontrolled
secondary
hyperparathyroidism
had
(143)
. Patients with
higher
FGF23
concentration compared to the patients with controlled secondary hyperparathyroidism as it is shown in figure 4.4 and there was a statistically significant difference between the two groups regarding their FGF23 concentration (P: <.001) according to Mann-Whitney U test. Figure 4.4:
Figure (4.4) shows the difference in FGF23 concentration between the hemodialysis patients with controlled secondary hyperparathyroidism and hemodialysis patients with uncontrolled secondary hyperparathyroidism. 51
Figure 4.5:
Figure (4.5) shows the Correlation between serum intact parathyroid hormone and serum intact FGF23 levels in the hemodialysis patients according to Spearman’s correlation test.
52
4.10.3. FGF23 and Vitamin D statues in the patients 6 of the hemodialysis patients had normal Vitamin D concentration (25-hydroxy vitamin D: 30-60 ng/ml)
(144)
, 25 patients had Vitamin D
insufficiency (25-hydroxy vitamin D: 16-29 ng/ml) (144), 21 of the patients had mild Vitamin D deficiency (25-hydroxy vitamin D: 5-15 ng/ml) (144). No patient had severe Vitamin D deficiency (25-hydroxy vitamin D: < 5 ng/ml) (144)
. Difference in FGF23 concentration between the 3 groups is shown in
figure 4.6. There was no statistically significant difference between the three groups regarding their FGF23log10 values according to ANOVA test (P: .26). Figure 4.6:
Figure (4.6) shows the difference in FGF23 concentration between the hemodialysis patients according to their Vitamin D statues. 53
Figure 4.7:
Figure (4.7) shows the Correlation between serum 25-hydroxy vitamin D and serum intact FGF23 levels in the hemodialysis patients according to Spearman’s correlation test.
54
4.10.4. FGF23 and serum total Calcium in the patients 22 hemodialysis patients had hypocalcemia (Ca: <8.5 mg/dl), 28 patients were normocalcemic (8.5-10.2 mg/dl) while 2 patients had hypercalcemia (Ca: > 10.2 mg/dl). Patients with hypocalcemia had higher FGF23 concentration compared to normocalcemic patients as shown in figure 4.8 however there was no statistically significant difference between the two groups regarding their FGF23 concentration according to MannWhitney U test (P: .1). Figure 4.8:
Figure (4.8) shows the difference in FGF23 concentration between the hemodialysis patients according to their Calcium concentration. 55
Figure 4.9:
Figure (4.9) shows the Correlation between serum total Calcium and serum intact FGF23 levels in the hemodialysis patients according to Spearman’s correlation test.
4.10.5. FGF23 level as a predictor for poor Phosphate homeostasis in hemodialysis patients The receiver operating characteristic (ROC) curve is shown in figure 4.10. According to the receiver operating characteristic curve analysis, FGF23 concentration above 4119.5 pg/ml was predicting poor Phosphate homeostasis (hyperphosphatemia + vitamin D deficiency + uncontrolled secondary hyperparathyroidism together) in the same hemodialysis patient 56
with 77% sensitivity and 89% specificity. The characteristics of the area under the curve are shown in table 4.10. Figure 4.10
Figure (4.10) shows the receiver operating characteristic (ROC) curve of intact FGF23 with poor Phosphate homeostasis as status variable.
Table 4.10: Table (4.10) shows the characteristics of the area under the curve according to receiver operating characteristic curve analysis: Area
0.835
Standard Asymptotic Asymptotic 95% confidence error Significance interval 0.084
0.002
Lower bound
Upper bound
0.67
0.999
57
4.11. FGF23 and anemia in the patients 21 hemodialysis patients had hemoglobin concentration below the target range (9-11.5 g/dl) (145), 26 of them had hemoglobin concentration within the target range while in 5 of them hemoglobin concentration was above the target range. Difference in FGF23 concentration between the two groups is shown in figure 4.11. No statistically significant difference was found between the patients with hemoglobin concentration below the target range and patients with hemoglobin concentration within or above the target range according to Mann-Whitney U test (P: .34). Figure 4.11:
Figure (4.11) shows the difference in FGF23 concentration between the hemodialysis patients according to the severity of their anemia. 58
Figure 4.12:
Figure (4.12) shows the Correlation between hemoglobin and serum intact FGF23 levels in the hemodialysis patients according to Spearman’s correlation test.
59
4.12. FGF23 and dialysis adequacy (Kt/V) Dialysis adequacy (Kt/V value) was 1.53 ± (0.15 SD). 42 hemodialysis patients had dialysis adequacy Kt/V value ≥ the target value (1.4) (133) while 10 of them had dialysis adequacy Kt/V value < the target value. Patients with better dialysis adequacy had lower FGF23 concentration compared to patients with lower dialysis adequacy as shown in figure 4.13 however there was no statistically significant differences between the two groups regarding their FGF23 concentration (P: .61) according to MannWhitney U test. Figure 4.13:
Figure (4.13) shows the difference in FGF23 concentration between the hemodialysis patients according to their dialysis adequacy (Kt/V). 60
Figure 4.14:
Figure (4.14) shows the Correlation between dialysis adequacy (Kt/V) and serum intact FGF23 levels in the hemodialysis patients according to Spearman’s correlation test.
61
4.13. FGF23 and dialysis vintage The median dialysis vintage in the hemodialysis patients was 29 ± (28 IQR) months. Patients with longer dialysis vintage had higher FGF23 concentration compared to patients with shorter dialysis vintage as shown in figure (4.15). There was a statistically significant difference between the 5 groups of patients (divided based on their dialysis vintage) regarding their FGF23Log10 values according to ANOVA test (<.001). Figure 4.15:
Figure (4.15) shows the difference in FGF23 concentration between the hemodialysis patients according to their dialysis vintage. 62
Figure 4.16:
Figure (4.16) shows the Correlation between dialysis vintage and serum intact FGF23 levels in the hemodialysis patients according to Spearman’s correlation test.
63
CHAPTER V DISCUSSION
DISCUSSION 5.1. Discussion Chronic kidney disease is an epidemic health problem that affects millions of people worldwide and it is associated with a high morbidity and mortality rates
(53,54)
. Chronic kidney disease- Mineral and bone disorder
(CKD-MBD) is one of the most common complications of CKD where there are various mineral, hormonal, skeletal and cardiovascular disorders
(21,53)
.
Many factors have a role in the development of CKD-MBD. Among these, FGF23 is considered one of the leading players (8). Previous studies have demonstrated the presence of numerous correlations between high FGF23 level with various complications in end stage renal diseases patients (21,81,86). In this study to assess the correlations of FGF23 with Phosphate hemostasis, hemoglobin and dialysis adequacy in end stage renal disease patients on hemodialysis in Sulaimani dialysis center, 78 participants were enrolled. 52 of them were patients on regular hemodialysis therapy for at least six months with median age of 55 years who were consisting of 26 males and 26 females. 26 healthy subjects with median age of 51.5 years who were consisting of 13 males and 13 females were also enrolled in the study. In addition to matching the cases and controls according to their age and sex, their ethnicity was also taken into consideration and they were matched accordingly with Arabs making 38.5% of both groups and the remainders were Kurds. As it is expected in patients with ESRD and in accordance with a studies in Egypt by Sliem et al.
(146)
64
, and by MohammedJumaah in
Kirkuk/Iraq (147), both serum urea and creatinine levels were elevated in the patients compared to the controls and reference range in our study (P: < .001). Our patients had elevated serum creatinine and urea by 4.6 and 10 folds respectively compared to the controls. This is usually due to accumulation of these waste products in the body from body metabolism and muscular activities with failure of the damaged kidneys to get the body rid of them. The ESRD patients in our study had altered mineral metabolism compared to the normal subjects and the reference ranges. In agreement with studies by Shimada et al. and Nishizawa et al. in Japan (148,149), Lima et al. in United States (150) and NasrAllah et al. in Egypt (151), our study demonstrated the presence of very high FGF23 level in the hemodialysis patients. FGF23 level has been multiplied by 87 folds compared to the normal subjects in our study (P: < .001). This is mainly due to the accumulation of Phosphate and reduction in expression of its cofactor Klotho which leads to resistance to FGF23 actions on its targets thereby uncontrolled secretion of FGF23. No statistically significant difference was found between the hemodialysis patients in their FGF23 concentration according to their demographic features including age and sex in agreement with Yuvaraj et al. study in India (152) and neither according to their race. In our study Kurds had higher FGF23 concentration compared to Arabs but the difference between the two groups was not statistically significant (P: .23). Multiple Studies have shown that there were differences in FGF23 level between Caucasians and Africans in normal and chronic kidney disease patients (153,154). However, to our knowledge there are no previous studies comparing Kurds and Arabs
65
on the basis of their FGF23 levels therefore this finding could be simply due to the small sample size of our study and needs further confirmations. In agreement with reports by United States National kidney foundation in 2016
(52)
, the three major etiologies for CKD among our
patients were diabetic (35%), hypertension (19%) and glomerulonephritis (12%), respectively. Other minor causes for CKD were polycystic kidney disease, obstructive and drug induced nephropathies which were responsible for over (11%) overall of the causes. In 23% of the patients, they etiology for their CKD was unknown. No statistically significant difference was found regarding the FGF23Log10 value between the three main etiologies of CKD in our patients and this is comparable to findings by Yuvaraj et al. (152) and Kojima et al. in Japan (155) where they also had similar findings. In agreement with Sliem et al. study
(146)
, serum Phosphorus was
increased while serum Calcium was decreased compared to the controls and the reference ranges in our study. Serum Phosphate level was increased by 42% in our patient compared to controls (P: < .001) due to failure of the kidneys to successfully excrete it and due to difficulties in Phosphate removal by the hemodialysis. Serum Calcium level was decreased just below the normal range and was reduced by 7% compared to the controls (P: .001) despite treatment and high PTH level. Low serum Calcium concentration is usually the result of several factors such as low vitamin D, high Phosphate and others. In parallel with Sliem et al. study (146), PTH was increased in the patients compared to the controls and the reference range in our study. Parathyroid hormone was increased by 8 folds compared to the control
66
group (P: < .001) which is usually resulting from the high Phosphate, low Calcium and vitamin D, inappropriate response to Calcium and vitamin D by the gland and down regulation of PTHR on the bone with development of resistance to the calcemic action of PTH with time. In agreement with studies by Mithal et al. in India (156) and Alwakeel et al. in Saudi Arabia
(157)
, 25-hydroxyvitamin D was decreased in the
patients compared to the controls and the reference range in our study. 25hydroxyvitamin D was decreased by 37% in our patients compared to the controls (P: .001) despite treatment. 25-hydroxyvitamin D deficiency is a common complication of CKD which is due to several factors such as reduced dietary intake, reduced sun exposure and leakage of 25hydroxyvitamin D bound to Vitamin D binding protein (DBP) with proteinuria. Serum Sodium level was maintained within the reference range in our patients in agreement with studies by Sliem et al. (147)
(146)
and by Munoz Mendoza et al. in United States
, MohammedJumaah
(158)
and was slightly
below the controls in our study. There was no statistically significant difference in serum Sodium level between the cases and the controls (P: .91) which suggests a successful salt and water restriction by the patients and proper response to the therapeutic agents by them in order to maintain predialysis Sodium level within normal range. In addition, the patients had an average dialysis adequacy above the target value which further confirms the successful Sodium and fluid control among them thereby the results regarding serum Sodium level in our study.
67
Anemia was common in the patients compared to the controls in our study. In parallel with Sliem et al. study (146), Hemoglobin level was below the reference range and was reduced by 30% compared to the controls in our study (P: < .001). Anemia is a common complication of CKD and its severity increases with decline in renal function. The etiology is usually multifactorial but mainly due to reduced erythropoietin production by the diseased kidneys (55). In accordance with a study by Agarwal et al. in United States (159), high blood pressure was common among the patients compared to the controls and the reference range in our study. Among the patients, 76% had high blood pressure with systolic blood pressure increasing by 17.5 mmHg (P: .001) and diastolic blood pressure increasing by 7.5 mmHg compared to controls (P: < .001). Hypertension is another common complication in patients with ESRD which is usually caused by volume expansion and increased systemic vascular resistance. Continuous fluid and electrolyte disturbance usually leads to development of hypertension in attempt to correct the problem and maintain homeostasis. After dividing the patients based on their FGF23 level into those with FGF23 level < the median value and those with FGF23 level ≥ the median value (because there is not clear cut-off point for FGF23), we noticed that the patients with lower FGF23 level had lower urea, creatinine, Phosphorus, PTH and dialysis vintage while they were having a higher Calcium, 25hydroxyvitamin D, hemoglobin, blood pressure and dialysis adequacy. In accordance with studies by Sliem et al. (146), Akalin et al. in Turkey (12)
and Imanishi et al. in Japan (160), serum FGF23 was positively correlated
68
with serum inorganic Phosphorus in our patients (P: < .001). As Phosphorus begins to build up in the body, FGF23 is secreted by the osteocytes in an attempt to facilitate excretion of the accumulated Phosphorus, but because of the failure of the damaged kidney to do so, extra amounts of FGF23 will be secreted continuously without having much effects to keep the serum Phosphorus down. Our study also confirmed the presence of a strong correlation between FGF23 and PTH in the hemodialysis patients. In agreement with Akalin et al. (12), Sliem et al. (146), and Imanishi et al.
(160)
studies, FGF23 was positively correlated with PTH in our study (P: < .001). The correlation coefficient (r) between FGF23 and PTH (r: 0.68) was higher than the one between FGF23 and serum Phosphorus level in our study (r: 0.62) which goes with the previous reports indicating PTH and bone remodeling to be the main regulator of FGF23 concentration in ESRD (26). In agreement with Sliem et al. study (146) where FGF23 was negatively correlated with serum Calcium, but in opposite to both Akalin et al. (12) and Imanishi et al.
(160)
studies where FGF23 was positively correlated to
Calcium and also in contrast to Jongbloed et al. study in Italy (11) where FGF23 was not correlated to serum Calcium, FGF23 in our study was negatively correlated with serum Calcium in our hemodialysis patients (P: .05). Furthermore, FGF23 in our patients was negatively correlated with 25hydroxyvitamin D level ((P: .01) in parallel to the Jongbloed et al. study (11) and in contrast to Hacıhamdioğlu et al. study in Turkey (13) where there was no correlation between the two. The negative correlations of FGF23 with Calcium could be due to the presence of high Phosphate (high Phosphate correlated with high FGF23) and so lower serum Calcium in those patients and the negative correlation of FGF23 with 25-hydroxy vitamin D could be 69
due to the inhibitory effect of FGF23 on 25-hydroxy vitamin D by enhancing its degradation pathway (similar to its effect on 1,25-dihydroxoy vitamin D). The noted differences among the different studies could be the result of the differences in dialysis vintage, residual renal function, nutritional statues, hypocalcemia/vitamin D deficiency treatment regimes, development of resistance and the difference in the patients’ ability to respond to the therapeutic agents. In agreement with studies by Isakova et al. in United States
(10)
,
Nishizawa et al. (149), NasrAllah et al. (151) and Imanishi et al. (160), longer dialysis vintage was correlated with higher FGF23 level in our hemodialysis patients (P: .001). This is the result of the loss of the residual renal function, poorer Phosphate control, more severe secondary hyperparathyroidism and gradual increase of resistance to the actions of FGF23 on its target organs with time. In addition, similar to studies by Hacıhamdioğlu et al. Nishizawa et al.
(149)
(13)
,
, and Cano et al. (161), but in contrast to the Imanishi et
al. (160) study where there was no correlation between FGF23 and dialysis adequacy (Kt/V), FGF23 was negatively correlated with dialysis adequacy (Kt/V) in our study (P: .02). This suggests that an effective dialysis might have a role in reducing FGF23 level hence reducing its off-target effects on the other organs in addition to the possibility of utilizing FGF23 as a clinical biomarker for determining dialysis adequacy in dialysis patients. In accordance with Imanishi et al. (160), FGF23 level was not correlated to blood pressure in our study. Also FGF23 was not correlated to serum urea, serum creatinine and serum Sodium in our study in agreement with Sliem et al. study (146).
70
In parallel with Sliem et al. and Imanishi et al. studies (146,160), but in opposite to the Akalin et al. (12) and Raafat et al. (162) studies; no statistically significant correlation was found between serum FGF23 and hemoglobin levels in our study (P: 0.39). The failure to find the potential correlation between FGF23 and hemoglobin in our study could be due to the small sample size, due to the effect of the confounders such as the difference in the dialysis vintage and residual renal function or could be due to the differences in therapies and/or the difference in patients’ response to the therapeutic agents. Nevertheless, the 21 patients with hemoglobin level below the target range had higher FGF23 level compared to the other 31 patients that were within or above the target hemoglobin range. Moreover, the 26 patients with FGF23 level below its median value had higher hemoglobin level compared to the patients with FGF23 level equal/above its median value. All these data suggest the likelihood of having a correlation between the two variables and calls for another study in our hemodialysis patients with larger sample size to confirm or reject the current findings. In a multivariate regression analysis to determine that factors that independently predict FGF23 level in our patients, serum Phosphorus (P: < .001), PTH (P: .002) and Calcium (P: < .05) were independently predicting FGF23Log10 value in our study in parallel with Imanishi et al. study (160) while dialysis vintage failed to be one of the independent FGF23Log10 predictors (p:.14) , in contrarily to the same study where it was predicting FGF23Log10 value along the other three mentioned factors. Receiver operating characteristic (ROC) curves was drawn in order to determine the sensitivity and specificity of FGF23 concentration in predicting poor Phosphate homeostasis in hemodialysis patients and we 71
found out that FGF23 concentration above 4119.5 pg/ml was predicting poor Phosphate homeostasis (hyperphosphatemia, Vitamin D deficiency and uncontrolled secondary hyperparathyroidism in the same hemodialysis patient) with 77% sensitivity and 89% specificity (P: .002). Unfortunately we could not find similar analyses in previous studies so that we could compare this particular finding.
5.2. Limitations of the study 1. The sample size was not large enough to be able to make generalization with certainty for all of our findings. 2. All the variables were measured once and there was not enough time for following up the patients. 3. Some useful laboratory tests such as serum Klotho and ReninAngiotensin-Aldosterone system activity were not available for the study. 4. The long duration of the disease made the patients unable to recall certain events in the past hence increased the likelihood of “recall bias”.
5.3. Strength of the study 1. According to our knowledge, it is the first study that has been done in Kurdistan region of Iraq about FGF23 and its effect and/or clinical importance in end stage renal disease patients. 2. All the necessary information for the questionnaire was taken from the patients through interviewing method.
72
CHAPTER VI CONCLUSION AND RECOMMENDATIONS
CONCLUSION AND RECOMMENDATIONS
6.1. Conclusions 1. Serum FGF23 level was markedly elevated in the hemodialysis patients compared to the normal subjects regardless of their age, sex, race or the etiology of their chronic kidney disease. 2. FGF23 was positively correlated with serum Phosphorus and PTH levels, but it was negatively correlated with serum Calcium and 25-hydroxy vitamin D levels. 3. FGF23 was not correlated to the hemoglobin level. 4. FGF23 was negatively correlated with dialysis adequacy (Kt/V). 5. FGF23 was positively correlated with dialysis vintage.
6.2. Recommendations 1. Further studies on larger samples, with more investigations and patient follow up in order to have more valuable results. 2. Measuring FGF23 level is better to be considered during clinical assessment of mineral metabolism in end stage renal disease patients after standardization of the measurement methods. 3. Clinical trials for FGF23 lowering agents according to their internationally recognized phases in clinical research.
73
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157. Alwakeel J, Usama S, Mitwalli A, Alsuwaida A, Alghonaim M. Prevalence of vitamin D deficiency in peritoneal dialysis patients. Saudi J Kidney Dis Transpl [Internet]. 2014 [cited 7 January 2017];25(5):981-985. Available from:http://www.sjkdt.org/article.asp?issn=13192442;year=2014;volume=25;issue=5;spage=981;epage=985;aulast=Alwakee l 158. Munoz Mendoza J, Sun S, Chertow G, Moran J, Doss S, Schiller B. Dialysate sodium and sodium gradient in maintenance hemodialysis: a neglected sodium restriction approach?. Nephrol Dial Transplant [Internet]. 2011 [cited 4 February 2017];26(4):1281-1287. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3108351/ 159. Agarwal R, Nissenson A, Batlle D, Coyne D, Trout J, Warnock D. Prevalence, treatment, and control of hypertension in chronic hemodialysis patients in the United States. AM J MED [Internet]. 2003 [cited 7 January 2017];115(4):291-297. Available from: https://www.researchgate.net/publication/10572717_Prevalence_treatment_a nd_control_of_hypertension_in_chronic_hemodialysis_patients_in_the_Unit ed_States 160. Imanishi Y, Inaba M, Nakatsuka K, Nagasue K, Okuno S, Yoshihara A et al. FGF-23 in patients with end-stage renal disease on hemodialysis. Kidney Int [Internet]. 2004 [cited 7 January 2017];65(5):1943-1946. Available from: https://www.researchgate.net/publication/8616300_FGF23_in_patients_with_end-stage_renal_disease_on_hemodialysis 161. Cano F, Freundlich M, Ceballos M, Rojo A, Azocar M, Delgado I et al. Longitudinal FGF23 and Klotho axis characterization in children treated with chronic peritoneal dialysis. Clin Kidney J [Internet]. 2014 [cited 4 February 2017];7(5):457-463. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4379333/ 162. Raafat M, Madkour M, Metwaly A, Nasr F, Mosbah1 O, El-Sheikh N. Clinical significance of FGF-23 in Chronic Kidney Disease Patients. Sch J App Med Sci [Internet]. 2016 [cited 4 February 2017];2015(3):741-750. Available from: https://www.researchgate.net/publication/289521235_Clinical_significance_ of_FGF-23_in_Chronic_Kidney_Disease_Patien
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APPENDIX
APPENDIX Questionnaire (Page 1) NO.: RACE
NAME: WEIGHT
AGE: DATE:
/
/
SEX: /
Etiology and duration of chronic kidney disease: How long he/she has CKD: How long he/she has been on hemodialysis: Has he/she been told the reason for his/her CKD: Risk factors for developing chronic kidney disease before the onset of the disease: Diabetes Hypertension Heart disease Urinary stone Urinary tract infection Lower urinary tract obstruction Chronic liver disease Autoimmune disease Family history of CKD History of long term drug use Smoking Drinking alcohol Drug abuse Obesity Low income
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
No No No No No No No No No No No No No No No
History of diabetes: Type: Duration: Medications:
Yes one
No Two
History of hypertension: Blood pressure: Duration: Medications:
Yes
No
History of chronic heart disease: Duration: Stroke: Medications: Cardiovascular surgery:
Yes
No
Yes
No
History of anemia: Duration: Blood coagulopathy: Duration: Medications for anemia or Coagulopathy:
Yes
No
Yes
No
Chronic kidney disease complications and related disorders:
96
Appendix (continued) Questionnaire (Page 2) History of one disease: Duration: How he/she was diagnosed: History of: Fracture: Family history of bone disease: Exercise: Deformation: Bone pain: Most common site: Joint pain: Most common site: Change in power of muscles: Difficulty in movement of muscles or joints: History of swellings near the joint: Pruritus: Medications: Musculoskeletal Surgery:
Yes
No
No
Yes Yes Yes Yes Mild
No No No No Moderate
Severe
No
Mild
Moderate
Severe
No No No No
Mild Mild Mild Mild
Moderate Moderate Moderate Moderate
Severe Severe Severe Severe
Yes Yes Yes
No No No
Yes
No
Yes Yes Yes Mild Mild
No No No Moderate Moderate
Yes Yes
No No
Dialysis adequacy: Duration of the session: Blood flow rate: Ultrafiltration volume: Dietary (Salt, phosphate etc.) restriction: Is he/she feels comfortable during dialysis: does he/she feel comfortable after the dialysis: If yes How long: Any history of recent Emergency dialysis: If yes how many times: History of recent: Weight loss: GIT disturbance: Sleep disturbance: Weakness, Tiredness: Dyspnea:
No No
Others: History of Thyroid disease: Lipid disorders: Other medications: Notes:
97
Severe Severe
حكومة اَّلقليم الكردستان/جمهورية العراق وزارة التعليم العالي والبحث العلمي جامعة السليمانية /كلية الطب قسم الفسيولوجي
دور عامل نمو األرومة الليفية )FGF23( 23كواصمة حيوية في المرضى المصابين بالفشل الكلوي المزمن في مرحلته النهائية ومتلقيين عالج الغسل الكلوي في مدينة السليمانية هذا البحث مقدم الى كلية الطب /جامعة السليمانية كجزء من متطلبات الحصول على درجة الماجستير في العلوم الفيسيولوجية
التحضير:
د.شهرام حمة علي عزيز M.B.Ch.B
اَّلشراف:
د.كاوه حسين امين M.B.Ch.B C.A.B.M
يناير 2017
الخالصة المقدمة :عامل نمو األرومة الليفية (Fibroblast Growth Factor 23) 23او ( )FGF23هو فرز من العظام وجائل في الدورة الدموية .ترتفع نسبة ( )FGF23الى مستويات عالية هورمون ُم َ شف ان هناك عالقة جدا عند التعرض لمرض )الفشل الكلوي المزمن( في مرحلته النهائية حيث اكت ُ َ تربط ارتفاع نسبة هذا الهورمون في الجسم مع كل من سوء عملية استقالب الفوسفات وشدة فقر الدم وانخفاض كفائة عملية الغسل. االهداف :يهدف هذا البحث الى اضهار اَّلرتباط بين FGF23مع كل من عملية استقالب الفوسفات في الجسم و نسبة الهيموغلوبين )خضاب الدم او )Hemoglobinو مدة العالج على الديال ( )dialysis vintageو كفائة عملية الغسل (.)Kt/V dialysis adequacy طريقة البحث :في هذه الدراسة التي هي من نوع (الحاَّلت والشواهد) ,ادرج اثنين وخمسين مريضا بالغا مصابين بمرض (الفشل الكلوي المزمن) في مرحلته النهائية متلقيين عالج غسل الكلوى بشكل منتظم لمدة َّل تقل عن ستة اشهر في مركز السليمانية لغسل الكلى .كما ادرج ستة وعشرين من األشخاص األصحاء في نفس الدراسة .في كلتا المجموعتين تم قياس نسبة ضغط الدم ونسبة اليوريا والكرياتينين والفوسفات والكالسيوم و FGF23والهرمون الجار درقي ()Parathyroid hormone و-25هيدروكسي فيتامين دال والصوديوم في مصل الدم ونسبة الهيموغلوبين في الدم باَّلضافة لحساب كفائة عملية الغسل الكلوي ) (Kt/Vفي المرضى. النتائج :نسبة FGF23في المرضى كان مرتفعة جدا حيث تصل الى 87ضعفا مقارنة مع الشواهد. كان هناك ارتباطا ايجابيا بين FGF23مع الفسفور)(P: <.001
والهرمون الجار درقي
) (P:<.001ومدة العالج على الديال ) ,(P: .001ولكن اَّلرتباط كان سلبيا بين FGF23مع -25
هيدروكسي فيتامين دال ) (P: .01وكالسيوم ) (P: .05و .(P: .02) Kt/Vلم يكن هنالك اية ارتباط بين FGF23و هيموغلوبين في المرضى. االستنتاجات FGF23 :عالمة بايولوجية سريرية مفيدة في المرضى المصابين بالفشل الكلوي المزمن في مرحلته النهائية حيث ارتفاع نسبة FGF23في اوَّلئك المرضى ترتبط بضعف عملية استتباب الفسفور في الجسم وبانخفاض قيمة Kt/Vوبطول مدة العالج على الديال ولكن تبقى الصلة بين نسبة FGF23وشدة فقر الدم مبهمة.
حكومةتي هةريَمي كوردستان/كؤماري عيَراق وةزارةتي خويَندني باالَ وليَكؤلَينةوةي زانسيت زانكؤي سليَماني/كؤليَذي ثزيشكي بةشي فيسؤلؤجي
رِؤلَي فايربؤبالست طرؤس فاكتةر ( )FGF23وةكو نيشاندةريَكي بايؤلؤجي لةو نةخؤشانةي كة تووش بوون بة شكسيت دريَذخايةني طورضيلة لة دوواين قؤناغيدا و لةسةر ضارةسةري ثاكذكردنةوةي خويَنن لة شاري سليَماني ئةم ليَكؤلَينةوةية ثيَشكةش كراوة بة كؤليَذي ثزيشكي/زانكؤي سليَماني وةكو بةشيَك لة دواكاري بةدةستهيَناني منرةي ماستةر لة زانسيت فيسيؤلؤجي
ئامادةكردني:
د.شةهرام محة علي عزيز M.B.Ch.B
بة سةرثةرشيت:
د.كاوة حسني امني M.B.Ch.B C.A.B.M
كانوني دووةم 2017
ثوختة ثاشخان :فايربؤبالست طرؤس فاكتةر )FGF23( 23بريتية لة هؤرِمؤنيَكي ناو سورِي خويَن كة لة ئيَسكةكانةوة دةردةدريَت .رِيَذةي FGF23بة شيَوةيةكي بةرضاو لة لةشدا زياد دةكات ثاش تووش بوون بة نةخؤشي (شكسيت دريَذخايةني طورضيلة) و طةيشنت بة دوواين قؤناغي ئةو نةخؤشية .لةم نةخؤشانةدا بينراوة كة بووني رِيَذةي بةرزي فايربؤبالست طرؤس فاكتةر 23 هةمةئاهةنطة لةطةلَ تيَكضووني هاوسةنطي فؤسفات و سةخيت كةمي ريَذةي خويَن و كةم بوونةوي كارايي دايلةزة (.)Kt/V ئاماجنةكان :مةبةست لةم ليَكؤلينةوةية بريتيية لة هةلسةنطاندني ثةيوةسيت نيَوان FGF23 لةطةل هةريةكة لة ثرؤسةي هاوسةنطي فؤسفات لة لةشدا و ريَذةي هيمؤطلؤبني و ماوةي دايلةزة (ثرؤسةي ثاكذكردنةوةي خويَين نةخؤشاني تووش بوو بة شكسيت دريَذخايةني طورضيلةكان) و كارايي دايلةزة ( )Kt/Vلة نةخؤشاني تووش بوو بة شكسيت دريَذخايةني طورضيلةكان لة دوايني قؤناغيدا. رِيَطةو شيَواز :لةم ليَكؤلَينةوةية لة جؤري (كةيس-كونرتول)ييةدا ,ثةجناو دوو نةخؤشي طةورةسالَي تووشبوو بة بة نةخؤشي شكسيت طورضيلة لة دوواين قؤناغيدا كة بة شيَوةيةكي ريَسابةندي ضارةسةري ثاكذكردنةوةي خويَنيان وةردةطرت ( )Hemodialysisبة اليةني كةم بؤ ماوةي 6مانط لة بنكةي دايةلةزةي سليماني وة هةروةها بيست و شةش كةسي تري ئاسايي ناونوس كران .بؤ هةردوو ثؤلةكة ,فشاري خويَن و ريَذةي يوريا وكرياتينني وفؤسفؤر وكاليسيؤم و
سؤديؤم و FGF23وهؤرمؤني تةنيشت لووي بن مل و-25هايدرؤكسي ظيتامني دال لة سريةمدا و ريَذةي هيمؤطلؤبني لة خويَندا ثيَورا .هةروةها بةهاي ( )Kt/Vلة نةخؤشةكاندا ئةذميَريدرا. ئةجنامةكان :ريَذةي FGF23بة شيَوةيةكي بةرضاو لة نةخؤشةكاندا زيادي كردبوو 87,قات زياتر بة بةراورد كردن لةطةل كةسة ئاساييةكاندا FGF23 .هاوثةيوةنديةكي ئةريَين هةبوو لةطةل فؤسفؤر ) ,(P: <.001هؤرمؤني تةنيشت لووي بن مل ) ,(P: <.001ماوةي وةرطرتين ضارةسةري دايلةزة ) )P: .001وة هاوثةيوةنديةكي نةريَين هةبوو لةطةل -25هايدرؤكسي ظيتامني دال ) (P: .01و كاليسؤم ) (P: 0.05و (P: 0.02) Kt/Vلة نةخؤشةكاندا .هيض هاوثةيوةنديةك نةبوو لة نيَوان FGF23لةطةل هيمؤطلؤبني لة نةخؤشةكاندا. دةرئةجنام FGF23 :هؤكاريَكي نيشاندةري بة سوودة لة نةخؤشاني تووشبوو بة شكسيت دريَذخايةني طورضيلة لة دوواين قؤناغيدا كة تيايدا ئاسيت بةرزي FGF23هاوثةيوةنديدارة لةطةل بةدهاوسةنطي فؤسفات لة لةشدا وة لةطةل نزمي كارايي دايلةزة ( )Kt/Vودريذي ماوةي وةرطرتين ضارةسةري دايلةزة بةالَم ثةيوةنديةكةي لةطةل سةخيت كةمي رِيَذةي خويَن روون نية.
