Age and sex effects on FGF23-mediated response to mild phosphate challenge

Abstract

Background:

During aging, there is a normal and mild loss in kidney function that leads to abnormalities of the kidney-bone metabolic axis. In the setting of increased phosphorus intake, hyperphosphatemia can occur despite increased concentrations of the phosphaturic hormone FGF23. This is likely from decreased expression of the FGF23 co-receptor Klotho (KL) with age; however, the roles of age and sex in the homeostatic responses to mild phosphate challenges remain unclear.

Methods:

Male and female 16-week and 78-week mice were placed on either normal grain-based chow or casein (higher bioavailable phosphate) diets for 8 weeks. Gene expression, serum biochemistries, micro-computed tomography, and skeletal mechanics were used to assess the impact of mild phosphate challenge on multiple organ systems. Cell culture of differentiated osteoblast/osteocytes were used to test mechanisms driving key outcomes.

Results:

Aging female mice responded to phosphate challenge by significantly elevating serum intact FGF23 (iFGF23) versus control diet; males did not show this response. Male mice, regardless of age, exhibited higher kidney KL mRNA with similar phosphate levels across both sexes. However, males and females had similar blood phosphate, calcium, and creatinine levels irrespective of age, suggesting that female mice upregulated FGF23 to maintain blood phosphorus, and compromised renal function could not explain the increased serum iFGF23. The 17β-estradiol levels were not different between groups, and in vivo bone steroid receptor (estrogen receptor 1 [Esr1], estrogen receptor 2 [Esr2], androgen receptor [Ar]) expression was not different by age, sex, or diet. Trabecular bone volume was higher in males but decreased with both age and phosphate challenge in both sexes. Cortical porosity increased with age in males but not females. In vitro studies demonstrated that 17β-estradiol treatment upregulated FGF23 and Esr2 mRNAs in a dose-dependent manner.

Conclusions:

Our study demonstrates that aging female mice upregulate FGF23 to a greater degree during a mild phosphate challenge to maintain blood phosphorus versus young female and young/old male mice, potentially due to direct estradiol effects on osteocytes. Thus, the control of phosphate intake during aging could have modifiable outcomes for FGF23-related phenotypes.

1.1. Introduction

Control of phosphorus homeostasis is mediated by the actions of the osteocyte-produced hormone Fibroblast growth factor-23 (FGF23). FGF23 acts in concert with its co-receptor, Klotho (KL), and fibroblast growth factor receptors (FGFRs), which form a heteromeric signaling complex[1]. Circulating concentrations of phosphorus are regulated through altering reabsorption by the kidneys[2–4]. FGF23 acts in the kidney to downregulate the sodium-dependent phosphate transporters (NPT2a and NPT2c) to reduce phosphate reabsorption[5,6]. In parallel, FGF23 reduces the expression of the anabolic vitamin-D metabolizing enzyme 25-OH vitamin D 1α-hydroxylase and upregulates the catabolic 1,25-OH vitamin D 24-hydroxylase to lower circulating 1,25(OH)₂ vitamin D (1,25D)[7].

As people age, a variety of physiological changes occur, including reduced cardiac function, muscle wasting, bone loss, and cognitive impairment. Many of these features have been recapitulated in Fgf23^−/− and Kl^−/− mice with premature aging phenotypes that are associated with the perturbation of phosphate homeostasis, causing hyperphosphatemia[8]. Studies have established that FGF23 levels increase while KL levels may decrease with age[9–12]. However, little is known about whether cellular mechanisms differ between young and old individuals and whether mild phosphate challenges induced through the consumption of a Western diet (higher than normal phosphate) further alter mineral metabolism.

Around age 45, females begin to maintain higher basal phosphate and FGF23 levels compared to males, which persists into old age[13]. Limited studies have focused on the mechanisms underlying these age-related sex effects. Given that the divergence in phosphate/FGF23 response between males and females is around the time of menopause, a role of estrogen seems likely. Indeed, estrogen depletion results in increased reabsorption and thus higher systemic phosphate levels, and estrogen is associated with increased FGF23 and KL in vivo[14–16]. Further, women receiving estrogen therapy had lower phosphate levels compared to untreated women but comparable levels to age-matched men[17], suggesting a role of estrogen in this bone-kidney regulatory axis.

Chronic elevations of FGF23 lead to several adverse physiological changes including increased risk of fracture and cardiovascular disease, both associated with higher mortality[9,18]. For example, patients with CKD undergoing dialysis are at 4–13 times higher risk of hip fractures than the general population, and elevated FGF23 is associated with six-fold higher odds for death in this patient population[19,20]. How conditions with which chronic elevations in FGF23 are impacted by sex and age is not fully understood, however.

The aim of the present study was to test the hypothesis that advanced age leads to divergent effects with respect to sex on FGF23, its regulatory pathways, and bone in response to a phosphate challenge, which may mimic what occurs in the elderly population.

1.2. Materials & Methods

1.2.1. Animal Studies:

Animal studies were approved by and performed according to the Institutional Animal Care and Use Committee (IACUC) at the Indiana University School of Medicine and comply with the NIH guidelines for the use of animals in research. Sixteen- and seventy-eight-week-old C57BL/6 wild-type male and female mice (n=6–8/age/sex) were purchased (JAX stock #000664, Bar Harbor, ME, USA) and acclimated prior to all studies. After one-week acclimatization to our facility, mice were placed on either a standard grain-based rodent diet (0.7% total phosphate and 0.4% non-phytate phosphorus, 2018SX, Harlan Teklad = Ch) or a casein-based rodent diet[21] (0.9% highly bioavailable phosphate and 0.6% calcium, TD.150303 Envigo; Madison, WI = Ca) for eight weeks (group sizes of 6–8). Diets and water were provided ad libitum throughout the study. Blood samples were collected from mice by facial vein bleed at days 0, 14, 28, and 42 of the study. Mice were euthanized by CO₂ inhalation followed by cervical dislocation, and blood was collected via cardiac puncture for endpoint serum and plasma biochemistries.

1.2.2. Serum Biochemistries:

Routine serum biochemistries were tested by the Core facility of the Clinical and Translational Sciences Institute (CTSI) of the Indiana University School of Medicine using an automated COBAS MIRA Plus Chemistry Analyzer (Roche Diagnostics; Indianapolis, IN). Serum iFGF23, cFGF23, intact PTH, and 17β-estradiol concentrations were assessed using commercial ELISAs for mouse/rat (Quidel, Inc.; San Diego, CA and Abcam; Cambridge, UK).

1.2.3. RNA Preparation and Quantitative RT-PCR (qPCR):

Kidney, liver, bone, and flushed bone marrow from long bones were harvested and homogenized in 1mL of Trizol reagent (Invitrogen/Life Technologies, Inc.; Grand Island, NY) according to the manufacturer’s protocol using a Bullet Blender (Next Advance, Inc.; Troy, NY), then further purified using the RNeasy Kit (Qiagen, Inc.; Germantown, MD). RNA samples were tested with intron-spanning primers (Applied Biosystems/Life Technologies, Inc; Grand Island, NY) specific for fibroblast growth factor 23 (Fgf23), klotho, vitamin D 24-hydroxylase (Cyp24a1), 1α-hydroxylase (Cyp27b1), IL-1β, IL-6, TNFα, estrogen receptor α (Esr1), estrogen receptor β (Esr2), and androgen receptor (AR) mRNAs (primers were purchased as pre-optimized reagents (Applied Biosystems/Life Technologies, Inc; Grand Island, NY); mouse actin was used as an internal control (IDT; Coralville, IA). The Taqman One-Step RT-PCR kit was used to perform qPCR. PCR conditions for all experiments were: 30min 48°C, 10min 95°C, followed by 40 cycles of 15s 95°C and 1min 60°C. The data was collected and analyzed by a StepOne Plus system (Applied Biosystems/Life Technologies, Inc.; Grand Island, NY). The expression levels of mRNA were calculated relative to the appropriate controls, and the 2^−ΔΔCT method was used to analyze the data[22].

1.2.4. Aortic Calcification Assay:

Segments of the aortic arch were isolated and incubated in 0.6N HCl for 48h, the supernatant was analyzed for calcium using the o-cresolphthalein complex 1 method (Pointe Scientific; Canton, MI), and the data was normalized by tissue dry weight as previously described[23].

1.2.5. Bone Microarchitecture:

The distal third of the femur was scanned using the Skyscan 1172 μCT (Bruker; Billerica, MA) using an isotropic voxel size of 8μm. Trabecular parameters were obtained from taking a 1mm sized region of interest (ROI) just proximal to the distal growth plate. Trabecular bone volume per tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N) were measured. Cortical parameters (porosity and thickness, Ct.Th) were obtained as the average of five consecutive slices from a region located ~2mm proximal to the most proximal region of the trabecular bone ROI. All analyses and nomenclature were in accordance with recommended protocols[24].

1.2.6. Bone Mechanics:

Tibial midshafts were scanned using the Skyscan 1176 μCT (Bruker; Billerica, MA) using an isotropic voxel size of 9μm. The total length and the medial-lateral diameter were measured with calipers. Bones were thawed and hydrated with saline, and tibiae were tested by four-point bending (ElectroForce; New Castle, DE) with the medial and lateral surfaces in compression and tension, respectively. Testing was carried out at a displacement rate of 0.025mm/sec. After the samples were tested until failure, the length of the distal fragment was measured to determine the site of fracture for later use in geometric calculations to determine material-level mechanical properties. A custom MATLAB code was used to generate geometric information, and a second MATLAB code was used for data collection[25]. Structural parameters include ultimate force, stiffness, displacement, and work; material parameters include stress, strain, modulus, and toughness. These properties were estimated using standardized equations that convert force and displacement into stress and strain, respectively, by accounting for bone geometry[26]. All mechanical parameters are presented using standard nomenclature[27].

1.2.7. In Vitro Studies:

MPC2 (mesenchymal stem cells that can be induced to osteocytes) cells were cultured in complete media (α-MEM supplemented with 10% FBS, 25mM L-glutamine, and 25mM penicillin/streptomycin) at 33°C until 80% confluent[28]. The cells were then transferred to osteogenic media (complete media supplemented with 4mM beta-glycerophosphate and 50μg/mL ascorbic acid) and differentiated at 37°C for 3 or 3.5 weeks. Cells were treated with either 0.01mM or 0.1mM estradiol (doses according to [15,29]) (Sigma-Aldrich; Burlington, MA) for 24h with and without 10nM 1,25(OH)₂D (Millipore Sigma; Cleveland, OH) for 24h. The cells were then harvested for RNA using the RNeasy Kit (Qiagen, Inc.; Germantown, MD) according to the manufacturer’s protocol for cells.

1.2.8. Statistical Analysis:

Murine data were analyzed as a 2×2 factorial ANOVA (diet-by-age) with main effects of diet, age, and diet-by-age interactions noted as well as effect sizes (ES); male and female data were analyzed separately. Effect sizes were used to compare and quantify the difference between males and females for respective categories. If the model 2×2 ANOVA was statistically significant (p<0.05), an all-groups Duncan post hoc analysis was applied to determine differences between groups. Intact FGF23 analysis was performed by repeated measures ANOVA followed by a Tukey post hoc test. In vitro data was analyzed as a one-way ANOVA; if the ANOVA was statistically significant (p<0.05), a Dunnett post-hoc analysis was applied to determine differences between vehicle and each respective group (SPSS Statistics 25; IBM, Armonk, NY, USA). All data are presented as mean ± standard deviation (SD).

1.3. Results

1.3.1. Under phosphate challenge, aging mice maintain normal biochemical regulation.

To assess the influence of dietary phosphate, young and aging male and female mice were placed on either a control (chow) or a casein-based diet that has higher phosphate bioavailability and thus mimics higher phosphate intake. At the end of the study, neither female nor male mice had differences in serum phosphate when assessed by age (young vs. old) and diet (chow vs. casein) (2×2 ANOVA p=0.177 and p=0.207, respectively; Figure 1A). For both sexes, the age/diet effects for serum calcium and creatinine were not different (Table 1), suggesting minimal changes in kidney function in the aging mice despite additional phosphate load.

Fig. 1.

Measures of chronic kidney disease (phosphate and parathyroid hormone (PTH), and intact FGF23 (iFGF23)). a Serum phosphate was not statistically different in male or female mice (p=0.177 and p=0.207, respectively). b PTH was elevated in older, male animals on casein diet as compared to those on the chow diet (diet p=0.019, ES=0.226) but there was no difference in female mice (p=0.541). c iFGF23 in male mice was elevated in aging animals and further elevated in those on casein diet (diet p=0.014, ES=0.265). d iFGF23 in female mice was higher overall as compared to male mice, with older animals on casein diet having the highest levels (diet p<0.0001, ES=0.788; age p<0.0001, ES=0.599; interaction p=0.004, ES=0.309). Group sizes of 6–8. Data plotted as mean and standard deviation with point plots representing individual animal data. Bars not sharing the same letter are statistically different.

Table 1.

Biochemical parameters.

	Male				Female
	16Ch (n=8)	16Ca (n=6)	78Ch (n=6)	78Ca (n=6)	16Ch (n=8)	16Ca (n=6)	78Ch (n=7)	78Ca (n=6)
Phosphate (mg/dL)	12.53±1.2	11.89±1.44	12.69±1.52	13.85±2.09	10.73±1.73	12.99±1.11	12.53±3.35	12.55±0.52
Calcium (mg/dL)	13.27±1.2	12.96±0.92	13.13±1.28	13.6±1.3	11.99±1.1	13.23±0.63	13.69±2.63	13.26±1.1
Alkaline Phosphatase (mg/dL)	67±5b	65±8b	78±33ab	100±23a	94±11	166±109	126±34	133±65
BUN (mg/dL)	21.61±4.77	25.53±3.1	23.53±3.56	24.34±3.81	25.21±3.38a	26.18±6.62a	22.77±7.83a	13.21±1.2b
Creatinine (mg/dL)	0.32±0.14	0.34±0.16	0.35±0.06	0.32±0.17	0.4±0.1	0.5±0.3	0.35±0.1	0.41±0.1
Serum Iron (mmol/L)	25.3±9.4a	16.8±3.7b	16±2.8b	14.8±7.7b	24.3±4.1	26.9±7.5	23.5±9.9	30.3±8.8
cFGF23 (pg/mL)	27.02±17.47	50.83±30.19	40.66±13.83	66.37±16.84	65.43±43.27	109.55±20.83	80.16±19.06	142.58±143.44
Serum PTH (pg/mL)	91.13±10.62b	184.24±70.56ab	120.46±28.73b	309.44±254.9a	80.14±27.18	117.8±88.51	82.68±34.54	109.52±68.48
Serum 17β-estradiol (pg/mL)	50.28±11.50	41.23±8.28	64.88±28.55	49.03±23.09	75.92±7.82	58.99±8.80	77.30±9.25	73.07±21.59

Ch – chow diet, Ca – casein diet. Data provided as mean and standard deviation. Columns not sharing the same letter are statistically significant (males and females analyzed separately).

1.3.2. Sex is a critical factor in determining maintenance of mineral metabolism following phosphate challenge in aging mice.

We next tested the hormones critical for maintaining phosphate homeostasis via the bone-kidney axis. There was a main effect of time for both male and female iFGF23 levels over the course of the study (p<0.0001) (Figure 1C and 1D). In both sexes, older mice fed the casein diet had higher levels of plasma iFGF23. Older females had higher iFGF23 (old > young, p<0.0001) with effect of diet (casein > chow, p<0.0001) and an age-by-diet interaction (p=0.004). Male mice fed the casein diet had higher iFGF23 (casein > chow, p=0.014, ES=0.265) without effect of age (p=0.065) or an interaction (p=0.095). FGF23 can also be processed into proteolytic fragments, therefore the ‘C-terminal’ or ‘total’ FGF23 ELISA was also used. There were no differences in final cFGF23 between groups for either females or males (Table 1). Females fed the casein diet had higher bone FGF23 mRNA expression (casein > chow, p=0.027, ES=0.221) without effect of age or an interaction (Table 2). There were no differences across groups for male mice. For marrow FGF23 mRNA expression, female and male mice showed no differences across groups (Table 2). Thus, with phosphate diet challenge, FGF23 was differentially increased in aging female mice.

Table 2.

Mineral metabolism gene expression data.

	Male				Female
	16Ch (n=8)	16Ca (n=6)	78Ch (n=6)	78Ca (n=6)	16Ch (n=8)	16Ca (n=6)	78Ch (n=7)	78Ca (n=6)
Bone Fgf23	1.199±0.72	1.045±0.72	2.125±1.58	1.045±0.59	1.105±0.49b	1.222±0.59b	1.374±0.84b	2.852±1.18a
Marrow Fgf23	1.259±0.94	0.523±0.45	1.046±0.68	0.800±0.97	1.189±0.82	1.282±0.71	2.125±2.59	2.426±2.46
Kidney Klotho	1.008±0.14	1.030±0.08	1.609±1.04	1.578±1.13	1.011±0.15a	0.773±0.06ab	0.677±0.10ab	0.530±0.12b
Kidney Cyp24a1	1.067±0.43	2.011±1.53	0.788±0.55	1.328±0.84	1.069±0.42b	0.848±0.56b	1.080±0.51b	1.933±0.72a
Kidney Cyp27b1	1.245±0.89	1.188±0.54	1.965±1.29	1.962±1.51	1.112±0.56b	3.028±1.90ab	2.933±2.23ab	5.130±2.52a

Ch – chow diet, Ca – casein diet. Data provided as mean and standard deviation. Columns not sharing the same letter are statistically significant (males and females analyzed separately).

Diet had a significant effect on PTH levels in male mice (p=0.019, ES=0.226) but no effect of age and no age-by-diet interaction (Figure 1B). Older male mice fed the casein diet had higher PTH compared to both young and old mice receiving the chow diet. Female mice had no differences in serum PTH across groups (Figure 1B). There were no differences in calcium levels across groups in female or male mice (2×2 ANOVA p=0.078 and p=0.813, respectively) (Table 1).

To begin exploring potential mechanisms underlying the biochemical findings, gene expression was assessed in several tissues. In older females fed the casein diet, elevated FGF23 mRNA expression was associated with elevated kidney KL, kidney Cyp24a1, and kidney Cyp27b1 mRNA expression (Table 2).

Older females exhibited increased kidney KL mRNA expression (old > young, p<0.0001, ES=0.643) with an effect of diet (casein > chow, p<0.0001, ES=0.444) but no age-by-diet interaction. Older females also exhibited increased kidney Cyp24a1 mRNA expression (old > young, p=0.02, ES=0.222) with no effect of diet (p=0.164) but an interaction (p=0.023, ES=0.215); kidney Cyp27b1 mRNA expression was also higher in older females (old > young, p=0.02, ES=0.233) with an effect of diet (casein > chow, p=0.015, ES=0.25) but no interaction effect (Table 2). There were no differences between groups in male mice for kidney KL, Cyp24a1, or Cyp27b1 mRNA expression (Table 2).

In addition to transcriptional changes in genes involved in mineral metabolism, increased phosphate intake can also be linked to markers of tissue inflammation and calcification. Indeed, the kidney showed increased IL-6 and TNFα mRNAs, and the liver showed increased IL-1β mRNA expression (Supplementary Table 1). Older mice had the highest levels of aortic calcification, but mild phosphate challenge did not contribute to worsening calcification over the time course tested (Supplementary Table 1).

1.3.3. Aging mice exhibit reduced trabecular bone volume, increased cortical porosity, and altered bone mechanical properties, which are independent of phosphate challenge.

To determine whether phosphate challenge influenced bone properties over the time course tested, several static and functional parameters were tested.

Trabecular bone volume and cortical bone thickness were higher in young mice regardless of sex. In this regard, young female mice had higher trabecular bone volume (young > old, p<0.0001, ES=0.767) without effect of diet or an interaction effect; young male mice also had higher trabecular bone volume (young > old, p<0.0001, ES=0.683) without effect of diet or an interaction effect (Figure 2A). There were no differences in trabecular thickness between groups of female and male mice (Supplementary Table 2). Female mice fed the casein diet exhibited greater trabecular separation (casein > chow, p=0.001, ES=0.394) without effect of age or an interaction; older male mice had greater trabecular separation (old > young, p<0.0001, ES=0.854) without effect of diet or an interaction (Supplementary Table 2). Female mice fed the casein diet had higher trabecular number (casein > chow, p=0.001, ES=0.394) without effect of age or an interaction; older male mice had higher trabecular number (old > young, p<0.0001, ES=0.696) without effect of diet or an interaction effect (Supplementary Table 2).

Fig. 2.

Bone parameters and mechanical properties. a Bone volume significantly decreased with age in both male and female mice (p<0.0001, ES=0.683 and p<0.0001, ES=0.767, respectively). b Cortical porosity increased significantly with age in both male and female mice (p=0.002, ES=0.348 and p=0.003, ES0.319, respectively). c Representative μCT images of female (top – 16Ch, 16Ca, 78Ch, 78Ca) mouse femurs. d Representative μCT images of male (bottom – 16Ch, 16Ca, 78Ch, 78Ca) mouse femurs. e Ultimate force significantly decreased with age in female mice (p=0.001, ES=0.406), but there were no differences in male mice. f Total work was significantly different with age in both male and female mice (p=0.041, ES=0.192 and p=0.005, ES=0.323). Group sizes of 6–8. Data plotted as mean and standard deviation with point plots representing individual animal data. Bars not sharing the same letter are statistically different.

Older males had increased cortical porosity (old > young, p=0.002, ES=0.348) without effect of diet or an interaction (Figure 2B). In female mice, increased porosity was affected by age (old > young, p=0.003, ES=0.319) and diet (casein > chow, p=0.048, ES=0.16) with a strong interaction between the variables (p=0.002, ES=0.347) (Figure 2B). Older female mice receiving the casein diet exhibited the highest levels of porosity of all the female groups.

To determine the bone mechanical properties across groups, 4-point bending of the tibia was performed. Young females had higher ultimate force (young > old, p=0.001, ES=0.406) without effects of diet or interaction; there were no differences in ultimate force between groups in male mice (Figure 2E). Young female mice exhibited higher total work (young > old, p=0.005, ES=0.323) without effect of diet or an interaction; young male mice also exhibited higher total work (young > old, p=0.041, ES=0.192) but no effect of diet or an interaction (Figure 2F). Young females had higher ultimate stress (young > old, p=0.002, ES=0.368) and resilience (young > old, p=0.007, ES=0.297), but ultimate stress and resilience were not different between groups of male mice. For female and male mice, there were no differences in total displacement, stiffness, or total strain (Supplementary Table 2). Thus, natural aging negatively influences normal bone properties, with females performing slightly worse than males.

1.3.4. Administration of exogenous estradiol upregulates Fgf23 and estrogen receptor levels in vitro.

We next tested potential differences in sex steroids across sexes. There were no differences in plasma 17β-estradiol concentration between groups in male and female mice (2×2 ANOVA p=0.256 and p=0.07, respectively), consistent with reports across all ages in humans[30] (Table 1). There were no differences across groups in male or female mice for bone mRNA expression of Esr1, Esr2, or Ar (Supplementary Table 3).

To examine mechanistic effects of estradiol in isolation on bone cells, we used a novel MSC line that can be differentiated into osteocytes[28], like primary MSCs[31]. The cells were treated at two stages of differentiation (3 and 3.5 weeks) with estradiol and/or 1,25(OH)₂D, hormones that potentially influence Fgf23 transcription. After 24h, there was a main effect of estradiol treatment on Fgf23 mRNA expression between groups at 3 weeks of differentiation (p=0.038, ES=0.631) (Figure 3A). Cells treated with the higher estradiol dose (0.1mM) alone exhibited statistically higher Fgf23 expression as compared to the vehicle control (p=0.033); there were no differences in cells treated with 1,25(OH)₂D or the lower estradiol dose (0.01mM). For Esr1 mRNA expression, there were no differences between groups (Figure 3B). For Esr2 expression, there was a main effect of treatment between groups (p<0.0001, ES=0.898) (Figure 3C); cells treated with the higher estradiol dose alone exhibited statistically higher Esr2 expression as compared to the vehicle (p=0.001). At 3.5 weeks of differentiation, there was no effect of treatment on Fgf23 expression or Esr1 expression; however, there was a main effect of treatment on Esr2 mRNA between groups (p=0.001, ES=0.855). Cells treated with the higher estradiol dose exhibited significantly higher Esr2 expression as compared to the vehicle-treated cells (p=0.002) (Figure 3C).

Fig. 3.

In vitro gene expression with estradiol treatment in osteoblast/osteocyte mesenchymal stem cell line. a FGF23 expression was significantly increased with 0.1mM estradiol treatment as compared to vehicle (p=0.033). Addition of 1,25-vitamin D did not produce any additional effect. b Esr1 expression was no different with any concentration of estradiol of 1,25-vitamin D treatment. c Esr2 expression was significantly increased with 0.1mM estradiol (p=0.001) and 0.1mM + 1,25-vitamin D treatment (p=0.002). Data plotted as mean and standard deviation with point plots representing individual animal data (n=3 for all treatments). *statistically different from all other groups (p<0.05).

In summary, our results show the presence of sex- and age-specific effects of moderate phosphate loading in mice. These collective data support that aging females employ mechanisms, potentially through the presence of estradiol, to increase FGF23 to maintain effective FGF23-KL signaling and thus normal serum phosphate concentrations, whereas male mice maintain KL expression. These changes may provide insight into differential mechanisms and regulation that are increasingly important during aging when phosphate clearance is reduced.

1.4. Discussion

Our study highlights sex, age, and diet differences in the management of phosphate metabolism. Both age and diet influenced iFGF23 in female mice, with older females fed the casein diet expressing the highest levels of iFGF23; although aging males fed the casein diet also expressed the highest iFGF23, the level was two-fold higher in older female mice. KL is widely reputed as the “anti-aging gene,” because its expression has been shown to decrease with age[10–12], and KL overexpression is associated with extended lifespan in both male and female mice[32]. Coincidentally, in the present study, KL mRNA levels decreased with age and diet only in females, whereas levels in male mice increased with age. Previous studies performed in human cohorts demonstrating elevations in kidney KL expression in both sexes may have been potentially confounded by combining male and female results[33,34] and/or normal versus various stages of CKD[35]. Considering that males and females may acquire CKD and proceed through the stages toward ESRD at different rates, gender variation in KL expression is especially interesting. Since males express less serum iFGF23 than females, it is plausible that KL expression is higher to facilitate more efficient FGF23-KL signaling. These results could indicate that older females use protective mechanisms, such as increasing FGF23 production, to maintain serum phosphate in the normal range, thus preventing mineral dysregulation. Although males upregulated kidney KL mRNA, these protective mechanisms may be somewhat compromised in older males. Although additional clinical studies are certainly required, the changes in KL could, in part, explain why males exhibit more severe kidney damage than females during progressive renal disease[36].

FGF23 is well-established as a hormone known to control phosphate metabolism via direct actions on the kidneys following its secretion from osteocytes. Indeed, disturbances in the FGF23-KL axis can lead to significant pathological consequences. The MrOS study found that when eGFR is below 60mL/min/1.73m² and renal function is considered poor, elevated FGF23 correlates with fracture risk in elderly men[37]. In our study, serum creatinine and BUN were not significantly elevated, supporting that there was not a major decline in kidney function following mild phosphate challenge, yet this did produce differential FGF23 expression to maintain similar phosphate levels. A previous study that evaluated biochemical parameters in an elderly population demonstrated that serum phosphate did not increase exponentially until eGFR dropped below 47mL/min/1.72m². This finding potentially illustrates that the mice in the current study maintained generally normal renal function, thus the observed patterns in FGF23 production and KL mRNA expression observed in human patients are likely due to other mechanisms, such as sex steroids, inflammation, or oxidative stress[38]. Indeed, we found that older female mice with normal renal function were able to respond to increased phosphate load by elevating FGF23.

Alterations in sex steroid signaling could be considered a likely mechanism for the observed gender differences in phosphate-sensitive responses, therefore in vivo and in vitro assays were performed. Estradiol was higher in females of each age and diet group as has been shown clinically, but older females did not exhibit the age-related drop in estrogens that are associated with human menopause. This could be due to difficulties in simulating human menopause as a result of a large proportion of female mice (60–70%) entering a state of constant estrus that can last for up to 100 days; in these cases, older female mice expressed stable estradiol levels, similar to observations in our study[39,40]. A previous study also found that administration of high amounts of estradiol caused increased Fgf23 and KL mRNA expression both in rats that had undergone ovariectomy and in UMR-106 rat osteosarcoma cells, respectively[15]. Our results recapitulated the increased Fgf23 mRNA expression with estradiol treatment in a novel mouse mesenchymal cell line as well as expansion into examining changes in estradiol receptor expression. This previous study did not investigate the downstream signaling effects of FGF23 upregulation and how this was influenced by sex differences, a notable strength of the present study.

There are several limitations to our study. First, the grain-based chow diet and the purified casein diet are not completely comparable. Although grain-based diets are relatively rich in phosphorus (0.7% in chow diet), the phosphorus is bound in the form of inositolphosphoric acids (phytate, “myo-inositol hexaphosphate”); previous studies have demonstrated that only a portion of the phosphorus is absorbed by the GI tract and is thus less bioavailable[41–44]. It is estimated that 0.4% may be bioavailable. In contrast, the casein diet contains casein-based protein and no phytate, so the phosphorus is almost completely bioavailable[45,46]. The two diets also utilize different protein sources/amounts of protein and amounts of fiber/different types of fiber. A future study could address diets with varied phosphorus availability as the sole difference between diets. Second, we did not measure glomerular filtration rate (GFR) in our mice, and thus minor changes in renal function may not have been identified. However, BUN was not different across groups, thus there were no signs of even mild CKD. Third, we did not utilize charcoal-stripped fetal bovine serum in our in vitro studies; thus, estradiol was likely present in the culture media. Our experiments did use similar estradiol concentrations as those reported as activating concentrations in bone cells, however we cannot rule out that our effects were seen at the highest doses due to the presence of physiological levels of this hormone. Indeed, a future study could be performed with MPC2 cells and charcoal-stripped serum. Key strengths of our study included paired sex and aged animals, and the fact that a slight increase in bioavailable phosphate through the casein diet did not induce CKD but rather mimicked potential increases in phosphate in an aging model. Further, the ability to test hormonal effects in a new in vitro system of cultured osteocytes that produce FGF23 demonstrated isolated effects of estrogen.

In conclusion, this study demonstrated that sex and age contribute to crossover effects on FGF23 and phosphate regulatory pathways. Aging was associated with increased aortic calcification, decreased trabecular bone mass, and increased cortical porosity, all of which are common signs of the aging process but were not FGF23-dependent. More importantly, our results demonstrate that the expression of KL mRNA differs between male and female mice, with males upregulating KL expression in aging compared to females. This observation could indicate that the sexes employ different mechanisms to maintain effective FGF23-KL signaling. Finally, controlling phosphate intake throughout aging could have modifiable outcomes for FGF23-related phenotypes.