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Abstract

Objectives

The aim of this study was to investigate serum fibroblast growth factor (FGF)-23 concentrations in young and mature adult cats with chronic kidney disease (CKD).

Methods

The present study retrospectively investigated the serum samples and medical records of 1–8-year-old clinically healthy cats (control group, n = 7) and cats with CKD (n = 54). Cats with CKD were divided into four stages based on serum creatinine concentrations, according to the International Renal Interest Society (IRIS) CKD guidelines. Serum FGF-23 concentrations were compared between cats in the control group, IRIS stages 1, 2 and 3–4 CKD. Continuous variables were analysed using correlations and multiple linear regression.

Results

Serum FGF-23 concentrations were significantly higher in cats with IRIS stages 1, 2 and 3–4 CKD, compared with those in the control group (P = 0.02, P = 0.002 and P = 0.002, respectively). Additionally, serum FGF-23 concentrations in cats with IRIS stages 3–4 CKD had higher serum FGF-23 concentrations than those with IRIS stages 1 and 2 CKD (P = 0.04 and P = 0.02, respectively). In the multiple linear regression analysis, serum urea nitrogen concentration, serum phosphorus concentration and blood ionised calcium concentration were independent variables predicting serum FGF-23 concentration.

Conclusions and relevance

Serum FGF-23 concentrations in younger cats with CKD increased in early-stage CKD and were associated with mineral metabolic markers, as also occurs in geriatric cats.

Keywords

CKD-MBD phosphate calcium biomarker renal

Introduction

Fibroblast growth factor (FGF)-23 is a phosphaturic hormone that is secreted from the osteocytes in response to increased blood phosphorus and calcitriol levels.¹ FGF-23 enhances urinary phosphate excretion via the downregulation of sodium-phosphate co-transporter in the kidney and the inhibition of calcitoriol synthesis.^2,3 FGF-23 has been shown to be involved in chronic kidney disease (CKD)-mineral and bone disorder (MBD).^4–6 In CKD, circulating FGF-23 levels increase to compensate for phosphate excretion dysfunction in declining glomerular filtration rate (GFR).^5–7 Plasma FGF-23 concentrations are increased in cats with CKD.^8,9 Additionally, plasma FGF-23 concentrations have been shown to increase in the early stages of CKD, compared with elevated phosphorus,⁸ and are associated with a shorter survival time in feline CKD.¹⁰ Therefore, FGF-23 is considered an early maker of CKD-MBD in cats.

CKD is a common cause of morbidity and mortality in geriatric cats.^11–14 Thus, most studies investigating FGF-23 in cats have targeted geriatric cats.^8,10,15–17 In humans, blood FGF-23 levels has been reported to increase in patients with CKD regardless of life stage,^18–24 whereas few studies have evaluated FGF-23 in younger cats with CKD. Thus, the purpose of this study was to investigate serum FGF-23 concentrations in young and mature adult cats with CKD.

Materials and methods

We retrospectively investigated the medical records and stored serum samples of 1–8-year-old client-owned cats diagnosed with CKD between August 2015 and December 2020 through the nephrology service of an animal medical centre at Nippon Veterinary and Life Science University. CKD was diagnosed based on persisting (⩾3 months) renal azotaemia, renal proteinuria and/or ultrasonographic renal abnormalities. Renal azotaemia was defined as a serum creatinine concentration >159 µmol/l (the upper limit of the reference interval at our institution) and urine specific gravity (USG) <1.035. Renal proteinuria was determined based on a urine protein:creatinine ratio ⩾0.4 without an apparent pre- and/or post-renal cause. Ultrasonographic renal abnormalities included a small-sized kidney, decreased corticomedullary differentiation and/or irregular renal contours. We excluded cats diagnosed with or suspected to have an acute kidney injury, tumour or hyperthyroidism. Cats with CKD were classified according to the 2019 International Renal Interest Society (IRIS) staging of CKD guidelines based on serum creatinine concentrations as stages 1–4.²⁵

The control group included 1–8-year-old clinically healthy cats owned by our laboratory or students. Cats in the control group were considered clinically healthy based on their history, physical examination, serum biochemical analysis, urinalysis and abdominal ultrasonography.

Information regarding age, breed, body weight, serum biochemical analysis, blood gas analysis, urinalysis and blood pressure were collected from medical records. Serum chemical analyses were performed using an automatic analyser (7180 Biochemistry Automatic Analyzer; Hitachi High-Technologies). Blood ionised calcium and bicarbonate concentrations were measured by a blood gas analysis (GEM Premier 3500; Instruments Laboratory). Blood pressure was measured using oscillometric methods (BP100D haemomanometer; Fukuda M-E Kogyo), according to the methodology of a previous report.²⁶

Blood samples for serum FGF-23 concentration analysis were collected with the informed consent of the cats’ owners. Blood samples in tubes containing serum separators were centrifuged at 1181 × g for 5 mins and the obtained serum samples were stored at −30°C for submission to an external laboratory (FUJIFILM VET Systems). Serum FGF-23 concentrations were measured using sandwich ELISA (MedFrontier FGF23; Hitachi Chemical Diagnostics Systems). The FGF-23 assay validation for feline serum samples is shown in the supplementary material.

Statistical analysis was performed using a commercial software package (SPSS 24 for Windows, IBM Japan) and a freely available software package (EZR 1.54 for Windows).²⁷ Data normality was evaluated using the Shapiro–Wilk test. The Steel–Dwass test was used to compare between groups. For the correlation analyses, variables with non-parametric distributions were log-transformed. Correlations were investigated using Pearson’s product-moment correlation coefficients. Variables that were significantly correlated with FGF-23 in the univariable analysis were included in the multiple linear regression analysis using stepwise forward selection to identify the independent predictors of FGF-23. P values <0.05 were considered statistically significant.

Results

The present study included 54 cats (1–8 years of age) in the CKD group. These cats were grouped according to IRIS stage: stage 1 (n = 7); stage 2 (n = 32); stage 3 (n = 13); and stage 4 (n = 2). Since there were few cats with stage 4, they were grouped together with those with stage 3 for statistical analysis. All cats in stage 1 were diagnosed with CKD based on ultrasonographic renal abnormality. The breed distribution among cats in the CKD group included: 21 domestic shorthair, eight American Shorthair, eight Munchkin, eight Scottish Fold, four Russian Blue and one each of Abyssinian, British Shorthair, Maine Coon, Persian and Somali. Eight cats had nephroliths and 24 had ureteroliths. Four cats were diagnosed with polycystic kidney disease. Two cats had a history of an acute kidney injury. Four cats were suspected to have chronic pyelonephritis. Based on the reference interval for serum phosphorus concentration at our institution (0.84–1.94 mmol/l), only two cats had hyperphosphataemia.

Seven cats were included in the control group. Distribution of breeds in the control group included five domestic shorthair, one Maine Coon and one Selkirk Rex.

Table 1 lists the variables for each group. Serum FGF-23 concentrations of cats with stages 1, 2 and 3–4 CKD were significantly higher than those of cats in the control group (P = 0.02, P = 0.002 and P = 0.002, respectively; Figure 1). In addition, cats with stages 3–4 had significantly higher concentrations of FGF-23 than those with stages 1 and 2 (P = 0.04 and P = 0.02, respectively), but there were no differences between cats with stages 1 and 2 (P = 0.97). Serum total calcium concentrations in stages 3–4 were higher than those of the control group (P = 0.03). Serum phosphorus and blood ionised calcium concentrations did not differ between groups.

Table 1

Median (range) and count of various characteristics of control cats and cats with chronic kidney disease (CKD)

	Control (n = 7)		CKD (n = 54)
			Stage 1 (n = 7)		Stage 2 (n = 32)		Stages 3–4 (n = 15)
	Median (range)	n	Median (range)	n	Median (range)	n	Median (range)	n
Sex (n)
Intact male/castrated	–	0/3	–	0/4	–	1/21	–	0/8
Intact female/spayed	–	4/0	–	0/3	–	2/8	–	0/7
Age (years)	3.8 (2.0–8.3)	7	4.2 (1.9–8.2)	7	5.8 (1.3–8.9)	32	6.0 (2.0–8.2)	15
Body weight (kg)	3.9 (2.5–5.3)	7	3.7 (2.5–6.1)	7	4.3 (1.9–7.9)	32	4.0 (2.4–6.1)	15
FGF-23 (pg/ml)	115 (88–194)^a	7	284 (119–746)^b	7	365 (94–8720)^b	32	1378 (182–14978)^c	15
UN (mmol/l)	8.2 (7.3–9.8)^a	7	8.5 (6.2–11.7)^a	7	8.3 (5.8–12.6)^b	32	19.2 (11.4–72.9)^c	15
Creatinine (µmol/l)	70 (56–104)^a	7	126 (111–138)^b	7	191 (143–244)^c	32	327 (248–1099)^d	15
Total calcium (mmol/l)	2.42 (2.30–2.58)^a	7	2.67 (2.32–3.27)^a,b	7	2.67 (2.32–3.72)^a,b	32	3.02 (2.37–3.32)^b	15
Phosphorus (mmol/l)	1.32 (1.10–1.45)	7	1.19 (0.97–1.45)	7	1.08 (0.77–1.87)	32	1.32 (0.52–5.55)	15
Ionised calcium (mmol/l)	–	0	1.28 (1.24–1.46)	6	1.31 (1.15–1.82)	28	1.33 (1.19–1.67)	15
HCO₃^- (mmol/l)	–	0	17.6 (17.2–23.2)	6	18.1 (13.0–23.7)	28	17.3 (12.7–24.2)	15
Haematocrit	41.0 (29.7–58.4)	6	41.0 (24.0–46.0)	7	34.5 (25.7–47.0)	28	31.0 (16.0–45.0)	14
USG	1.046 (1.040–1.063)^a	7	1.023 (1.017–1.032)^b,c	6	1.025 (1.010–1.052)^b	25	1.015 (1.009–1.024)^c	12
UPC (g/gCre)	0.13 (0.06–0.18)	7	0.27 (0.10–0.64)	4	0.09 (0.01–4.44)	19	0.04 (0.02–4.12)	6
SBP (mmHg)	127 (116–142)^a	6	130 (110–142)^a	6	138 (84–161)^a	18	150 (133–221)^b	12
Renal diet	–	0	–	3	–	13	–	9

Different superscript letters within rows indicate statistical differences

FGF = fibroblast growth factor; UN = urea nitrogen; USG = urine specific gravity; UPC = urine protein:creatinine ratio; SBP = systolic blood pressure

Figure 1

Serum fibroblast growth factor-23 concentrations in control cats and cats with chronic kidney disease. Boxes represent the 25th and 75th percentiles and the central lines in the boxes represent the median values. Whiskers extend from the minimum to the maximum values. Circles represent outliers. Significant values are indicated with asterisks.

Table 2 shows the correlation analysis results. When variables that significantly correlated with log FGF-23 were included in the multiple linear regression analysis, log urea nitrogen, log phosphorus and log ionised calcium independently predicted log FGF-23 (Table 3).

Table 2

Univariable correlations between (log-transformed) fibroblast growth factor-23 and other parameters in control cats and cats with chronic kidney disease

Variable	Correlation coefficient	P value
Age	0.09	0.491
Log body weight	−0.240	0.061
Log urea nitrogen	0.670	<0.001
Log creatinine	0.648	<0.001
Log total calcium	0.479	<0.001
Log phosphorus	0.502	<0.001
Log ionised calcium	0.448	0.001
Bicarbonate	0.242	0.094
Haematcrit	−0.573	<0.001
Log urine specific gravity	−0.586	<0.001
Log urine protein:creatinine ratio	0.317	0.06
Log systolic blood pressure	0.415	0.008

Table 3

Results of a multivariable regression analysis to predict (log) fibroblast growth factor-23

Independent variable	Unstandardised coefficient		Standardised cofficient	P values
	B	SE
Constant	−1.157	0.552		0.049
Log (urea nitrogen [mmol/l])	1.433	0.334	0.411	<0.001
Log (phosphorus [mmol/l])	1.656	0.520	0.328	0.004
Log (ionised calcium [mmol/l])	6.121	1.155	0.544	<0.001

Adjusted R² = 0.786; Durbin-Watson ratio = 1.986B = coefficient; SE = standard error

Discussion

The present study demonstrated that serum FGF-23 concentrations in 1–8-year-old cats with CKD were significantly higher than those in the control group. Additionally, urea nitrogen, phosphorus and ionised calcium were independent predictors of FGF-23 in cats aged 1–8 years.

In humans with CKD, blood FGF-23 levels increase while GFR decreases; this is true not only for middle-aged and geriatric patients,^17–20 but also in paediatric and younger patients with CKD.^21–24 The results of our study, in which serum FGF-23 concentrations increased with advanced stage CKD, are consistent with those of previous studies assessing geriatric or various-aged cats.^8,9 Additionally, in our study, serum FGF-23 concentrations of cats with stage 1 were significantly higher than those of the control group. This result is in line with those of a previous study, which demonstrated that plasma FGF-23 concentration increases in cats with non-azotaemic CKD.¹⁵ Moreover, in the present study, although most cats with CKD had normophosphataemia and serum phosphorus concentrations did not differ between each group, cats with CKD had higher serum FGF-23 concentrations than control cats. As such, FGF-23 may act as an early marker of CKD-MBD, regardless of the cat’s life stage.

The median (range) of serum FGF-23 concentrations in the control group was 115 pg/ml (88–194 pg/ml) and lower than the upper limit of reference interval (700 pg/ml) reported in geriatric cats.⁸ A recent study investigated the association between a cat’s life stages and serum FGF-23 concentrations and showed that there was no effect of age on FGF-23 in healthy cats.²⁸ The difference in FGF-23 levels of healthy cats between the studies can be caused by variation of FGF-23 assay rather than age. Thus, the reference interval of feline FGF-23 should be determined per FGF-23 assay.

In the present study, ionised calcium was associated with FGF-23. A previous study of geriatric cats did not evaluate ionised calcium, which the authors described as a study limitation.⁸ Our study demonstrated that FGF-23 levels were associated with a calcium metabolic marker having biological activity.²⁹ At this time, the detailed mechanism for the regulation between FGF-23 and calcium remains unknown.¹ A study in mice reported that dietary calcium supplementation increased serum FGF-23 levels independently of vitamin D receptors.³⁰ A similar association between FGF-23 and calcium was revealed in rats using continuous intravenous calcium loading.³¹ These rodent studies suggest that blood calcium levels can directly increase circulating FGF-23 levels. Thus, the association between FGF-23 and ionised calcium observed in our study may be explained by the direct effect of blood calcium on FGF-23. Further studies to determine the role of calcium on FGF-23 in cats are needed.

The present study had various limitations. First, the sample size was small. This may have contributed to our inability to detect a statistical difference. Additionally, the study included a limited number of cats with stage 4 CKD or hyperphosphataemia. Second, this study was retrospective in design and was thus unable to assess causation. Information for ionised calcium in the control group could not be obtained and affected the result of a multiple linear regression analysis. Third, parathyroid hormone levels were not measured; as such, the causes of hypercalcaemia could not be identified.

Conclusions

Serum FGF-23 concentrations of young and mature adult cats with CKD increased in early stage and were associated with mineral metabolic markers. Thus, FGF-23 may be an early indicator of CKD-MBD in younger cats, as geriatric cats.

Supplemental Material

Appendix 1

Feline FGF-23 assay validations.

Footnotes

Acknowledgements

We thank FUJIFILM VET Systems for the analysis of FGF-23. In addition, the authors would like to thank Enago () for English language review.

Accepted: 22 July 2021

Supplementary material

The following file is available online:

Appendix 1: Feline FGF-23 assay validations.

Conflict of interest

The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Yuichi Miyagawa is in receipt of speaker honoraria from FUJIFILM VET Systems.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Ethical approval

This work involved the use of non-experimental animals only (including owned or unowned animals and data from prospective or retrospective studies). Established internationally recognised high standards (‘best practice’) of individual veterinary clinical patient care were followed. Ethical approval from a committee was therefore not specifically required for publication in JFMS.

Informed consent

Informed consent (either verbal or written) was obtained from the owner or legal custodian of all animal(s) described in this work (experimental or non-experimental animals) for the procedures undertaken (prospective or retrospective studies). No animals or humans are identifiable within this publication, and therefore additional informed consent for publication was not required.

ORCID iD

Hirosumi Miyakawa

Mizuki Ogawa

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Supplementary Material

Please find the following supplemental material available below.

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Serum fibroblast growth factor-23 concentrations in young and mature adult cats with chronic kidney disease

Abstract

Objectives

Methods

Results

Conclusions and relevance

Keywords

Introduction

Materials and methods

Results

Discussion

Conclusions

Supplemental Material

Appendix 1

Footnotes

Acknowledgements

Supplementary material

Conflict of interest

Funding

Ethical approval

Informed consent

ORCID iD

References

Supplementary Material