Iron Sucrose: A Double‑Edged Sword in High Phosphate Media‑Induced Vascular Calcification
Ping Wang1 · Chengkun Guo1 · Hui Pan1 · Wangshan Chen1 · Dan Peng2

Received: 7 August 2020 / Accepted: 29 December 2020
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021

Abstract
The high incidence of vascular calcification (VC) in patients with chronic kidney disease (CKD) has become an important clinical subject. Hyperphosphatemia is a primary cause of CKD-related VC. Intravenous iron sucrose (IS) is commonly used to treat anemia in CKD patients, and is effective and well tolerated worldwide. However, the interaction between iron and VC remains controversial, and the underlying mechanisms are yet to be clarified. In the present study, ex vivo normal rat aortic rings were cultured with various concentrations of phosphate and IS, and the levels of calcium and iron depositions, oxidative injury, as well as phenotypic marker genes were detected. To the best of our knowledge, the present study is the first to report that IS is a double-edged sword in high phosphate media-induced VC which not only alleviates VC in a dose- dependent manner but also leads to iron overload in vasculature when in high concentration. IS is a promising agent for VC prevention in patients with hyperphosphatemia and iron deficiency. Meanwhile, the appropriate blood concentration of IS in patients with hyperphosphatemia needs to be explored clinically.
Keywords Iron sucrose · Vascular calcification · Hyperphosphatemia · Type III sodium-dependent phosphate cotransporter-1 · Oxidative injury

Introduction
There is a high incidence of vascular calcification (VC) in patients with chronic kidney disease (CKD), especially those with end-stage renal disease (ESRD), and VC is considered to be an independent risk factor for cardiovascular disease (CVD) [1, 2]. VC is harmful to the heart and vascular tis- sues, which is associated with the development of life-threat- ening cardio-cerebral vascular diseases such as myocardial infarction, stroke and malignant arrhythmia [3]. The molec- ular mechanisms and corresponding treatments for CKD- related VC are of great significance in medical research.

VC is generally believed to be an active process mediated by vascular smooth muscle cells (VSMCs), and includes various pathophysiological features such as VSMC pheno- typic transition, dysregulation of calcification-promoting and
-inhibiting factors, apoptosis and extracellular matrix dys- function [4]. Hyperphosphatemia is a common complication of CKD and a primary cause of CKD-related VC, resulting in a higher mortality rate in CKD patients [5–7]; however, the underlying mechanisms are far from clarified.
Iron deficiency is extremely common in CKD patients, which is the result of numerous pathophysiological fac- tors, including recurrent blood loss, reduced gastrointes- tinal absorption of dietary iron and an increased demand

for iron secondary to the use of erythropoiesis-stimulating

Ping Wang and Chengkun Guo have contributed equally to this work.
 Dan Peng
[email protected]
1 Nephrology Department, The First People’s Hospital of Jingmen, Jingmen 448000, Hubei, China
2 Neonatology Department, The First People’s Hospital of Jingmen, Jingmen 448000, Hubei, People’s Republic of China
agent (ESA). These factors promote and then make the repeating and obstinate happen of iron deficiency anemia (IDA), especially in those undergoing hemodialysis [8, 9]. Furthermore, oral iron supplementation is often insufficient for CKD patients [10]. Consequently intravenous (IV) iron is a necessary recommendation in the management of IDA [9, 11–13]. However, in CKD patients, the side effects of iron supplementation on CVD outcome have long prompted controversy [14–17]. Therefore, assessing the advantages

and disadvantages of iron supplementation is a key factor for dose optimization.
In clinical practice, available IV iron formulations are low molecular weight iron dextran (LMWID), sodium fer- ric gluconate (FG), iron sucrose (IS), ferric carboxymalt- ose (FCM), ferumoxytol (FMX), and iron isomaltoside 1000 (IIM) [18, 19]. IS is a commonly used form of IV iron worldwide, which is effective, well-tolerated and has been employed in many large-scale clinical investigations, such as the FACT and PIVOTAL trials [20, 21]. The results of the PIVOTAL trial were recently published online, which revealed that in patients undergoing hemodialysis, the use of a proactively administered high-dose regimen of IS (median monthly dose, 264 mg) resulted in a significantly lower risk of mortality or major non-fatal cardiovascular events, com- pared with that observed with a reactive low-dose (median monthly dose, 145 mg) [22]. This provides compelling evidence that IS has direct cardiovascular benefits in CKD patients with iron deficiency, though the underlying mecha- nisms remain unknown. Additionally, iron-containing agents were found to suppress VC in a number of rat CKD models [23–25]. Therefore, we speculate that IS may protect the cardiovascular system in CKD patients by relieving hyper- phosphatemia-induced calcification.
In the present study, ex vivo normal rat aortic rings were
cultured with various concentrations of phosphate and IS, and the levels of calcium and iron depositions, oxidative injury, as well as phenotypic marker genes were detected to investigate the effects and underlying mechanisms of IS on high phosphate media-induced VC.

Methods
Aortic Tissue Culture and Calcification Model

A total of twenty 8–10-week-old male Sprague–Dawley rats (280–300 g) were purchased from the Hubei Provincial Center for Disease Control and Prevention (Wuhan, China). The rats were adaptively fed for one week with a stand- ard balanced diet, and free access to both food and water. The thoracic aortas of the rats were harvested and cultured after anesthesia and decapitation as previously described [26]. Na2HPO4·12H2O and NaH2PO4·2H2O (in DMEM
(HyClone); 2.5 mM; pH 7.2–7.4) were used to create high phosphate media and induce subsequent calcification as previously described [26]. The aortic rings were divided into five groups: (i) Control group (CNT), normal condi- tions with 0.9 mM inorganic phosphorous (Pi); (ii) high Pi group (HP), normal conditions with 2.5 mM Pi; (iii) high Pi with low dose IS group (HPLFe), normal conditions with 2.5 mM Pi and 2 μg/ml IS; (iv) high Pi with medium dose IS group (HPMFe), normal conditions with 2.5 mM
Pi and 5 μg/ml IS; and (v) high Pi with high dose IS group (HPHFe), normal conditions with 2.5 mM Pi and 10 μg/ml IS. The corresponding media were replaced every 3 days. The aortic rings were cultured for a total of 14 days, and subsequently embedded in paraffin for storage.
Intracellular Calcium, Iron and Malondialdehyde (MDA) Content, and Superoxide Dismutase (SOD) Activity

Following harvest, the rat aortic rings were washed with PBS, treated with 0.6 N HCl overnight at 4 °C, and then lysed in RIPA lysis buffer as previously described [26]. Intracellular calcium, iron and MDA content, as well as SOD activity were colorimetrically detected using commer- cial kits purchased from Nanjing Jiancheng Bioengineer- ing Institute, according to the manufacturer’s protocol. The results were normalized to the respective cellular protein content and detected using a commercial BCA kit (Aspen Biotechnology Co).
Reverse Transcription‑Quantitative Polymerase Chain Reaction (RT‑qPCR)

Total RNA was isolated from the aortic rings using TRI- zol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The temperature protocol of the RT reaction, the qPCR conditions and the primers for the ɑ-SMA (α-smooth mus- cle actin), Runx2 (runt-related transcription factor 2), Pit1 (type III sodium-dependent phosphate cotransporter-1), and GAPDH (glyceradehyde-3-phosphate dehydrogenase) genes were as previously described [26]. The primer sequences for FGF23 (fibroblast growth factor-23) are as follows: 5′-TCA CATCAGAGGATGCTGGCT-3′ (forward) and 5′-CACCAG
GTAGTGATGCTTCGG-3′ (reverse). The expression levels of the genes were detected using the 2−ΔΔCq method [27].
Western Blotting

The western blot procedure, the primary antibodies for the Runx2, ɑ-SMA and GAPDH proteins, and the second- ary antibody were as previously described [26]. The pri- mary antibodies for Pit1 and FGF23 were rabbit anti-Pit1 (1:1,000; 12423-1-AP; ProteinTech Group, Inc.,) and rabbit anti-FGF23 (1:500; bs-5768R; Bioss).
Von Kossa Staining

The rat aortic rings were analyzed using standard Von Kossa staining as previously described [26]. Black or brown stain- ing, as viewed under a light microscope (Olympus Corpo- ration; magnification, × 200) indicates positive staining of calcium nodules.

Perls’ Prussian Blue Staining

To detect the presence of iron, the vascular rings were stained using Perls’ Prussian blue stain according to the manufacturer’s protocol (AS1105; Aspen). Samples exhib- iting a characteristic blue color under a light microscope (Olympus; magnification, × 200) were considered to be posi- tively stained.
Intracellular Reactive Oxygen Species (ROS) Production

Intracellular ROS production was examined using the fluo- rescent probe-dihydroethidium (DHE) assay, according to the manufacturer’s protocol (KGAF019; Nanjing KeyGen Biotech Co., Ltd.). The nuclei were stained with 4′,6-diamid- ino-2-phenylindole (AS1075; Aspen) for 5 min at room tem- perature, and the samples were visualized under an inverted fluorescence microscope (IX51; Olympus Corporation) with an imaging system (MicroPublisher; QImaging) at × 200 magnification. Blue fluorescence indicates the nuclei and the red fluorescence intensity represents relative ROS content.
Statistical Analysis

The data were presented as the mean ± standard deviation, and each experiment was conducted at least three times. Statistical analyses were performed using SPSS 13.0 soft- ware (SPSS, Inc). Significant differences between groups were evaluated using one-way ANOVA followed by Tukey’s post hoc test, and P < 0.05 was considered to be statistically significant.

Results
Alterations in Calcium Depositions

Von Kossa staining and calcium detection indicate the alter- nations of calcium deposition in rat aortic rings (Fig. 1). The results demonstrate that obvious VC occurs in the HP group, which is dose-dependently alleviated by IS. Compared with the HP group, the calcium content differs significantly in the HPMFe group (P < 0.05), but not in the HPLFe group. The difference between the HPHFe and HPLFe groups is also significant (P < 0.01). These results confirm that 5 μg/ ml or more concentration of IS can effectively inhibit high phosphate media-induced VC.
Alterations in VSMC Oxidation Levels

To determine the specific role of oxidative stress in high phosphate media-induced VC, ROS generation, MDA

Fig. 1 Alterations in calcium deposition. Brown staining in Von Kossa staining indicate calcium deposits (magnification × 200). The calcium detection were used to examine the quantitative calcium dep- osition in aortic rings. Data shown are mean ± SD (n = 3). Identical results were obtained in a replicate experiment. **, P < 0.01 vs. CNT;
∆, P < 0.05 vs. HP; ∆∆, P < 0.01 vs. HP; #, P < 0.05 vs. HPLFe.
CNT, normal conditions with 0.9 mM Pi; HP, normal conditions with
2.5 mM Pi; HPLFe, normal conditions with 2.5 mM Pi and 2 μg/ml iron sucrose; HPMFe, normal conditions with 2.5 mM Pi and 5 μg/ml iron sucrose; HPHFe, normal conditions with 2.5 mM Pi and 10 μg/ ml iron sucrose. Ca, calcium; Pi, inorganic phosphorous

content and SOD activity were investigated. In the fluo- rescent probe-DHE assay, the brightest red fluorescence is observed in the HP group, and then declines with increasing concentrations of IS (Fig. 2). The changes in MDA content and SOD activity are displayed in Fig. 3; the highest MDA content and the lowest SOD activity (indicating the most severe oxidative injury) are observed in the HP group, and are both reversed by IS in a dose-dependent manner. No significant difference is revealed between the HP and the HPLFe groups. However, at ≥ 5 μg/ml IS, there is a signifi- cant difference in both the MDA content and SOD activity between the HP and HPMFe groups (P < 0.05), as well as between the HPHFe and HPLFe groups (P < 0.01). Taken together, we conclude that at a concentration ≥ 5 μg/ml, IS inhibits high phosphate media-induced oxidative injury in VSMCs in a dose-dependent manner.
Alterations in Phenotype

To examine the phenotypic transition in VSMCs, RT-qPCR analysis was used to detect the mRNA expression levels of

Fig. 2 Alterations in ROS production. The fluorescent probe-DHE assay was used to detect ROS production. The brightness of red fluorescence under fluorescence microscope reflects the amount of ROS, and blue fluorescence indicates the nuclei stained with DAPI (magnification × 200). CNT, normal conditions with 0.9 mM Pi; HP, normal conditions with 2.5 mM Pi; HPLFe, normal conditions with
2.5 mM Pi and 2 μg/ml iron sucrose; HPMFe, normal conditions with
2.5 mM Pi and 5 μg/ml iron sucrose; HPHFe, normal conditions with
2.5 mM Pi and 10 μg/ml iron sucrose. DHE, dihydroethidium; DAPI, 4′,6-diamidino-2-phenylindole; ROS, reactive oxygen species. Pi, inorganic phosphorous

ɑ-SMA, Runx2, Pit1 and FGF23 (Fig. 4). Significant down- regulation of ɑ-SMA and upregulation of Runx2, Pit1 and FGF23 mRNA genes are observed in the HP group, com- pared with the CNT (P < 0.01), which are reversed by IS in a dose-dependant manner. The differences between gene expressions in the HPLFe and the HPHFe groups are also significant (P < 0.05). The findings indicate that the pheno- typic transition of high phosphate media-induced VSMCs is dose-dependently inhibited by IS.
Alterations in Protein Expressions

To verify the phenotypic transition in VSMCs observed in gene expressions, the relative protein expression levels of ɑ-SMA, Runx2, Pit1 and FGF23 were detected by western blot analysis (Fig. 5). The results demonstrate that the alter- nations in protein expressions in all experimental groups are consistent with the gene expressions. Collectively, we
Fig. 3 Alterations in MDA content and SOD activity. The MDA content and SOD activity were detected using chemistry colorimet- ric method. Data shown are mean ± SD (n = 3). Identical results were obtained in a replicate experiment. *, P < 0.05 vs. CNT; **, P < 0.01 vs. CNT; ∆, P < 0.05 vs. HP; ∆∆, P < 0.01 vs. HP; ##, P < 0.01
vs. HPLFe. CNT, normal conditions with 0.9 mM Pi; HP, normal conditions with 2.5 mM Pi; HPLFe, normal conditions with 2.5 mM Pi and 2 μg/ml iron sucrose; HPMFe, normal conditions with 2.5 mM Pi and 5 μg/ml iron sucrose; HPHFe, normal conditions with 2.5 mM Pi and 10 μg/ml iron sucrose. MDA, malondialdehyde; SOD, super- oxide dismutase. Pi, inorganic phosphorous

conclude that IS can inhibit high phosphate media-induced VSMCs’ phenotypic transition in a dose-dependent manner.
Alterations in Iron Depositions

To explore the side effects of IS to vasculature, we exam- ined the iron deposition in rat aortic rings (Fig. 6). The iron content analysis indicates that significant iron deposition occurs with ≥ 5 μg/ml IS (P < 0.05), which promotes iron overload. Perls’ Prussian blue staining show that no obvious blue points are found until the concentration of IS reached 10 μg/ml. Our findings indicate that, when in a high concen- tration, IS will lead to iron overload in aortic rings.
Differences Between MFe and HPMFe Conditions

Based on the findings above, HPMFe group acted effec- tively in preventing VC and safely in avoiding iron overload. Therefore, we examined the differences of Ca/Fe deposition and oxidative stress between MFe and HPMFe conditions (Fig. 7). There is a significant difference in the MDA content (P < 0.05), SOD activity (P < 0.01) and calcium deposition

Fig. 4 Alterations in phenotype. RT-qPCR analysis was used to detect the relative mRNA expression levels of ɑ-SMA, Runx2, Pit1 and FGF23. Data shown are mean ± SD (n = 3). Identical results were obtained in a replicate experiment. *, P < 0.05 vs. CNT; **, P < 0.01 vs. CNT; ∆, P < 0.05 vs. HP; ∆∆, P < 0.01 vs. HP; #, P < 0.05
vs. HPLFe. HPLFe. CNT, normal conditions with 0.9 mM Pi; HP, normal conditions with 2.5 mM Pi; HPLFe, normal conditions with

(P < 0.01) between the two groups, but not in the iron depo- sition. The results indicate that high phosphate media can exacerbate oxidative injury and VC in media with iron, but could not change the iron deposition.

Discussion
The mechanisms of VC are complex [28, 29]. Iron is gener- ally regarded as an oxidant, and oxidative stress is reported to be an important initiating factor in the formation of VC [30, 31]. However, currently the effects of iron on VC remain controversial [32]. By treating uremic rats with iron dextran, Seto et al. found that the development of VC was suppressed by iron overload; however, the underlying mechanisms were not conclusively studied [23]. Ciceri et al. reported that iron citrate could reduce hyperphosphatemia-induced VC by inhibiting apoptosis [24], and Phan et al. reported that sucroferric oxyhydroxide, a novel iron based noncal- cium phosphate binder, could effectively control hyperphos- phatemia and VC [25]. Contrary to these findings, several studies considered the pro-calcification properties of iron [33, 34]. In the present study, normal rat aortic rings cultured with high phosphate media exhibited significant increases in markers of oxidative stress and calcification, which is in line
2.5 mM Pi and 2 μg/ml iron sucrose; HPMFe, normal conditions with 2.5 mM Pi and 5 μg/ml iron sucrose; HPHFe, normal conditions with 2.5 mM Pi and 10 μg/ml iron sucrose. RT-qPCR, reverse tran- scription-quantitative polymerase chain reaction; ɑ-SMA, α-smooth muscle actin; Runx2, runt-related transcription factor 2; Pit-1, type III sodium-dependent phosphate cotransporter-1; FGF23, fibroblast growth factor-23. Pi, inorganic phosphorous

with previous reports [30, 31]. Furthermore, ROS and MDA generation, and a high phosphate media-induced decrease in SOD activity, were dose-dependently reversed by IS; this is a marked departure from the common belief that iron induces oxidative stress [35–37]. There are some reasonable expla- nations for the lower oxidative stress of iron. Firstly, in our research, the oxidative stress was induced by high phosphate media but not iron. In line with our findings, a report indi- cated that IS could reduce anemia-induced oxidative stress [38]. That is to say, iron has the potential to reduce oxidative stress which is induced by other predisposing factors. Our novel findings also indicate that there may be a competitive reaction between iron and phosphorous on the induction of oxidative stress. Secondly, different complexes can bring different effects. Sucrose may be a reason for the diversity. Toblli et al. reported that repeat-dose administration of LMWID, FMX, and IIM to healthy rats was associated with significant increases in lung oxidative stress, however, FCM and IS administration was not associated with the increases of it [39]. This finding supports our judgement. Lastly, the time and dosage are important factors. Iron deposits in the vascular wall were found in the group with large dosage of IS. The iron-induced oxidative stress may occur as the time and dosage go on, however, it is out of our interests and we did not investigate it. Its detailed mechanism is still in need

Fig. 5 Alterations in protein expressions. Western blot analysis was used to detect the protein expression levels of ɑ-SMA, Runx2, Pit1 and
FGF23 normalized to GAPDH.
Data shown are mean ± SD (n = 3). Identical results were
obtained in a replicate experi- ment. *, P < 0.05 vs. CNT; **, P < 0.01 vs. CNT; ∆, P < 0.05 vs. HP; ∆∆, P < 0.01 vs. HP; ##, P < 0.01 vs. HPLFe. CNT,
normal conditions with 0.9 mM Pi; HP, normal conditions with
2.5 mM Pi; HPLFe, normal conditions with 2.5 mM Pi and 2 μg/ml iron sucrose; HPMFe, normal conditions with 2.5 mM Pi and 5 μg/ml iron sucrose; HPHFe, normal conditions with
2.5 mM Pi and 10 μg/ml iron sucrose. ɑ-SMA, α-smooth muscle actin; Runx2, runt- related transcription factor 2; Pit-1, type III sodium-depend- ent phosphate cotransporter-1; FGF23, fibroblast growth fac- tor-23; GAPDH, glyceradehyde- 3-phosphate dehydrogenase. Pi, inorganic phosphorous

for further research. Mokas et al. reported that ROS genera- tion in VSMCs is essential for hypoxia inducible factor-1 (HIF-1) stabilization during hyperphosphatemia-induced calcification, and sustained HIF-1 activation can promote an osteogenic phenotypic switch and subsequent calcifica- tion in VSMCs [40]. Moreover, Ratcliffe et al. found that ROS inhibited the catalytic activity of the prolyl hydroxylase domain and led to HIF-1α stabilization and activation of HIF transcriptional programs [41]. However, iron is an important cofactor for HIF-1α hydroxylases [42]. Taken together, these findings indicate that iron may intensify HIF-1α hydroxyla- tion, reduce ROS generation and subsequently relieve oxi- dative stress-associated calcification in VSMCs. In other words, the iron/HIF-1/ROS interaction may be a significant signaling pathway, though additional studies are required to support this hypothesis. Nevertheless, to the best of our knowledge, this is the first report that IS dose-dependently
protects VSMCs from calcification by inhibiting high phos- phate media-induced oxidative stress. Somehow, we believe that our novel findings will help clinicians revise their rigid viewpoints on iron and oxidative stress.
Due to an overwhelming lack of evidence, the time, type and dose of iron, as well as patient suitability and an effective monitoring index for administration, are cur- rently under debate [14–16, 43, 44]; this also indicates the complex action of iron in the human body. In an observa- tional study of 58,058 hemodialysis patients, Kalantar- Zadeh et al. found that monthly doses of ≤ 400 mg IV iron were associated with improved survival, whereas monthly doses above this tended to be associated with higher rates of mortality [45]. In the PIVOTAL trial, a total of 2141 participants were divided into a high-dose group, with a median monthly iron dose of 264 mg (interquartile range [25th to 75th percentile], 200–336), and a low-dose group,

Fig. 6 Alterations in iron deposition. Blue points in Perls’ Prussian blue staining indicate the iron deposits (magnification × 200). The iron detection were used to examine the quantitative iron deposition in aortic rings. Data shown are mean ± SD (n = 3). Identical results were obtained in a replicate experiment. *, P < 0.05 vs. CNT; **, P < 0.01 vs. CNT; ∆, P < 0.05 vs. HP; ∆∆, P < 0.01 vs. HP; #,
P < 0.05 vs. HPLFe; ##, P < 0.01 vs. HPLFe. CNT, normal conditions with 0.9 mM Pi; HP, normal conditions with 2.5 mM Pi; HPLFe, nor- mal conditions with 2.5 mM Pi and 2 μg/ml iron sucrose; HPMFe, normal conditions with 2.5 mM Pi and 5 μg/ml iron sucrose; HPHFe, normal conditions with 2.5 mM Pi and 10 μg/ml iron sucrose. Fe, iron; Pi, inorganic phosphorous

with a 145-mg dose (interquartile range, 100–190); the median follow-up time was 2.1 years. Encouragingly, increased efficacy in anemia improvement and a lower rate of fatal or nonfatal cardiovascular events were observed in the high-dose group, and the incidence of infection did not differ significantly [22]. We therefore consider high- dose iron to be safe and beneficial in patients, and that its effects are partially attributed to its protection from VC. In the present study, obvious iron deposition was observed in VSMCs at concentrations of IS ≥ 5 μg/ml, which emulates the occurrence of iron overload in vascular tissues. Iron overload is harmful to the cardiovascular system [46, 47] and should be proactively prevented. Our findings indicate that the blood concentration of IS is crucial. Considering both the effect on IDA and the protective effect on high phosphate media-induced VC, we advocate more exten- sive applications and higher doses of IS in patients, espe- cially those with hyperphosphatemia and iron deficiency. However, rigorous clinical studies should be carried out to investigate more appropriate indications and optimal dosing.
Fig. 7 Differences between MFe and HPMFe conditions. The MDA content, SOD activity, calcium and iron deposition were detected. Data shown are mean ± SD (n = 3). Identical results were obtained in a replicate experiment. *, P < 0.05 vs. MFe; **, P < 0.01 vs. MFe. MFe, normal conditions with 0.9 mM Pi and 5 μg/ml iron sucrose; HPMFe, normal conditions with 2.5 mM Pi and 5 μg/ml iron sucrose. MDA, malondialdehyde; SOD, superoxide dismutase; Ca, calcium; Fe, iron; Pi, inorganic phosphorous

Our findings suggest that IV iron may interfere with the transportation, combination or clearance of Pi, which ought to change its serum concentration. However, the relation between IV iron and hypophosphatemia is also under debate. Bellos et al. found that the occurrence of hypophosphatemia is common after the administration of intravenous FCM, but not IIM, IS, iron dextran, or FMX [48]. Muras-Szwedziak et al. considered that intravenous iron supplementation may only result in hypophosphatemia at the commencement of iron therapy [49]. Recently, a clinical study, named FER- WON-IDA trial, which enrolled a total of 1512 patients with iron deficiency anemia, suggested that the frequency of hypophosphatemia was low in both IIM and IS groups [50]. Taken together, we hold that IS is not likely to impact Pi serum concentration obviously. Due to the extreme com- plexity of human body, the exact effect should be studied thoroughly.
The central role for VSMC phenotypic transition in hyperphosphatemia-induced VC has been confirmed [4]. In the present study, the upregulation of RUNX2, a typical osteoblast-specific gene, and the downregulation of ɑ-SMA, a characteristic VSMC differentiation marker gene, exhibits the VSMC phenotypic transition clearly which is consistent with others’ and our previous studies [26, 51, 52]. Mean- while, we also found that IS is effective in inhibiting the phenotypic transition of VSMC in a dose-dependent manner.

FGF23 is a phosphaturic hormone produced by osteocytes which directly affects sodium-phosphate cotransporters in response to phosphate overload [53–55]. The overexpres- sion of FGF23 gene and protein induced by high phos- phate media in the present study are consistent with prior reports [53–57]. Pit1, the predominant sodium-phosphate cotransporter in human VSMCs, is essential for hyperphos- phatemia-induced VC, which transports Pi into cells and then triggers the downstream molecular pathways to induce calcification [56, 57]. The results of our experiments indicate that the overexpression of Pit1 gene and protein induced by high phosphate media are inhibited by IS dose-dependently, which implies that iron may be a competitive inhibitor of Pi transportation and alleviate hyperphosphatemia-induced oxidative stress in VSMC by interfering with intracellular Pi transport. However, there is no relative report on this view- point and further studies are required to clarify the underly- ing relationship between iron and Pi.
Interestingly, our findings suggest that the calcium-iron product (Ca*Fe) in rat aortic rings remains stable in the pres- ence of IS (data not shown), which implies that the product of calcium and iron deposition in VSMC may be a con- stant in patients with iron supplementation. Hydroxyapa- tite [Ca10(PO4)6(OH)2] deposition in VSMC is considered significant to VC development [28]. Scialla et al. reported that iron oxide/nano-hydroxyapatite nanocomposites could drive bone growth, and differential dextran-grafted iron oxide/nano-hydroxyapatite ratios showed diverse biomimetic potential to osteoblast-like cells [58]. Hence, we consider that iron may consume active oxygen induced by hyper- phosphatemia and then produces iron oxide/hydroxyapatite compounds to inhibit the phenotypic transition of VSMCs. This subject requires further investigation.
Compared with in vivo models, the ex vivo normal rat aortic rings used in the present study had a well-preserved extracellular matrix, mimicked hyperphosphatemia, and were not susceptible to the possible effects of other organ systems, such as intact parathyroid hormone (iPTH), anemia, advanced glycation end products (AGEs) and bone morpho- genetic protein-2 (BMP2). However, the current model does not fully emulate in vivo conditions. When evaluating the effect of IS on the human body, race differences may also result in inconsistencies.
In conclusion (and to the best of our knowledge), the present study is the first to report that IS is a double-edged sword in high phosphate media-induced VC which not only protects VSMCs from calcification by inhibiting high phos- phate media-induced oxidative injury, in a dose-dependent manner, but also leads to iron overload in vasculature when in high concentration. In addition to its effect on anemia, IS is also a promising agent for VC prevention in patients with hyperphosphatemia and iron deficiency. Considering the effect on VC and the risk of iron overload, the blood
concentration of IS in patients with hyperphosphatemia is significant. Further studies with a higher dose range in experimental models and humans are required to confirm the underlying mechanisms of VC, and to determine the optimal dose of IS.

Author Contributions All authors conceived and designed the research. PW drafted the manuscript. PW, CG, HP and DP performed the experi- ments. DP analyzed the data and revised the manuscript. WC inter- preted the data and discussed the manuscript. All authors read and approved the final manuscript.

Funding The present study is supported by the Jingmen City Science and Technology Project (Grant No. 2018YFYB023).

Data Availability All data generated and analyzed during the cur- rent study are available from the corresponding author on reasonable request.
Compliance with Ethical Standards
Conflict of interest Ping Wang, Chengkun Guo, Hui Pan, Wangshan Chen and Dan Peng declares that they have no competing interests.
Ethical Approval Ethical approval was granted by the Ethical Commit- tee of the First People’s Hospital of Jingmen (Jingmen, China) and the study protocols conformed to the National institute of Health (NIH) guidelines for the care and use of laboratory animals.

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