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. Author manuscript; available in PMC: 2026 Feb 22.
Published in final edited form as: Eur J Pharm Sci. 2025 Sep 9;214:107265. doi: 10.1016/j.ejps.2025.107265

Distinct roles of ferric carboxymaltose and ferric derisomaltose on phosphate homeostasis in iron deficiency anemia

Jessica A Dominguez Rieg a,b,c, Monika Domenech Acevedo a, Jennifer Nogueira Coelho a, Monica Stevens a, Linto Thomas a,b, Timo Rieg a,b,c,*
PMCID: PMC12924155  NIHMSID: NIHMS2141103  PMID: 40935025

Abstract

Intravenous (IV) iron-carbohydrate nanoparticles like ferric carboxymaltose (FCM) and ferric derisomaltose (FDI) are used to treat iron deficiency anemia (IDA); however, they differ in their side-effects (e.g., hypo-Phosphatemia). We compared the effects of FCM and FDI in a newly generated mouse model of IDA and determine their efficacy in resolving IDA and effects on mineral homeostasis. Eight-week-old female C57Bl/6J mice were fed an iron-deficient diet for 5 weeks followed by IV bleeding (0.7 % of body weight) for 3 consecutive days to establish IDA. On day 1 and 7 after induction of IDA, mice were injected with vehicle, FCM or FDI (both 20 mg kg−1). On day 14, blood, urine and tissues were collected. Compared to baseline, all mice developed microcytic hypochromic anemia. FCM and FDI treatment resolved IDA, reversed thrombocytosis, and prevented the development splenomegaly and cardiomegaly observed in vehicle-treated anemic mice. Plasma iron increased to a greater extent with FCM versus FDI. Plasma iron showed an inverse relationship with intact fibroblast growth factor 23 (iFGF23) and C-terminal FGF23 (cFGF23). The ratio of iFGF23:cFGF23 increased in all groups but for different reasons: vehicle (iFGF23↑, cFGF23↔); FCM and FDI (iFGF23↔, cFGF23↓). The ratio was ~1.8-fold greater in FDI versus FCM at the end of the experimental period. Only FCM caused hypophosphatemia, despite the abundance of the renal Na+-Pi cotransporter 2a being similarly lower (~30 % versus vehicle) with FDI. Our results demonstrate that while FCM and FDI are equally effective at resolving IDA, hypophosphatemia is not solely caused by renal mechanisms.

Keywords: Anemia, DMT1, Intravenous iron, Kidney, Npt2a, Phosphate

1. Introduction

Iron is an important component of various cellular processes (e.g., DNA synthesis, cell proliferation, production, respiration, oxygen transfer, energy transfer). In adults, erythrocytes utilize the bulk of the body’s iron stores (~70 % of the 3–5 g total), while the remainder is primarily stored in the liver. Given the low daily absorption of dietary iron (~2 mg day−1), the body relies on reticuloendothelial macrophages to recover the majority of iron required (~30 mg day−1) from senescent erythrocytes in the spleen (Galy et al., 2024). Notably, iron deficiency or overload can have detrimental effects on the body. Iron overload, also called hemochromatosis, can be hereditary or induced by repeated blood transfusions. Conversely, iron deficiency anemia (IDA) is a global health problem and a significant health concern. Treatment for IDA entails iron store replenishment; oral iron supplementation is typically the first-line choice, with intravenous iron administered in cases of severe deficiency or when there is a poor response or adverse events from oral supplementation.

Intravenous iron is delivered via colloids that consist of spheroidal iron-carbohydrate nanoparticles where the iron-oxyhydroxide core is surrounded by a carbohydrate shell (Danielson, 2004). Based on the molecular weight of the carbohydrate shell, these agents can be further subcategorized into low and high molecular weight compounds. Currently approved intravenous iron preparations include: ferric carboxymaltose (FCM), ferric derisomaltose (FDI), iron sucrose, low molecular weight iron dextran, ferric gluconate, and ferumoxytol (Auerbach and Ballard, 2010). Globally, FCM currently holds the largest market share (~40 %), followed by iron sucrose (~30 %), iron dextran (~14 %), with the remaining market share (~16 %) distributed among other formulations (Hambley et al., 2020).

Notably, variations in nanoparticle core size and carbohydrate shell chemistry are critical determinants of the pharmacological profile and biological effects of intravenous iron formulations, including but not limited to, systemic clearance, rate and extent of iron release, onset of iron bioactivity, maximum tolerated dose, and maximum infusion rate. A growing body of evidence highlights hypophosphatemia as an increasingly recognized adverse effect associated with FCM administration. In severe cases, this hypophosphatemia can persist for several months (Fierz et al., 2014) and has been implicated in the development of osteomalacia and insufficiency fractures (Bartko et al., 2018; Callejas-Moraga et al., 2020; Tozzi and Tozzi, 2020). The reported incidence of FCM-induced hypophosphatemia exhibits variability depending on the underlying treated condition and can reach ~70 % in female patients with IDA secondary to heavy menstrual bleeding (Seid et al., 2017; Van Wyck et al., 2009). The proposed mechanism involves an increase in the phosphaturic hormone fibroblast growth factor 23 (FGF23); however, the precise molecular mechanism remains incompletely understood (Boots and Quax, 2022). The observed supraphysiological levels of FGF23 are hypothesized to induce internalization of the renal sodium-phosphate cotransporter 2a (Npt2a), consequently leading to phosphaturia (Baum et al., 2005; Fenton et al., 2014).

In the current study, we aimed to compare the effectiveness of intravenous FCM and FDI in resolving IDA and the development of hypophosphatemia in a novel mouse model of hemorrhage-induced IDA. In this model, no controlled studies have systematically investigated the effects of FCM and FDI on hormones involved in Pi homeostasis, transporters involved in iron and Pi transport, and biomarkers of bone metabolism. Our data demonstrate that intravenous iron administration quickly replenishes iron homeostasis independent of the iron preparation used. However, development of hypophosphatemia is only observed with FCM treatment. We further provide evidence that extended periods of untreated IDA, where plasma iron concentrations are the lowest, are associated with the highest plasma FGF23 levels. This study has important clinical implications for patients with IDA as well as the preference on choice of drugs for resolution of IDA.

2. Materials and methods

2.1. Animals and ethics

All animal experimentation was conducted in accordance with the Guide for Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD) and was approved by the local Institutional Animal Care and Use Committee (protocol #11,592). Age-matched 8-week-old female C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Female mice were used because studies in humans have demonstrated that the prevalence of iron-deficiency anemia is higher in females compared to males (Wolf et al., 2020). Mice were housed under a 12:12-hour light-dark cycle in isolated ventilated cages with free access to standard rodent chow (TD.2018; Envigo, Madison, WI) and tap water. All experiments outlined below were performed at the same time (9:00) of the day.

2.2. Studies on standard and iron-deficient diet

2.2.1. Blood collection on standard rodent diet

After 2 weeks of acclimatization, all mice underwent spontaneous urine collections and were subsequently anesthetized with isoflurane for baseline blood collections from the retrobulbar plexus. Blood and urine samples were analyzed as described below.

2.2.2. Low dietary iron and induction of IDA

After baseline blood collections, mice were placed on iron-deficient diet (4 ppm iron; TD.80396, Envigo, USA). After 5 weeks on the iron-deficient diet, mice were anesthetized with isoflurane and IDA was induced by venous bleeding (0.7 % of body weight [b.w.]) for 3 consecutive days. On the last day of bleeding (day 0; “anemia”), blood was kept for analysis and spontaneously voided urine was collected.

2.3. Intravenous FCM and FDI administration to mice with IDA

One day after induction of IDA (day 1), mice were randomized to vehicle (saline, 2 μL g−1 b.w.; n = 16), FCM (20 mg kg−1; n = 12, American Regent, Shirley, NY), or FDI (20 mg kg−1; n = 12, Pharmacosmos Therapeutics, Morristown, NJ) treatment via retroorbital injection. Treatments were repeated on day 7. The investigator administering FCM, FDI, or vehicle was blinded to treatment identity. A small (<10 μL) blood sample was collected daily (from day 1 to day 13) from the tail in minicaps® (Hirschmann Laborgeräte, Eberstadt, Germany) to monitor hematocrit measured by a micrometer caliper.

2.4. Terminal organ, blood, and urine collections

On day 14, spontaneous voided urine was collected, and mice were subsequently anesthetized with isoflurane for terminal blood collection. Under isoflurane anesthesia, spleen, liver, intestine, and heart were harvested and weighed. Spleen and half the liver were processed for iron measurements (see below). Kidneys were snap frozen and processed for immunoblot analysis as described below. Intestine and the remaining liver were fixed in 4 % paraformaldehyde and processed for immunofluorescent staining as described below.

2.4.1. Clinical chemistry, blood and urine analyses

Complete blood counts were performed using a Vetscan HM II (Abaxis, Parsipanny, NJ). All clinical chemistry was performed using commercially available assays that were modified to work with small volumes (Blanco et al., 2025; Thomas et al., 2019). Urinary creatinine was determined using Pointe Scientific Creatinine Reagent (Canton, MI, USA), phosphate (Pi) and Ca2+ in plasma and urine were determined using Inorganic Pi Reagent and Calcium Arsenazo III Reagent (Pointe Scientific), respectively, and iron was analyzed by Iron Reagent (Pointe Scientific). Plasma hormones were measured using enzyme-linked immunosorbent assays (ELISA) for PTH (cat. #60–2305, Quidel, San Diego, CA), intact FGF23 (cat. #60–6800, Quidel) and C-terminal FGF23 (cat. #60–6300, Quidel) (Xue et al., 2022). Bone resorption and formation markers were measured using ELISAs for tartrate-resistant acid phosphatase isoform 5b (TRAcP 5b, cat. #8033, Quidel), osteocalcin (cat. #8001, Quidel), procollagen type 1 N-propeptide (P1NP, cat. #AC–33F1, Immunodiagnostic Systems, Gaithersburg, MD), and type 1 Collagen Cross-Linked C-Telopeptide (CTX-1, cat. #AC–02F1, Immunodiagnostic Systems).

2.5. Immunoblot analysis

Kidney tissue was homogenized in dissection buffer (250 mmol L−1 sucrose and 10 mmol L−1 triethanolamine) containing protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany) and Halt phosphatase inhibitor cocktail (Thermo Fisher Scientific, Middletown, VA) as described previously (Poulsen et al., 2025; Thomas et al., 2024). The homogenate was centrifuged at 1000 g for 15 min, and the resultant supernatant was further centrifuged at 17,000 g for 30 min. Pellets were resuspended and used for immunoblotting. Equal lane loading (30 μg) was achieved using a Bio-Rad DC Protein assay (Bio-Rad Laboratories, Hercules, CA). Samples were resolved on NuPAGE 4 %– 12 % or 12 % Bis-Tris gels in MOPS. Gel proteins were transferred to PVDF membranes and immunoblotted with rabbit polyclonal antibodies against type II Na+-phosphate cotransporter (Npt2a, dilution 1:1000, (Fenton et al., 2014)), divalent metal transporter 1 (DMT1, dilution 1:500, courtesy of François Canonne-Hergaux, Université de Toulouse, France; (Canonne-Hergaux et al., 1999)), and β-actin (dilution 1:30,000, mouse, Sigma-Aldrich, St. Louis, MO). Detection was performed with secondary antibodies against rabbit (IRDye® 800CW donkey anti-rabbit IgG, dilution 1:5000) or mouse (IRDye® 680RD donkey anti-mouse IgG, dilution 1:5000) and detected with an Odyssey® CLx (LI-COR Biosciences, Lincoln, NE). Densitometric analysis was performed using Image Studio Lite (LI-COR Biosciences).

2.6. Immunofluorescent staining and quantification

Intestine and liver were fixed in 4 % paraformaldehyde in PBS overnight and embedded in paraffin. Sections were deparaffinized, rehydrated, and incubated in 3 % H2O2 for 10 min. Slides were then placed in citrate buffer for antigen retrieval and heated in a rice cooker for 45 min, incubated in 0.2 % Triton-X for 15 min, blocked with 10 % normal goat serum, and then rabbit anti-mouse primary antibodies against ferroportin (10 μg mL−1, MTP11-A, Alpha Diagnostic International, San Antonio, TX, USA) or hepcidin (10 μg mL−1, HEPC11-A, Alpha Diagnostic International) were applied in blocking buffer and incubated overnight at 4 °C. The specificity of the ferroportin (Schwartz et al., 2019) and hepcidin (Malerba et al., 2020) antibodies has been reported previously. Slides were then incubated with 0.2 % Triton-X for 10 min and washed with PBS. The secondary antibody (Texas Red™ goat anti-rabbit, 10 μg mL−1; Vector Laboratories, Newark, CA, USA) was applied in a dilution of 1:200 in blocking buffer and incubated for 1 h at room temperature in the dark. The slides were washed with PBS before being mounted with VECTASHIELD® Antifade Mounting Medium with DAPI (Vector Laboratories, Newark, CA). Slides were analyzed and pictures were taken on an Olympus BX53 digital upright microscope (Center Valley, PA, USA).

Tissue slides were fully scanned using an Olympus VS120 slide scanner under automatic exposure settings to ensure uniform image acquisition across all slides. Image quantification was performed in ImageJ (NIH, Bethesda, MD) using a customized macro script designed to minimize operator-dependent variability. At the beginning of the analysis, a user-defined region of interest position was set, and the macro automatically adjusted it to a fixed rectangular region of 6000 × 3000 pixels to standardize measurements across images. The macro then split the channels, subtracted background fluorescence (using fixed offset values for each channel), applied thresholding (Shanbhag image threshold for Texas Red™ and default threshold for DAPI), and measured integrated signal intensity. Quantification was expressed as the Texas Red™/DAPI ratio, representing relative protein expression per nucleus. The investigator performing this analysis was blinded to treatment identity.

2.7. Determination of tissue iron content

After spleen and liver harvesting, the collected tissues were dried for 24 h at 50 °C. Following the drying process, the weight of each tissue was determined. Next, the tissues were incinerated at a temperature of 560 °C for 12 h in a muffle furnace (Thermolyne F48015–60, Thermo Fisher Scientific). The ashes from the incineration were dissolved in 0.75 mol L−1 HCl. The concentration of iron in the dissolved samples was determined by iron reagent (Pointe Scientific).

2.8. Statistical analyses

The data are expressed as mean ± S.E.M. and analyzed with paired Student’s t-test, one- and two-way ANOVA or two-way mixed-effects ANOVA followed by the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli or Tukey multiple comparison tests, as indicated in the figure legends (all data analyzed via GraphPad Prism or SigmaPlot, San Jose, CA, USA). Significance was considered at P < 0.05.

3. Results

3.1. Characterization of the IDA model

In preliminary studies, we were not able to observe IDA when mice were subjected solely to a prolonged (up to 6 weeks) iron-deficient dietary regimen (data not shown). To establish a robust model of IDA, we then studied mice that were on an iron-deficient diet for 5 weeks in combination with intravenous bleeding. Consistent with the induction of IDA, this model showed hypochromic microcytic anemia compared to baseline (Fig. 1a-d). Other significant changes included increased thrombocytes (Fig. 1f), increased plasma Pi (Fig. 1g), and a decrease in PTH (Fig. 1i). In contrast, white blood cell count (WBC, Fig. 1e), plasma Ca2+ (Fig. 1h), and intact FGF23 (iFGF23; Fig. 1j) were not significantly affected by induction of IDA. Notably, C-terminal FGF23 increased ~2.8-fold in anemic mice (Fig. 1k), consequently resulting in a significant reduction in the intact:C-terminal FGF23 ratio (Fig. 1l).

Fig. 1. Induction of IDA affects plasma Pi and PTH levels.

Fig. 1.

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After induction of IDA, (A) hematocrit, (B) number of red blood cells, (C) hemoglobin, and (D) MCV significantly decreased. (E) The number of white blood cells was unaffected, but (F) thrombocyte numbers significantly increased. (G) Plasma Pi significantly increased, but (H) plasma Ca2+ was unaffected. (I) PTH also decreased in response to induction of IDA, (J) no effect was observed on intact FGF23, but (K) C-terminal FGF23 significantly increased. Consequently, there was a significant reduction in the intact:C-terminal FGF23 ratio (L). In addition to individual data, mean ± S.E.M. are shown. Data were analyzed by a paired Student’s t-test. n = 40 female mice. #P < 0.05 versus baseline.

3.2. Effects of FCM and FDI treatment on resolution of IDA

Anemic mice were randomly assigned to treatment with either vehicle, FCM, or FDI which were administered on day 1 and 7. A time course of the hematocrit changes is shown in Fig. 2a. Recovery of hematocrit started immediately after administration of the first FCM or FDI dose, and after 8 days (1 day after the 2nd injection), reached levels comparable to those observed in non-anemic mice on a standard control diet. As evidence of resolution of IDA, red blood cell count (Fig. 2b), hemoglobin (Fig. 2c), and mean corpuscular volume (MCV, Fig. 2d) significantly increased after FCM and FDI treatment. In contrast, in vehicle-treated anemic mice, these parameters further decreased during the 14-day experimental period. WBC count (Fig. 2e) significantly decreased in vehicle-treated mice, which was prevented in anemic mice treated with FCM and FDI. Of note, vehicle-treated mice developed significant thrombocytosis (~1.7-fold increase) at the end of the experimental period (Fig. 2f); in contrast, FCM and FDI treatment slightly reduced thrombocyte numbers.

Fig. 2. FCM and FDI are equally effective in resolving IDA.

Fig. 2.

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(A) Daily time-course of hematocrit changes in vehicle-, FCM-, or FDI-treated anemic mice. (B) Red blood cell count, (C) hemoglobin levels, and (D) MCV recovered similarly in response to FCM and FDI treatment. FCM and FDI treatment prevented the decrease in (E) white blood cell count observed in vehicle-treated mice and decreased (F) thrombocyte count. Inset shows the correlation between iron concentration and thrombocyte numbers. Data are expressed as mean ± S.E.M and were analyzed by a repeated measures mixed-effects model followed by Tukey’s multiple comparisons test. n = 12–16 female mice/group. *P < 0.05 versus vehicle, #P < 0.05 versus baseline, §P < 0.05 versus FCM.

3.3. Effects of FCM and FDI treatment on iron, minerals, and hormones

As expected, FCM and FDI treatment (Fig. 3a) replenished iron stores, leading to a ~2–3-fold increase in plasma iron levels. Notably, at the end of the experimental period on day 14, plasma iron levels were ~1.5-fold greater in FCM- versus FDI-treated mice. In vehicle-treated anemic mice, iron levels continued to decline over the course of the 14-day experimental period. Plasma Pi levels were not significantly affected by vehicle or FDI treatment but significantly decreased in response to FCM treatment (Fig. 3b). Plasma Ca2+ levels significantly increased in all 3 treatment groups (Fig. 3c). The major hormones that regulate Pi and Ca2+ homeostasis are PTH and FGF23. In vehicle-treated anemic mice, PTH was significantly decreased on day 14 compared to day 0 (Fig. 4a), whereas no significant changes in PTH were observed in anemic mice treated with FCM or FDI. Intact FGF23 increased (~3-fold) in vehicle-treated anemic mice compared to FCM- or FDI-treated mice (Fig. 4b), where FGF23 remained unchanged on day 14. C-terminal FGF23 (Fig. 4c) remained unchanged in vehicle-treated mice, while mice treated with FCM and FDI exhibited significantly reduced C-terminal FGF23 at the end of the experimental period. The ratio of intact:C-terminal FGF23 (Fig. 4d) increased independent of treatment but for different reasons in vehicle-treated (intact FGF23 ↑, C-terminal FGF23 ↔) compared to FCM- or FDI-treated mice (intact FGF23 ↔, C-terminal FGF23 ↓). Notably, the ratio at the end of the experimental period was ~1.8-fold greater in FDI-compared to FCM-treated mice. Plotting iron concentrations versus intact and C-terminal FGF23 levels showed an inverse relationship between these parameters: at the end of the experimental period, vehicle-treated mice showed the highest FGF23 levels in the presence of the lowest iron concentrations (Fig. 4e, f).

Fig. 3. FCM and FDI replenish plasma iron but only FCM causes reductions in plasma Pi levels.

Fig. 3.

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(A) Plasma iron levels significantly increased in FCM- and FDI- treated mice after 14 days, although levels were higher with FCM compared to FDI treatment. (B) A Pi lowering effect was only seen with FCM treatment. (C) Total plasma Ca2+ increased in all groups regardless of treatment. Data are expressed as mean ± S.E.M. and were analyzed by a repeated measures mixed-effects model followed by Tukey’s multiple comparisons test. n = 12–16 female mice/group. *P < 0.05 versus vehicle, #P < 0.05 versus baseline, §P < 0.05 versus FCM.

Fig. 4. FCM treatment caused hypophosphatemia despite similar levels of PTH and FGF23 in FCM- and FDI-treated mice.

Fig. 4.

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(A) At the end of the experimental period, PTH was significantly lower in vehicle-treated anemic mice, whereas FCM and FDI treatment did not affect PTH levels. (B) Intact FGF23 significantly increased in vehicle-treated mice, but no effect was seen in FCM- and FDI-treated mice. (C) C-terminal FGF23 remained unchanged in vehicle-treated mice, while FCM- and FDI-treated mice had significantly reduced C-terminal FGF23. (D) The ratio of intact:C-terminal FGF23 increased independently of treatment but was highest in FDI-treated mice. There is an inverse correlation between plasma iron levels and (E) intact FGF23 levels and (F) C-terminal FGF23 levels. Data are expressed as mean ± S.E.M. and were analyzed by a repeated measures mixed-effects model followed by Tukey’s multiple comparisons test. n = 12–16 female mice/group. *P < 0.05 versus vehicle, #P < 0.05 versus baseline, §P < 0.05 versus FCM.

3.4. Effect of resolution of IDA on bone formation and resorption markers

Iron can directly (via affecting proliferation, differentiation, etc.) or indirectly (via changes in PTH/FGF23) affect bone health (von Brackel and Oheim, 2024). The bone formation markers osteocalcin (Fig. 5a) and P1NP (Fig. 5b) significantly decreased in vehicle-treated anemic mice; however, the levels of these markers were maintained in mice treated with FCM or FDI. In contrast, the bone resorption markers TRAcP 5b (Fig. 5e) and CTX-1 (Fig. 5f) significantly decreased irrespective of treatment. A dose-response effect and correlation were found between plasma iron concentrations and osteocalcin (Fig. 5c) and P1NP (Fig. 5d), respectively. No correlations were found between plasma iron concentrations and TRAcP 5b (Fig. 5g) or CTX-1 (Fig. 5h).

Fig. 5. Resolution of IDA affects bone formation and resorption markers.

Fig. 5.

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Prolonged IDA resulted in a significant decrease in bone formation markers (A) osteocalcin and (B) P1NP, an effect prevented by FCM or FDI treatment. A dose-response effect and correlation were found between plasma iron concentrations and (C) osteocalcin and (D) P1NP, respectively. (E) All groups showed a significant decrease in the bone resorption marker TRAcP 5b at the end of the experimental period compared to baseline. (F) Bone resorption marker CTX-1 levels significantly decreased in vehicle-treated mice, an effect prevented by FCM and FDI treatment. No correlations were found between plasma iron concentrations and (G) TRAcP 5b or (D) CTX-1. Data are expressed as mean ± S.E.M. and were analyzed by a repeated measures mixed-effects model followed by Tukey’s multiple comparisons test. n = 12–16 female mice/group. *P < 0.05 versus vehicle, #P < 0.05 versus baseline.

3.5. Renal expression of Npt2a and DMT1

In terms of overall homeostasis, the kidney is the major organ regulating the fine-tuning of excreted minerals and electrolytes. FGF23 is a major regulator of renal Npt2a abundance. Therefore, protein abundances of the iron transporter DMT1 and the Pi transporter Npt2a were compared at the end of the experimental period on day 14. Both FCM and FDI treatment resulted in a significantly greater DMT1 abundance (~1.3-fold) compared to vehicle treatment (Fig. 6a). Protein abundance of Npt2a was ~30 % lower in FCM- and FDI-treated mice compared to vehicle-treated anemic mice (Fig. 6b).

Fig. 6. Resolution of IDA by FCM and FDI causes greater DMT1 and lower Npt2a expression in the kidney compared to vehicle-treated anemic mice.

Fig. 6.

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At the end of the experimental period, kidney immunoblots of renal membrane preparations showed (A) greater DMT1 expression in FCM- and FDI-treated mice compared to vehicle-treated mice. (B) Expression of Npt2a was significantly lower in FCM- and FDI-treated mice compared to vehicle-treated mice. Data are expressed as mean ± S.E.M. and were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. n = 10 for DMT1 groups, n = 8 for Npt2a groups. *P < 0.05 versus vehicle.

3.6. Effects of FCM and FDI treatment on spleen, liver, heart, and intestine

In vehicle-treated anemic mice, harvested spleens were macroscopically grossly enlarged, with spleen weights being ~3-fold greater compared to FCM- and FDI-treated mice (Fig. 7a). Iron content in both FCM- and FDI-treated mice was ~3-fold and ~8-fold greater in liver (Fig. 7b) and spleen (Fig. 7c), respectively, compared to vehicle-treated mice, consistent with these organs being major iron storage compartments. No significant differences were observed between FCM- and FDI-treated groups. IDA can cause an increase in cardiac mass due to volume overload (Rakusan et al., 2001). Consistent with this, vehicle-treated anemic mice showed greater heart-to-body weight ratios, which was prevented in FCM- and FDI-treated mice (Fig. 7d). No significant differences in body weights were observed between groups at the end of the experimental period (vehicle: 21 ± 0.4 g, FCM: 22 ± 0.5 g, FDI: 21 ± 0.4 g). Plasma iron levels are regulated by the hepcidin-ferroportin axis. In the small intestine, ferroportin is required for basolateral iron export from enterocytes (Donovan et al., 2005), which is inversely regulated by hepatic hepcidin (Nicolas et al., 2001). Quantification of ferroportin fluorescence intensity showed there was significantly stronger expression in the basolateral membrane along duodenal villi of vehicle-treated mice compared to FCM- and FDI-treated mice (Fig. 8). No differences in staining intensity were observed between FCM and FDI treatment. Correspondingly, hepcidin fluorescence intensity was significantly weaker in the liver of vehicle-treated mice, while expression in FCM- and FDI-treated mice was stronger (Fig. 9). No differences in staining intensity were observed between FCM and FDI treatment.

Fig. 7. FCM and FDI increase splenic and hepatic iron content and prevent IDA-induced cardiomegaly.

Fig. 7.

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(A) Vehicle-treated anemic mice had significant splenomegaly which was absent in FCM- and FDI-treated mice. (B) Spleen and (C) liver iron content was significantly greater with FCM and FDI treatment compared with vehicle-treated mice. (D) Vehicle-treated mice had larger heart-to-body weight ratios indicating cardiomegaly, which was prevented in mice treated with either FCM or FDI. Data are expressed as mean ± S.E.M. and were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. n = 6 female mice/group for B and C, n = 12–16 female mice/group for A and D. *P < 0.05 versus vehicle.

Fig. 8. Basolateral ferroportin labeling is absent in duodenal villi of FCM- and FDI-treated mice.

Fig. 8.

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At the end of the experimental period, ferroportin labeling (red fluorescence) in the small intestine of FCM- and FDI-treated mice was visually absent compared to vehicle-treated mice. Nuclei are stained with the marker DAPI (blue fluorescence). Representative images are shown which were taken with identical laser settings. Scale bar: 100 μm. Quantitative analysis of immunofluorescent labeling showed a significantly lower fluorescence intensity in FCM- and FDI-treated mice compared to vehicle-treated mice. Data are expressed as mean ± S.E.M. and were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. n = 3–4 female mice/group *P < 0.05 versus vehicle.

Fig. 9. Resolution of IDA results in enhanced liver hepcidin labeling.

Fig. 9.

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At the end of the experimental period, hepcidin labeling (red fluorescence) in the liver of FCM- and FDI-treated mice was visually stronger compared to vehicle-treated mice. Nuclei are stained with the marker DAPI (blue fluorescence). Representative images are shown which were taken with identical laser settings. Scale bar: 100 μm. Quantitative analysis of immunofluorescent labeling showed a significantly greater fluorescence intensity in FCM- and FDI-treated mice compared to vehicle-treated mice. Data are expressed as mean ± S.E.M. and were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. n = 3–4 female mice/group *P < 0.05 versus vehicle.

4. Discussion

IDA poses a significant global health challenge. While oral iron supplementation is often the first approach in treatment, its efficacy can be limited. Consequently, intravenous iron administration is frequently necessary to effectively resolve iron deficiency and alleviate debilitating symptoms like drowsiness, fatigue, and lethargy. To our knowledge, this is the first in vivo study directly comparing effects of intravenous FCM and FDI in a novel mouse model of IDA. Importantly, this study directly compares equivalent doses of FCM and FDI. This is a crucial distinction, as prior clinical studies (Wolf et al., 2020) were criticized for using a higher total dose of FCM (1500 mg from two consecutive 750 mg injections) compared to a single 1000 mg injection of FDI. This could have confounded observations regarding a higher incidence of hypophosphatemia and associated hormonal changes with FCM. While our findings demonstrate comparable efficacy between FCM and FDI in resolving IDA, we observed differences in the final plasma iron concentrations, intact:C-terminal FGF23 ratios, and plasma Pi concentrations. Consistent with our findings, a clinical study using 20 mg kg−1 of either FCM or FDI (Emrich et al., 2020) reported a higher prevalence of hypophosphatemia after FCM administration: ~75 % of women treated with FCM developed plasma Pi levels <0.65 mmol L−1, compared to only 8 % in the FDI treatment group. The occurrence of severe hypophosphatemia (<0.6 mmol L−1) following FCM treatment has also been observed in other clinical studies (Adkinson et al., 2018; Schaefer et al., 2016; Wolf et al., 2020).

Phosphate homeostasis is primarily regulated by PTH and FGF23. In our study, vehicle-treated mice exhibited a decrease in PTH by the end of the experimental period, which could be attributed to elevated plasma Ca2+ levels. Interestingly, despite similarly elevated plasma Ca2+ in FCM- and FDI-treated mice, no corresponding changes in PTH were noted. In patients with chronic kidney disease, circulating Ca2+ levels were strongly linked to anemia independent of PTH (Boronat et al., 2017). This suggests that other factors, such as vitamin D3, may influence Ca2+ concentrations in IDA. Our study comprehensively analyzed the changes of intact (bioactive) FGF23 and C-terminal FGF23 (capturing intact and cleaved FGF23), in IDA and following its resolution with FCM or FDI. In IDA, it is known that C-terminal FGF23 levels rise while intact FGF23 levels remain relatively stable, consequently leading to a low intact:C-terminal FGF23 ratio and plasma Pi levels that are minimally affected (Edmonston and Wolf, 2020). In our study, induction of IDA also resulted in a low intact:C-terminal FGF23 ratio. However, by the end of the experimental period, vehicle-treated anemic mice showed a doubling of intact FGF23 levels without impacting levels of C-terminal FGF23. This suggests that furin-mediated cleavage of FGF23 (Al Rifai et al., 2021) might have reached a maximum capacity by day 14, supporting increased FGF23 production from osteocytes (Farrow et al., 2011). FCM-induced hypophosphatemia is hypothesized to result from a transient increase in intact FGF23, a mechanism thought to be influenced by the carbohydrate moieties of the iron preparation (Emrich et al., 2020; Wolf et al., 2013). A meta-analysis of 42 clinical trials showed that hypophosphatemia was more common with FCM (47 %) versus FDI (4 %) (Schaefer et al., 2021). While initial responses of intact and C-terminal FGF23 to FCM or FDI treatment were comparable, our study revealed a greater intact:C-terminal FGF23 ratio in FDI-treated mice at the end of the experimental period. Consistent with similar reductions of C-terminal FGF23 in response to FCM and FDI treatment, the major renal Pi transport protein, Na+-Pi cotransporter Npt2a, was equally reduced (~35 %) compared to vehicle treatment. Based on the timing of the blood collections in the current study, it might be possible that a transient change of intact FGF23 shortly after FCM or FDI administration might have been missed. Regarding the influence of iron status on FGF23, our study demonstrates an inverse relationship between plasma iron and the levels of both intact and C-terminal FGF23. This finding corroborates previous data from healthy humans and humans with autosomal dominant hypophosphatemic rickets (Imel et al., 2011). Consequently, this implies that contributions from other organs (e.g., intestine and bone) may account for the observed differences in plasma Pi between FCM and FDI treatment.

As anticipated, our study demonstrated significantly lower iron levels in the liver and spleen of vehicle-treated anemic mice compared to those treated with FCM or FDI. Furthermore, vehicle-treated mice developed significant splenomegaly, likely a consequence of extramedullary hematopoiesis (Rivera-Torruco et al., 2024), and a greater heart-to-body weight ratio, which were not observed in the FCM- or FDI-treated mice. However, the heart-to-body weight ratio only provides an indirect measure of cardiac hypertrophy compared to tibia length-corrected ratios. These findings are consistent with previous studies in mice lacking intestinal DMT1, which also exhibit hypochromic-microcytic anemia alongside splenomegaly and cardiomegaly (Shawki et al., 2015).

Replenishing plasma iron levels not only resolved red blood cell abnormalities but also normalized WBC counts and thrombocytosis. Reactive thrombocytosis is a common occurrence in IDA, stemming from increased megakaryopoietic differentiation (Evstatiev et al., 2014). This condition affects ~33 % of IDA patients, and deep vein thrombosis or pulmonary embolism in patients with IDA and concurrent thrombo cytosis was present in ~16 % of cases (Song et al., 2020). Our results confirm the development of severe thrombocytosis in IDA and demonstrate that FCM and FDI treatment reverse the development. Furthermore, our results revealed an inverse relationship between plasma iron concentration and thrombocyte numbers: the lowest plasma iron levels corresponded with the highest thrombocyte counts. Interestingly, despite comparable improvements in other hematological parameters (e.g., hematocrit, hemoglobin, red blood cells, MCV), FCM treatment led to significantly greater plasma iron concentrations compared to FDI.

This study, to our knowledge, is the first to directly compare changes in bone formation and resorption markers in IDA and following intravenous iron treatment. Notably, vehicle-treated anemic mice showed reductions in all measured bone formation (osteocalcin and P1NP) and resorption markers (TRAcP 5b and CTX-1). This aligns with previous studies in rats (Katsumata et al., 2006) confirming that IDA is associated with significantly lower osteocalcin levels. P1NP levels mirrored the changes seen with osteocalcin. P1NP is considered the most promising marker of bone formation in osteoporosis due to its minimal circadian and biological variation, lack of influence from food intake, and stability after blood collection (Schini et al., 2023). We found that the reduction in bone formation markers was prevented when IDA mice were treated with either FCM or FDI. These findings are consistent with a study in women with IDA, which demonstrated that osteocalcin levels were maintained by FCM administration over the two-week study period (Frazier et al., 2020). In contrast, in patients with IDA secondary to inflammatory bowel disease, both FCM and FDI treatments were associated with a temporary decrease in P1NP (Zoller et al., 2023). Interestingly, the bone resorption marker TRAcP 5b appears to have no clear diagnostic implications in IDA, as our study found its levels were neither sustained nor corrected by iron treatment; instead, vehicle-, FCM-, and FDI-treated mice with IDA all showed a significant decrease in TRAcP 5b at the end of the experimental period. The reason for this treatment-independent decrease remains elusive but could imply that TRAcP 5b levels are governed by other factors (etiology of anemia, presence or absence of inflammation and others). One previous study showed that iron deficiency increased TRAcP 5b and CTX-1; however, despite minor hematocrit differences between the studied groups, hematocrit in the iron deficiency groups was ~50 % (= normal range) indicating that this model did not develop IDA (Scott et al., 2022). Consequently, the presence or absence of anemia might have opposing effects on TRAcP 5b and CTX-1 and could be a possible predictor if anemia will develop in the face of iron deficiency in the context of bone density changes. Our study did not analyze bone structure or bone mineral density; however, the changes in bone formation and resorption markers may indicate an overall reduction in bone formation, warranting further investigation. While a correlation between iron intake and bone density has been observed in postmenopausal women (Harris et al., 2003), it’s important to consider that many other factors can influence these findings, and observational studies often extrapolate conclusions from correlations.

For the kidney and intestine, the iron content in the diet inversely correlates with DMT1 abundance (Qatato et al., 2022; Wareing et al., 2003; Zoller et al., 2001). Our study was not designed to perform a comparison of DMT1 abundance in iron deficient versus normal iron diet. All mice were on an iron deficient diet, and thus we would expect DMT1 abundance to be increased prior to any treatments. However, we found that FCM and FDI treatment resulted in even greater DMT1 expression compared to vehicle treatment. Likewise, patients with hereditary hemochromatosis show inappropriately greater duodenal DMT1 mRNA expression despite iron overload (Stuart et al., 2003; Zoller et al., 1999). This suggests that total body iron stores may exert opposing effects on intestinal DMT1 abundance compared to dietary iron content and may explain why intravenous iron repletion (e.g., with FCM or FDI) has similar effects on DMT1 as in iron overload. Currently, studies investigating renal DMT1 in this context are lacking. Consistent with increased total body iron following FCM and FDI treatment, our findings show enhanced hepatic hepcidin expression and attenuated enterocyte ferroportin expression compared to vehicle-treated anemic mice. This reciprocal regulation of hepcidin and ferroportin is well established, where elevated hepcidin levels can block iron export by occlusion or degradation of ferroportin (Nemeth and Ganz, 2021). Accumulation of iron in the liver was shown to upregulate hepcidin expression (Nicolas et al., 2001) and knockout of ferroportin in intestinal epithelial cells resulted in marked iron accumulation in duodenal enterocytes (Donovan et al., 2005). If these changes contribute to the previously reported differences in the microbiome in response to FCM and FDI (Rieg et al., 2023) remains to be determined.

5. Conclusion

In summary, our data indicate that FCM and FDI are equally effective in resolving IDA as well as common associated pathologies (leukopenia, thrombocytosis, splenomegaly, and cardiomegaly). A key discussion point in the field concerns the interchangeability of these intravenous iron-carbohydrate nanoparticle solutions (Di Francesco et al., 2020, 2019). Despite their classification as iron polymaltose complex “similars”, it is increasingly clear that their side-effect profiles differ significantly. Interestingly, while we observed difference in plasma Pi and the ratio of intact:C-terminal FGF23 between FCM and FDI, there were no differences in the expression of renal Npt2a. This suggests that the distinct effects on plasma Pi are not solely mediated through Npt2a regulation. Therefore, further studies are needed to elucidate the specific mechanisms contributing to changes in plasma Pi, given the otherwise highly similar hormonal and transporter responses to FCM and FDI.

Funding

This work was supported by a NIH R25DK134324 (to J.D.R. and T.R.), a VA Merit Review Award IBX004968A (to T.R.), an American Heart Association Career Development Award (24CDA1271666 to L.T.), and a USF Hypertension and Kidney Research Center Multi-PI Pilot Award (to T.R.). The contents do not represent the views of the U.S. Department of Veterans Affairs or the U.S. Government.

Footnotes

Declaration of competing interest

The authors declare that there are no competing interests associated with the manuscript.

CRediT authorship contribution statement

Jessica A. Dominguez Rieg: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Monika Domenech Acevedo: Writing – review & editing, Investigation, Formal analysis, Data curation. Jennifer Nogueira Coelho: Writing – review & editing, Visualization, Software, Investigation, Formal analysis. Monica Stevens: Writing – review & editing, Investigation, Formal analysis, Data curation. Linto Thomas: Writing – review & editing, Investigation, Formal analysis, Data curation. Timo Rieg: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Data availability

Data will be made available on request.

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