Phosphonoformic acid reduces hyperphosphatemia-induced vascular calcification via Pit-1

Abstract

Objective

This study aimed to examine the mechanism of hyperphosphatemia-induced vascular calcification (HPVC).

Methods

Primary human aortic smooth muscle cells and rat aortic rings were cultured in Dulbecco’s modified Eagle’s medium supplemented with 0.9 mM or 2.5 mM phosphorus concentrations. Type III sodium-dependent phosphate cotransporter-1 (Pit-1) small interfering RNA and phosphonoformic acid (PFA), a Pit-1 inhibitor, were used to investigate the effects and mechanisms of Pit-1 on HPVC. Calcium content shown by Alizarin red staining, expression levels of Pit-1, and characteristic molecules for phenotypic transition of vascular smooth muscle cells were examined.

Results

Hyperphosphatemia induced the upregulation of Pit-1 expression, facilitated phenotypic transition of vascular smooth muscle cells, and led to HPVC in cellular and organ models. Treatment with Pit-1 small interfering RNA or PFA significantly inhibited Pit-1 expression, suppressed phenotypic transition, and attenuated HPVC.

Conclusions

Our findings suggest that Pit-1 plays a pivotal role in the development of HPVC. The use of PFA as a Pit-1 inhibitor has the potential for therapeutic intervention in patients with HPVC. However, further rigorous clinical investigations are required to ensure the safety and efficacy of PFA before it can be considered for widespread implementation in clinical practice.

Introduction

Chronic kidney disease (CKD) is characterized by a progressive deterioration in renal function resulting from underlying primary kidney disorders or other contributing risk factors, such as hypertension, diabetes, autoimmune diseases, and obesity. ¹ CKD is currently acknowledged as one of the most widespread chronic ailments worldwide.^2,3 CKD ultimately progresses to end-stage kidney disease, thereby limiting treatment options to life-sustaining measures, such as renal transplantation and dialysis, which pose substantial societal burdens.^4–6 Therefore, the development of therapeutic strategies aimed at extending the survival rate and improving quality of life among individuals affected by CKD has become an urgent priority. ⁷

Cardiovascular disease is the most serious complication of CKD and remains the leading cause of mortality in patients with CKD.^8–10 Vascular calcification (VC), which is an independent risk factor for cardiovascular disease, has a high incidence among patients with CKD, and can lead to fatal complications, such as myocardial infarction, myocardial dysfunction, and arrhythmia. The cellular and molecular mechanisms underlying VC induced by CKD pose important challenges in current medical research. VC was originally believed to progress passively through mineral deposition. However, recent evidence suggests that VC is an active cell-mediated process involving phenotypic transition of vascular smooth muscle cells (VSMCs), dysregulation of calcification-promoting factors and inhibitors, apoptosis, and extracellular matrix dysfunction.^11,12 Hyperphosphatemia-induced VC (HPVC) represents a common critical factor contributing to CKD-related VC. ¹³

The intracellular delivery of phosphate plays a pivotal role in the occurrence and progression of HPVC, which is primarily mediated by sodium-dependent inorganic phosphorus cotransporters. ¹⁴ In human VSMCs, type III sodium-dependent phosphate cotransporter-1 (Pit-1) serves as the major sodium-dependent inorganic phosphorus cotransporter, playing a crucial role in HPVC and facilitating phenotypic transition from VSMCs to osteoblast-like cells.^15,16 Inhibition of Pit-1 has emerged as a promising strategy for controlling HPVC.^17,18 Our previous studies showed a strong correlation between Pit-1 expression and HPVC in human aortic smooth muscle cells (HASMCs) and rat aortic rings.^19,20 However, the precise role and underlying mechanisms of action of Pit-1 in HPVC remain unclear. We conducted the present study to address this knowledge gap. Our findings have great potential for innovative and useful therapeutic interventions targeting patients with HPVC.

Methods

Cells and aortic ring culture and the HPVC model

HASMCs were obtained from Procell Life Science and Technology Co., Ltd. (Wuhan, China). HASMCs were cultured in Dulbecco’s modified Eagle’s medium ([DMEM] Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% (v/v) fetal bovine serum (Hyclone; GE Healthcare Life Sciences) and 1% streptomycin/penicillin under a humidified atmosphere of 5% CO₂ at 37°C. Cells at passages four to six were used for subsequent experimentation. ¹⁹ Thoracic aortic rings were harvested from male 10-week-old Sprague–Dawley rats that were purchased from the Hubei Provincial Center for Disease Control and Prevention (Wuhan, China). The rats were acclimatized for 1 week under specific pathogen-free conditions with a controlled temperature (20 ± 2°C), relative humidity (50%–70%), and a 12-hour light/dark cycle along with ad libitum access to standard diet and water. The rats were then euthanized and their aortas were sectioned into multiple 3 to 4-mm rings for culturing purposes. ²⁰ The culture conditions for the rings were the same as those of HASMCs. To simulate the hyperphosphatemia condition, DMEM was supplemented with Na₂HPO₄·12H₂O and NaH₂PO₄·2H₂O at a concentration of 2.5 mM inorganic phosphorus (Pi) while maintaining pH within the range of 7.2 to 7.4, as previously described.^19,20 The cells and aortic rings were cultured for up to 1 or 2 weeks, and media changes were performed every 2 days.

Quantification of VSMC calcification

Calcium concentrations of the cells and rat aortic rings were quantified using a commercially available Calcium Assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), by following previously established protocols.^19,20 Briefly, after the cells and aortic rings were harvested and washed with phosphate-buffered saline, they were treated with 0.6 N HCl overnight at 4°C. Calcium concentrations in the supernatant were determined by the o-cresolphthalein complexone method. A Bicinchoninic Acid Protein Assay kit (Aspen Biotechnology Co., Ltd., Wuhan, China) was used to evaluate protein concentrations to normalize calcium concentrations.^19,20

Alizarin red staining

The HASMCs and rat aortic rings were subjected to standard Alizarin red staining to show calcium content by following the protocol previously described by our group. ¹⁹ Briefly, after the cells and aortic rings were harvested and washed with phosphate-buffered saline, they were fixed with 10% (v/v) formaldehyde for 10 minutes at room temperature. Subsequently, the slides of cells and aortic rings were washed three times with phosphate-buffered saline and then exposed to 1% (w/v) Alizarin red for 30 minutes at 37°C. The slides were then washed with 0.2% (v/v) acetic acid. Finally, the stained samples were observed under a light microscope (Olympus Corporation, Tokyo, Japan) to detect the presence of crimson or violet pigmentation. ¹⁹

Quantitative reverse transcription polymerase chain reaction

Total RNA from tissue was isolated using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s protocol.

Complementary DNA was prepared using a PrimeScript™ RT reagent kit with gDNA eraser by following the manufacturer’s protocol (Takara Bio, Inc., Otsu, Japan). The temperature protocol for reverse transcription consisted of incubation at 37°C for 15 minutes followed by heat inactivation at 85°C for 15 s. Quantitative polymerase chain reaction (PCR) was performed on a StepOne™ Real-Time PCR system (Thermo Fisher Scientific, Inc.) using a SYBR® Premix Ex Taq™ reagent kit (Takara Bio, Inc.). The PCR conditions included an initial denaturation step at 95°C for 1 minute, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 20 s, and extension at 72°C for 45 s. A melting curve analysis was conducted by gradually increasing the temperature from 60°C to 95°C with a ramp rate of 0.05°C/s.^19,20 The primers for Pit-1, Runt-related transcription factor 2 (Runx2), smooth muscle 22 alpha (SM22ɑ), and glyceraldehyde-3-phosphate dehydrogenase (internal reference) were procured from Genecreate Bioengineering Co., Ltd. (Wuhan, China). The primer sequences used were as follows: Pit-1, 5′-ACATCCTACACCATGGCAATAT-3′ (forward) and 5′-CACTTCAGGCTTATCCTGATCAT-3′ (reverse); Runx2, 5′-TACTCTGCCGAGCTACGAAATG-3′ (forward) and 5′-TGAAACTCTTGCCTCGTCCG-3′ (reverse); SM22ɑ, 5′-ATCCAAGCCAGTGAAGGTGC-3′ (forward) and 5′-ACTCCCTCTTATGCTCCTGGG-3′ (reverse); and glyceraldehyde-3-phosphate dehydrogenase, 5′-CGCTAACATCAAATGGGGTG-3′ (forward) and 5′-TTGCTGACAATCTTGAGGGAG-3′ (reverse). The expression levels of these genes were determined using the 2^−ΔΔCq method. ²¹

Western blotting

A western blot analysis was conducted in accordance with previously established procedures by our group.^19,20 Briefly, protein extraction was performed using radioimmunoprecipitation assay lysis buffer (cat. no. as1004; Aspen Biotechnology). The total protein concentration was quantified using bicinchoninic acid commercial reagents. Protein samples were separated by 8% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline with 0.1% Tween 20 for 1 hour at room temperature. The membranes were then immunoblotted overnight at 4°C with primary antibodies against rabbit anti-α-tubulin (1:2000; ANT014; Antgene, Wuhan, China) and rabbit anti-Pit-1 (1:2000; cat. no. ab177147; Abcam, Cambridge, UK). The immunoreactive signals of antibody–antigen complexes were visualized using enhanced chemiluminescence reagents (ECL prime western blotting detection reagent; cat. no. as1059; Aspen Biotechnology) after incubation with the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG [1:10,000]; cat. no. as1107; Aspen Biotechology Co., Ltd.). The density of each band was detected using a chemiluminescence imaging system (AlphaEaseFC software version 3.3.0; ProteinSimple, San Jose, CA, USA). Pit-1 protein expression levels were normalized to α-tubulin levels.

Small interfering RNA transfection

Custom-designed small interfering RNAs (siRNAs) targeting Pit-1 and a negative control siRNA (scramble siRNA) were synthesized by Santa Cruz Biotechnology, Inc. (sc-106414; Dallas, TX, USA). Transfections were performed using HiPerFect Transfection Reagent (Qiagen, Dusseldorf, Germany) according to the manufacturer’s instructions. Normal cells and cells transfected with scramble siRNA were included as experimental controls.

Statistical analysis

The experiments were conducted in triplicate and consistently yielded reliable results. Data are presented as the mean  ±  standard deviation. Statistical analysis was carried out using SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). Group comparisons were evaluated using one-way analysis of variance followed by the post hoc Fisher protected least-significant difference test to determine any significant differences at a significance level of P < 0.05.

Ethics approval

The study adhered to the National Institute of Health guidelines for the care and use of laboratory animals, and an exemption for approval of the study protocol was obtained from the institutional review board of Jingmen Central Hospital.

Results

Downregulation of Pit-1 protein expression levels by Pit-1 siRNA

To determine the specific role of Pit-1 in HPVC, RNA interference was used to suppress Pit-1 gene expression levels. Subsequently, Pit-1 protein expression levels were evaluated 24 hours after Pit-1 siRNA transfection (Figure 1). We found that relative protein expression levels of Pit-1 were significantly inhibited following treatment with Pit-1 siRNA compared with scramble siRNA controls (P < 0.01). These results confirmed the efficacy of the selected Pit-1 siRNA, thus establishing a solid foundation for subsequent experimental data.

Figure 1.

Human aortic smooth muscle cells were cultured in Dulbecco’s modified Eagle’s medium and transfected with Pit-1 or negative control siRNA. After 24 hours of siRNA transfection, Pit-1 protein expression levels were examined using western blotting analysis and normalized to α-tubulin levels. The data are expressed as the mean  ±  standard deviation (n = 3). Consistent findings were obtained in independent replicate experiments. **P < 0.01. Pit-1, type III sodium-dependent phosphate cotransporter-1; siRNA, small interfering RNA.

Effects of Pit-1 siRNA on VSMC phenotypic transition and HPVC

The HASMCs were divided into four groups: i) control (CNT) group, which consisted of normal HASMCs treated with Pi at a concentration of 0.9 mM; ii) high Pi (HP) group, which consisted of normal HASMCs treated with high Pi concentrations of 2.5 mM; iii) siRNA (SI) group, which consisted of Pit-1 siRNA-transfected HASMCs treated with normal Pi concentrations; and iv) siRNA and high Pi group, which consisted of Pit-1 siRNA-transfected HASMCs treated with high Pi concentrations. These cells were cultured for up to 7 days. The mRNA expression levels of Runx2, SM22α, and Pit-1 are shown in Figure 2a. Hyperphosphatemia significantly altered mRNA expression levels of Runx2, SM22α, and Pit-1 compared with the control condition (CNT vs HP, all P < 0.01). Under normal conditions, silencing Pit-1 gene expression using siRNA effectively suppressed its expression levels, but had no significant effect on Runx2 or SM22α mRNA expression levels compared with control cells without siRNA treatment (CNT vs SI, P = 0.03). However, high Pi silencing Pit-1 gene expression using siRNA significantly attenuated changes in Pit-1 (HP vs siRNA and high Pi, P < 0.05) and SM22α mRNA expression levels (HP vs siRNA and high Pi, P < 0.01). Additionally, in the high Pi condition, silencing Pit-1 gene expression using siRNA tended to reduce Runx2 mRNA expression levels (HP vs siRNA and high Pi, P = 0.09). Calcium deposits and calcium concentrations are shown in Figure 2b and 2c, respectively. Phenotypic transition induced by Pit-1 and VC under hyperphosphatemic conditions was alleviated by silencing Pit-1 gene expression as shown by reduced calcium deposition levels (P < 0.05, HP vs SIHP). This finding indicated that phenotypic transition and VC induced by Pit-1 only occurred under a hyperphosphatemic condition rather than under normophosphatemia.

Figure 2.

HASMCs were divided into four groups: normal HASMCs treated with 0.9 mM Pi (CNT), normal HASMCs treated with 2.5 mM phosphorous (HP), Pit-1 siRNA-transfected HASMCs treated with 0.9 mM phosphorous (SI), and Pit-1 siRNA-transfected HASMCs treated with 2.5 mM phosphorous (SIHP). The cells were cultured for up to 7 days. (a) Relative mRNA expression levels of Pit-1, Runx2, and SM22α in the four groups as determined by reverse transcriptase-quantitative polymerase chain reaction analysis. (b) Calcium deposits in the four groups as visualized by Alizarin red staining (original magnification, ×400). (c) Calcium concentrations in the four groups. The data are expressed as the mean  ±  standard deviation (n = 3). Consistent findings were obtained in independent replicate experiments. *P < 0.05, **P < 0.01. HASMCs, human aortic smooth muscle cells; Pit-1, type III sodium-dependent phosphate cotransporter-1; RUNX2, Runt-related transcription factor 2; SM22α, smooth muscle 22 alpha.

Inhibition of Pit-1 protein expression levels by PFA

To further determine the role of Pit-1 in HPVC, rat aortic rings were cultured in DMEM medium for 14 days and divided into the following groups: normal Pi concentrations (0.9 mM) (CNT group), high Pi concentrations (2.5 mM; HP group), and high Pi concentrations supplemented with 0.5 mM PFA (PFAHP group). Western blotting analysis showed that hyperphosphatemia induced upregulation of Pit-1 protein expression (CNT vs HP, P < 0.05), which was effectively inhibited by PFA treatment (HP vs PFAHP, P < 0.01, Figure 3).

Figure 3.

Rat aortic rings were cultured in Dulbecco’s modified Eagle’s medium containing 0.9 mM phosphorous (CNT group), 2.5 mM phosphorous (HP group), or 2.5 mM phosphorous supplemented with 0.5 mM phosphonoformic acid (PFAHP group) for 14 days. Western blotting was conducted to assess protein expression levels of Pit-1, which were subsequently normalized to α-tubulin levels (lower panels). The data are expressed as the mean  ±  standard deviation (n = 3, upper panel). Consistent findings were obtained in independent replicate experiments. *P < 0.05, **P < 0.01.

Effects of PFA on VSMC phenotypic transition and HPVC

In rat aortic rings, hyperphosphatemia significantly upregulated mRNA expression levels of Runx2 and SM22α (CNT vs HP, both P < 0.05, Figure 4a). However, only Runx2 mRNA expression levels were inhibited by PFA treatment (HP vs PFAHP, P < 0.05). Calcium deposits in the three groups are shown in Figure 4b. Calcium concentrations were significantly higher in the HP group than in the CNT and PFA groups (both P < 0.01, Figure 4c). Notably, calcium concentrations in the PFAHP group were similar to those in the CNT group. These findings suggest that PFA effectively attenuated HPVC by reducing Pit-1 expression.

Figure 4.

Rat aortic rings were cultured in Dulbecco’s modified Eagle’s medium containing 0.9 mM phosphorous (CNT group), 2.5 mM phosphorous (HP group), or 2.5 mM phosphorous supplemented with 0.5 mM phosphonoformic acid (PFAHP group) for 14 days. (a) Relative mRNA expression levels of Runx2 and SM22α in the three groups as determined using reverse transcriptase-quantitative polymerase chain reaction analysis. (b) Calcium deposits in the three groups as visualized by Alizarin red staining (original magnification, ×400). (c) Calcium concentrations in the three groups. The data are expressed as the mean  ±  standard deviation (n = 3). Consistent findings were obtained in independent replicate experiments. *P < 0.05, **P < 0.01. RUNX2, Runt-related transcription factor 2; SM22α, smooth muscle 22 alpha.

Discussion

Hyperphosphatemia is a prevalent clinical manifestation in patients with CKD, irrespective of the underlying etiology. Previous studies have demonstrated that hyperphosphatemia can accelerate VC in the laboratory setting and in living organisms,^22,23 but the precise mechanisms remain unclear.^24,25 Pit-1, which acts as the major sodium-phosphate cotransporter in human VSMCs, has become the focus of attention for its role in HPVC.^15,26 Our previous research indicated that spironolactone mitigated HPVC by downregulating Pit-1 expression. This finding led us to hypothesize that serum Pit-1 concentrations are associated with the severity of HPVC. ²⁰ In this study, we found that inhibiting Pit-1 using Pit-1 siRNA significantly alleviated phenotypic transition and VC induced by hyperphosphatemia. Furthermore, our findings suggest that, while Pit-1 can induce phenotypic transition and VC under conditions of hyperphosphatemia, it does not exert similar effects under normal serum Pi concentrations. Our findings show the crucial role of Pit-1 in the occurrence and progression of HPVC.

PFA, which is a phosphonocarboxylic acid analogous to inorganic pyrophosphate, is clinically used as an antiviral medication. ²⁷ The adverse effects of PFA are primarily gastrointestinal discomfort, hypersensitivity reactions, renal impairment, neurotoxicity, and cardiotoxicity. PFA functions as a sodium-dependent phosphate cotransporter inhibitor.^28,29 Villa-Bellosta et al. first showed the efficacy of PFA in preventing HPVC through the inhibition of mineralization and hydroxyapatite formation. ³⁰ However, their study was confined to cellular research and did not investigate the underlying mechanisms extensively, necessitating further investigations for robust validation. In the current study, we observed significant downregulation of Pit-1 expression in cellular and organ models treated with PFA, further supporting its therapeutic potential for preventing HPVC. Collectively, these findings suggest that PFA mitigates VSMC mineralization and hydroxyapatite formation via the suppression of Pit-1 expression. Consequently, PFA is promising as a prospective therapy for HPVC.

Phenotypic transition from smooth muscle cells to osteoblast-like cells is a critical stage in the progression of VC. SM22α serves as an important marker gene for VSMC differentiation, while Runx2 is commonly recognized as an osteoblast-specific gene. Our previous studies have convincingly shown that changes in SM22α and Runx2 expression effectively reflect VSMC phenotypic transition.^19,20,31 In this study, we observed that alterations in SM22α and Runx2 expression accurately represented phenotypic transition when experimental samples were cultured under hyperphosphatemic conditions. Interestingly, we observed that PFA inhibited the upregulation of Runx2, but had no effect on SM22α expression in ex vivo rat aortic rings. This finding is in contrast to the effects observed with Pit-1 siRNA in in vitro HASMCs. This discrepancy in findings may be attributed to differences in experimental models, specifically variations in extracellular matrix composition. Therefore, PFA may impede the recovery of SM22α by modulating a specific signaling pathway within the extracellular matrix. Further investigations are warranted to determine the underlying mechanisms involved.

This study has a limitation. This study was constrained by the ex vivo experimental model, which may not have fully replicated in vivo conditions, despite yielding rational and satisfactory outcomes.

In conclusion, our findings suggest that Pit-1 plays a pivotal role in the development of HPVC. The upregulation of Pit-1 expression in VSMCs serves as an initiating factor for this pathological process. The use of PFA as an inhibitor of Pit-1 expression has the potential for therapeutic intervention in patients with HPVC. However, further rigorous clinical investigations are required to ensure the safety and efficacy of PFA before it can be considered for widespread implementation in clinical practice.