Interaction between serum FGF-23 and PTH in renal phosphate excretion, a case-control study in hypoparathyroid patients.

BACKGROUND
phosphate homeostasis is mediated through complex counter regulatory feed-back balance between parathyroid hormone, FGF-23 and 1,25(OH)2D. Both parathyroid hormone and FGF-23 regulate proximal tubular phosphate excretion through signaling on sodium- phosphate cotransporters IIa and IIc. However, the interaction between these hormones on phosphate excretion is not clearly understood. We performed the present study to evaluate whether the existence of sufficient parathyroid hormone is necessary for full phosphaturic function of FGF-23 or not.


METHODS
In this case-control study, 19 patients with hypoparathyroidism and their age- and gender-matched normal population were enrolled. Serum calcium, phosphate, alkaline phosphatase,parathyroid hormone, FGF-23, 25(OH)D, 1,25(OH)2D and Fractional excretion of phosphorous were assessed and compared between the two groups, using SPSS software.


RESULTS
The mean serum calcium and parathyroid hormone level was significantly lower in hypoparathyroid patients in comparison with the control group (P < 0.001 and P < 0.001, respectively). We found high serum level of phosphate and FGF-23 in hypoparathyroid patients compared to the control group (P < 0.001 and P < 0.001, respectively). However, there was no significant difference in Fractional excretion of phosphorous or 1,25OH2D level between the two groups. There was a positive correlation between serum FGF-23 and Fractional excretion of phosphorous just in the normal individuals (P < 0.001, r = 0.79).


CONCLUSIONS
Although the FGF-23 is a main regulator of urinary phosphate excretion but the existence of sufficient parathyroid hormone is necessary for the full phosphaturic effect of FGF-23.

, which is an osteocytes derived hormone. FGF-23 decreases serum PO 4 by inhibiting renal PO 4 reabsorption through FGF-23-Klotho (coreceptor) signaling on NaPi II a and II c at proximal tubule of the kidney. FGF-23 also suppresses 1,25-dihydroxyvitamin D (1,25(OH)2 D) production by decreasing 1αhydroxylase expression [12][13][14]. PTH promotes PO 4 excretion by suppressing NaPi-II in the kidney [15]. PTH also enhances calcium absorption through the direct effect on bones and kidneys, and indirectly increases intestinal calcium and PO 4 absorption via the stimulation of 1α -hydroxylase activity and increase 1, 25(OH)2 D production [16][17][18]. The same site of action of FGF-23 and PTH in NaPi II a and II c at proximal tubule of kidney might raise the question that whether there is an overlapping effect between these hormones or not?
Hypoparathyroidism is a rare endocrine disorder characterized by inappropriately low or absent levels of PTH associated with hypocalcemia and hyperphosphatemia [17,19]. Hypoparathyroidism might occur as a primary congenital defect or might be due to a secondary cause. The most common cause of secondary hypoparathyroidism is the incidental destruction of parathyroid glands during anterior neck surgeries. Other causes are autoimmune disorders, radiation to the neck and infiltrative disorders of the parathyroid glands [20][21][22].
In the present study, we aim to evaluate the role of FGF-23 on PO 4 hemostasis in state of low or insufficient PTH in human. Hence, we conducted this casecontrol study to evaluate whether the renal excretion of PO 4 by serum FGF-23 in patients with hypoparathyroidism was different from normal population or not.

Patients and method
A total of 38 participants including 19 patients with hypoparathyroidism and their healthy controls were enrolled in this study. The study was performed at Shiraz University of Medical Sciences affiliated endocrine clinics in Fars province, southern Iran, from October 2017 till March 2018. Both groups were matched for age and gender. Hypoparathyroidism was diagnosed on the basis of hypocalcemia (serum calcium less than 8.5 mg/ dl) accompanied with documented PTH levels below the lower limit of the normal range.
All hypoparathyroid patients were follow up by an expert endocrinologist. Patients received proper doses of calcium carbonate (500 mg tablet, manufactured Toliddaru pharmaceutical, Tehran, Iran), and calcitriol (0.25 μg capsule, manufactured Zahravi pharmaceutical, Tehran, Iran) to maintain albumin-corrected serum calcium in the lownormal range (8-9 mg/dl) [23]. The exclusion criteria in both groups were renal failure (Glomerular filtration rate less than 60 ml/min), liver failure, other metabolic bone disease (e.g., rickets), hyperthyroidism, and diabetes mellitus. None of the patients received phosphate binder resins during the study.

Laboratory tests
All the samples were taken after 8 h overnight fasting. Blood samples were centrifuged for 15 min at 3000 rpm and the plasma was collected and stored at − 70°C till further analysis. All the biochemical studies were performed at the endocrinology and metabolism research center laboratory of Shiraz University of Medical Sciences. Colorimetric assays were used to measure calcium (mg/dL), phosphorus (mg/dL), albumin (g/dL) and alkaline phosphatase (ALP) (IU/L) levels, by using Biosystem SA auto-analyzer, made in Spain. Serum PTH (pg/ml) and 25(OH)D (ng/ml) levels were assessed by Electrochemiluminescence methods produced by Roche company in Germany with Sensitivity, intra-and inter-assay CVs 3.3 and 5.1%, respectively. ELISA method was used to determine the serum intact FGF-23 (pg/ ml) and 1, 25(OH)2D (pmol/l) using Bioassay technology laboratory kit. Intra-and inter-assay CVs for 1,25(OH)2D and FGF-23 were < 8 and < 10%, respectively. Normal references for serum calcium, phosphorus, ALP, PTH, 25(OH)D and 1,25(OH)2D were 8.5-10.5 mg/ dL, 3.5-5.5 mg/ dL, 44-147 IU/L, 10-65(pg/ml), 20-100 ng/ml, and 20 to 45 pg/ml, respectively. Initial morning urine collection was done to determine renal PO 4 clearance. Urinary PO 4 and creatinine concentrations were determined by digital flame spectrophotometer. Fractional excretion of phosphorous (FE PO 4 ) was done using the following formula:

Ethical statement
An informed written consent form was obtained from the participants after explaining the aim, method and goal of the study. Shiraz University of Medical Sciences local Ethics Committee and Vice-Chancellor of research at SUMS approved this study with number 1396-01-01-15,805.

Statistics
SPSS statistical software (version 22, IBM) were used to perform Statistical analysis. Data are mentioned as mean ± SD. Shapiro-Wilk was used to evaluate the normality of data distribution. Normally distributed data were compared using Student's t-test, and the Mann-Whitney test was used to compare non-normally distributed ones. Pearson's test and Spearman's ranking test were used to evaluate the correlations between normally distributed parameters and non-normal distributed ones, respectively. P value less than 0.05 was considered to be statically significant.

Results
A total of 38 participants were enrolled in this study, 19 with hypoparathyroidism as case group and 19 volunteers with normal parathyroid function as the control group. Mean age in the case and control groups was 43.6 ± 17 years and 46.7 ± 15.9 years, which was not statistically significant (P = 0.57). Both case and control groups included 5 male and 14 female. Also, there were no significant differences in weight and BMI between case and control groups.
In the case group, 9 patients had hypoparathyroidism due to previous neck thyroidectomy and 10 patients were case of primary hypoparathyroidism. General characteristic of patients and controls are summarized in Table 1.
The mean serum calcium and PTH level was significantly lower in the case group in comparison with the control group (P < 0.001 and P < 0.001, respectively). In patients with hypoparathyroidism serum PO 4 was significantly higher than the control group (P < 0.001). Serum FGF-23 was higher in patients with hypoparathyroidism in comparison with the control group (P = 0.001). However, there was no significant difference between the case and control groups with respect to the mean serum level of 1,25(OH)2D, 25(OH)D, ALP, and FE PO 4 (P = 0.25, P = 0.11, P = 0.23 and P = 0.08, respectively).
As shown in Table 2 there was a strong positive correlation between FGF-23 and FE PO 4 in the control; however, this correlation was not observed amongst hypoparathyroid patients. There was no correlation between FE PO 4 , serum PO 4 , PTH and 1, 25(OH)2 D in both case and control groups. Figure 1 shows the correlation between values of FE PO 4 and serum FGF-23 in both control and case groups (Spearman rho =0.79, P < 0.001). In addition, we have done an analysis in our hypoparathyroid patients evaluating the association of FGF23 and serum calcium in 2 separate group of low calcium and normal calcium level. It showed that there was no correlation between calcium and FGF23 in low calcium and normal calcium level groups (P = 0.598 and P = 0.054, respectively).

Discussion
Maintaining serum PO 4 homeostasis necessitates a complex counter regulatory feed-back balance between PTH, FGF-23 and 1,25(OH)2 D [24][25][26]. FGF-23 and PTH are probably the most important phosphaturic hormones in human [1]. FGF-23 is mainly produced by osteoblasts and osteocytes. Local expression of FGF-23 coreceptor (Klotho) is necessary for its function at the renal proximal tubules [27]. It inhibits renal Phos reabsorption through inhibitory effects on NaPi II a and II c at proximal renal tubules [28]. However, there are still controversies about the action site of FGF-23 in the kidney [29]. previous studies had showed that klotho is essentially expressed in distal renal tubules, and alteration in the extracellular signal-regulated kinase (ERK) phosphorylation in distal tubules occurs soon after FGF-23 injection [30,31]. Therefore, it still remain unclear as how FGF-23 could affect proximal tubules to suppress phosphate reabsorption. Data suggest that FGF-23 might require other factors such as PTH for signal transduction pathway at the proximal tubules [32].
PTH also increases renal PO 4 excretion at proximal tubule of the kidney by reducing apical membrane NaPi II a and II c [14,33,34]. Moreover, PTH increases FGF-23 gene expression [35]. In addition to kidneys, parathyroid gland also express considerable amount of klotho and FGF-23 receptor [36]. On the other hand, FGF-Klotho complex could activate the MAPK pathway leading to decreased PTH mRNA and PTH secretion [37][38][39]. Olena et al. showed that the phosphaturic actions of PTH, are blunted by FGF-23 or Klotho deficiency.  Hence, FGF-23 might be an important modulator of PTH signaling in the kidney [40]. Although the regulatory counterbalance between FGF-23 and PTH secretion was investigated, there is still insufficient information about the role of PTH on phosphaturic function of FGF-23 in human. Study of phosphaturic effect of FGF-23 in normal human physiology might be confounded by the fact that PTH and FGF-23 have some overlapping effects on PO 4 excretion. Hence, the present study on hypoparathyroid patients and normal population provides an opportunity to observe whether the phosphaturic effect of FGF-23 is independent of PTH or not.
In the present study, we detected high serum level of PO 4 and FGF-23 in hypoparathyroid patients compared to the control group; however, we found no significant difference in FE PO 4 or 1,25(OH)2 D level between the two groups. Also, we found a strong positive correlation between serum FGF-23 and FE PO 4 in the control population, but this correlation was absent in hypoparathyroid patients. These findings could suggest that although the FGF-23 is one of the main regulator of urinary PO 4 excretion, the existence of intact PTH is necessary for the full phosphaturic effect of FGF-23. However, further relevant human studies are warranted.
Previous studies showed that mean serum PO 4 levels in hypoparathyroid patients remained above the normal range, even in the presence of high serum FGF-23 level [53]. Yamashita et al. showed that in transient hypoparathyroidism high serum level of FGF23 and hyperphosphatemia will be normalized only after parathyroid recovery [54]. Animal studies also showed that PTH-null mice experienced high PO 4 in spite of high circulating FGF-23, resembling participants with hypoparathyroidism in the present study [55]. In another study on hypoparathyroid patients, treatment with rhPTH could reduce serum PO 4 level from the upper normal range to the normal values parallel with increased urinary PO 4 excretion [56][57][58]. These findings also support our hypothesis about the importance of PTH in phosphaturic action of FGF-23. Another explanation by Gracia-Iguacel et al. was that PTH may have effect on phosphaturic function of FGF-23 through the serum calcium level [59]. Also, some studies showed that FGF23 have positive correlation with serum calcium [60,61]. In our study, we found no correlation between calcium and FGF23 in low calcium and normal calcium level hypoparathyroid patients. However, the number of hypoparathyroid patients in low Ca group was just 7, which could affect the results. Another important issue in this regards was that normal renal function is necessary for this association. In some patients with chronic kidney disease, high serum phosphate was observed in spite of high serum PTH and FGF23 level [62]. Also, it was shown that in chronic kidney disease patients treated with hemodialysis, FGF23 could predict the progression of secondary hyperparathyroidism. Interestingly, in these patients total parathyroidectomy could decrease high serum FGF-23 level to normal values [63,64]. In spite of many strengths of this study that evaluated FGF23 function in hypoparathyroid patients, we had some limitations. This study was a case-control cross sectional study, which could be better if we design an interventional clinical trial to evaluate the effect of PTH in hypoparathyroid patients in the future.

Conclusion
The present study could suggest that although the FGF-23 is one of the main regulator of urinary PO 4 excretion, the existence of sufficient parathyroid hormone is necessary for the full phosphaturic effect of FGF-23. We hypothesized that PTH might play a role in PO 4 excretory signal pathway of FGF-23. However, further in vivo and in vitro studies are necessary to determine the mechanism of action of parathyroid hormone on PO 4 excretory function of FGF-23. Authors' contributions FS: design, data gathering, Analysis, preparing the manuscript. AS: design, data gathering, preparing the manuscript. SRK: design, data gathering, preparing the manuscript. GHRO: design, data gathering, preparing the manuscript and the correspondence. All authors have read and approved the manuscript.

Funding
There is no financial support.

Availability of data and materials
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate Shiraz University of Medical Sciences local ethic committee and vicechancellor of research at SUMS approved this study with number 1396-01-01-15805. All the patients signed a written informed consent form after a session explaining the aim, method and goal of the study for each participant.

Consent for publication
All the patients signed a written informed consent form to publish their data in manuscript after a session explaining the aim, method and goal of the study.

Competing interests
Gholamhossein Ranjbar Omrani, Azita Salehifar, Seyed Reza Kassaee and Forough Saki declare that they have no conflict of interest.