# Oral pyrophosphate protects Abcc6-/- mice against vascular calcification induced by chronic kidney disease

Elise Bouderlique, Jennifer Kervadec, Ellie Tang, Jeremy Zaworski, Amélie Coudert, Isabelle Rubera, Christophe Duranton, Edmat Khan, Jean-Philippe Haymann, Georges Leftheriotis, et al.

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## To cite this version:

Elise Bouderlique, Jennifer Kervadec, Ellie Tang, Jeremy Zaworski, Amélie Coudert, et al. Oral pyrophosphate protects Abcc6-/- mice against vascular calcification induced by chronic kidney disease. Journal of Molecular Medicine, 2024, 102 (10), pp.1217-1227. 10.1007/s00109-024-02468-y. hal-05217474

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<figure><img src="image_2.png" /></figure>
Oral pyrophosphate protects Abcc6-/- mice against vascular calcification induced by chronic kidney disease

Elise Bouderlique,<fnref n="1" />,<fnref n="2" /> Jennifer Kervadec,<fnref n="1" />,<fnref n="2" /> Ellie Tang,<fnref n="1" />,<fnref n="2" /> Jeremy Zaworski,<fnref n="1" />,<fnref n="2" /> Amélie Coudert,<fnref n="4" /> Isabelle Rubera,<fnref n="5" />
Christophe Duranton,<fnref n="5" /> Edmat Khan,<fnref n="1" />,<fnref n="2" /> Jean-Philippe Haymann,<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> Georges Leftheriotis,<fnref n="5" />,<fnref n="6" /> Michel Daudon,<fnref n="1" />,<fnref n="2" />,<fnref n="3" />
Emmanuel Letavernier<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

AUTHOR INFORMATION

**Correspondence:** Emmanuel LETAVERNIER, Service des Explorations Fonctionnelles Multidisciplinaires, Hôpital TENON, 4 rue de la Chine, 75020 Paris (France). Phone 33 1 56 01 67 73; Fax 33 1 56 01 70 03. E-mail: emmanuel.letavernier@aphp.fr

**Running Head:** Pyrophosphate and vascular calcification

**Word Count:** 3821 words

**Keywords:** Pyrophosphate (PPi); arterial calcification; chronic kidney disease (CKD); calcium; phosphate; ABCC6; TNAP; alkaline phosphatase (ALP)

<footnote n="1">¹ Sorbonne Université, UMR S 1155, F-75020, Paris, France</footnote>

<footnote n="2">² INSERM, UMR S 1155, F-75020, Paris, France</footnote>

<footnote n="3">³ Physiology Unit, AP-HP, Hôpital Tenon, F-75020, Paris, France</footnote>

<footnote n="4">⁴ UFR d'odontologie (Département des Sciences Biologiques), Université Paris Diderot BIOSCAR - INSERM U1132, Hôpital Lariboisière 75010 PARIS</footnote>

<footnote n="5">⁵ University Côte d'Azur, CNRS UMR-7370, Laboratoire de Physiomédecine Moléculaire, Labex ICST, Nice, France</footnote>

<footnote n="6">⁶ PXE Consultation Center, FAVAMULTI Sud Competence Center for Rare Arterial Calcifying Diseases, Nice University Hospital, 06000, Nice, France</footnote>
**Abstract**

One of the hallmarks of chronic kidney disease (CKD) is the development of vascular calcification. Inorganic pyrophosphate is a potent inhibitor of calcification, and previous studies have reported low plasma pyrophosphate levels in hemodialysis patients. A long-term mouse model of CKD-accelerated vascular calcification was developed to study pyrophosphate metabolism and to test whether oral pyrophosphate supplementation attenuates the propensity for arterial calcification. CKD was induced by repeated injections of aristolochic acid in wild-type and Abcc6⁻/⁻ mice, which tend to develop vascular calcifications. CKD accelerated the development of vascular calcifications in Abcc6⁻/⁻ mice, in the aorta and small renal arteries, and decreased circulating pyrophosphate levels. Oral pyrophosphate supplementation for 6 months attenuated CKD-induced vascular calcification in this model. These results show that oral pyrophosphate may be of interest in preventing vascular calcification in patients with CKD.
**Introduction:**

Cardiovascular events are particularly common in patients with chronic kidney disease (CKD)[1, 2]. A specific feature of vascular lesions promoted by CKD is the development of the calcification in the media of small and large arteries. Calcium load in the arteries of patients with CKD correlates with cardiovascular prognosis and mortality[3]. The development of medial calcification is accelerated by uremic toxins and low-grade inflammation, inducing changes in the phenotype of vascular wall smooth muscle cells [4]. SMCs develop osteoblastic activity and express osteogenic genes such as *RUNX2* or *BMP2*[5, 6]. Furthermore, elevated serum phosphate levels in CKD patients increase calcium phosphate supersaturation and apatite precipitation in tissues, particularly in the vascular wall[7]. While the role of serum phosphate levels in cardiovascular outcomes has been widely studied, the role of calcification inhibitors in CKD has received less attention. Some studies have suggested that fetuin A and osteopontin play a role in the pathogenesis and progression of vascular calcification in CKD, but one of the most important inhibitors may be inorganic pyrophosphate (PPi) [8–13]. CKD patients, particularly those on renal replacement therapy, show reduced circulating levels of PPi [14, 15]. PPi has been known for decades as a potent inhibitor of calcification, even at nanomolar concentrations, but its major role in the clinical context has emerged in recent years. In fact, several monogenic disorders, including pseudoxanthoma elasticum (PXE), are characterized by calcification of the vascular media relatively similar to the lesions seen in CKD, and recent reports have shown that these diseases are characterized by decreased PPI plasma levels[16–18]. Patients with PXE have mutations in the *ABCC6* gene, which is expressed in the liver and in proximal tubular cells of the kidney. ABCC6 is a basolateral transporter that facilitates the efflux of adenosine triphosphate (ATP) into the blood by unknown mechanisms. ATP is then hydrolyzed by ectonucleotide pyrophosphatase phosphodiesterase-1 (ENPP1), producing PPi and adenosine monophosphate (AMP).

Another major determinant of PPi homeostasis is the Tissue-nonspecific Alkaline phosphatase (TNAP) which degrades inorganic pyrophosphate (PPi) to inorganic phosphate and thus promotes biomineralization. An increase in alkaline phosphatase activity has been described in the serum of CKD and hemodialysis patients[15, 19]

The first aim of our studies was to test whether a long-term murine model of mild CKD could accelerate vascular calcification in *Abcc6*⁻/⁻ mice that are predisposed to developing calcification. The second objective was to study PPi and TNAP homeostasis and test whether oral PPi could attenuate the propensity for CKD-enhanced arterial calcification.
**Methods:**

**Animal studies:**

*Abcc6* knock-out mice were generated on a 129/Ola background and backcrossed onto a C57BL/6J background more than 10 times at the Netherlands Ophthalmic Research Institute (Amsterdam, the Netherlands) as previously described[20]. These mice are designated *Abcc6*⁻/⁻ in the present study. The animals were maintained at the Mouse Facility of the INSERM's Unité Mixte de Recherche S 1155 (Paris, France) with a 12-hour dark/light cycle. *Abcc6*⁻/⁻ mice were regularly crossed with C57BL/6J wild type animals to avoid genetic drift. All mice were fed a standard rodent diet (catalogue number 3432, Kliba Nafag, Kaiseraugst, Switzerland) and had access to drinking water ad libitum. The study was carried out in accordance with national and European Union directives on animal experimentation and ARRIVE guidelines. Ethics committee approval was obtained (Comité d'Éthique en Expérimentation Animale Charles Darwin, France; APAFIS #27030). All animals were 12 weeks old females at the start of the protocols. Only female mice were used to limit the number of animals, in accordance with ethical guidelines.

CKD was induced by intra peritoneal injections of aristolochic acid (A5512, SigmaAldrich, Saint Quentin Fallavier, France), at a dose of 1mg/kg, on three consecutive days, at the start of the experiment and one month later (i.e. 6 injections/mouse). A total of 72 female mice were required to carry out the protocol: 36 mice were sacrificed 3 months after the first injection of aristolochic acid and 36 mice were sacrificed 6 months later. Mice were given a euthanizing dose of sodium pentobarbital (100 mg/kg) prior to exsanguination. Mice were divided into subgroups of 6 animals as follows: (1) *Abcc6*⁺/⁺ control (no CKD), (2) *Abcc6*⁺/⁺ + CKD, (3) *Abcc6*⁻/⁻ control, (4) *Abcc6*⁻/⁻ + CKD, (5) *Abcc6*⁻/⁻ + PPi and (6) *Abcc6*⁻/⁻ + PPi + CKD. The “6 months” protocol was carried out first and the “3 months” protocol was carried out later in view of the initial results, notably to assess alkaline phosphatase activity (not initially planed), and to obtain more urine and plasma samples for PPi quantification.

Tetrasodium PPi (P8010, Sigma Aldrich, Saint Quentin Fallavier, France) was diluted in drinking water to 10mM.

Control mice without PPi received sodium chloride water with the same equimolar concentration of sodium as the tetrasodium PPi solution (NaCl, S9888, Sigma Aldrich, Saint Quentin Fallavier, France). Drinking water containing either PPi or sodium chloride was changed twice a week.

**Biological samples and biochemistry**

Blood samples were analyzed for calcium (total), phosphate, urea, and creatinine at the time of sacrifice: M3 or M6 (n=6/group unless the blood sample was insufficient, e.g. in some animals at 3 months). Dry tubes and tubes
containing lithium heparinate were used for serum and plasma collection, respectively, except for PPi (citrate tube followed by ultracentrifugation with Amicon 0.5 ml ultracel ® 50K). After centrifugation, plasma and serum samples were frozen. Plasma and urine PPi concentrations were assessed only in animal sacrificed at 3 months (inappropriate pre-analytical conditioning at 6 months). Urine was collected in metabolic cages the week before sacrifice. Creatinine, urea and phosphate levels were analyzed on the IDS-iSYS automat (Immunodiagnostic Systems Holdings PLC, Pouilly-en-Auxois, France). Calcium levels were measured on a PerkinElmer 3300 atomic absorption spectrometer (PerkinElmer, Villebon-sur-Yvette, France). PPi concentrations in plasma and urine were measured as previously described <sup>15</sup>: In brief, ATP sulfurylase (M0394; New England Biolabs, Évry, France) was used to convert PPi to ATP in the presence of excess adenosine 5'phosphosulfate (A5508; SigmaAldrich, Saint-Quentin Fallavier, France). The ATP generated then was quantified using the ATP Determination Kit (ATPlite 6016941; PerkinElmer).

## Histology and immunochemistry

Kidney and liver tissues were fixed in formaldehyde and embedded in paraffin.

Four-micron tissue sections were stained with Sirius red to quantify fibrosis. Morphometric analysis of the kidney tissue surface was performed using Image J software (NIH) on the entire kidney surface (photo mosaic) of 2 representative sections at ×100 magnification under polarized light. Four-micron sections were stained with periodic acid Schiff and Masson trichrome to characterize structural lesions of the kidney.

For immunochemistry, tissue was deparaffinized and the antigen retrieval was made with 10mM citric acid pH6 (target retrieval solution, Agilent, Les Ulis, France) at 95°C. Endogenous peroxidase activity was blocked with peroxidase blocking solution (Dako Agilent, Les Ulis, France). Sections were permeabilized with PBS containing 10% BSA and 0.1% triton. Sections were then incubated with the specific primary antibody anti-TNAP (1/200, ab108337, Abcam, Cambridge, United Kingdom). An HRP-coupled secondary antibody was used for detection (Histofine, Brignais, France) and was revealed by AEC (Dako Agilent, Les Ulis, France) or DAB (K3468, Dako Agilent, Les Ulis, France).

## Quantitative RT-PCR
mRNA was extracted from kidney and liver by using Trizol solution (Life Technologies BRL, Gaithersburg, MD, USA). RNA concentration was measured using the NanoDrop1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). RT-PCR was performed using SYBR green and specific probes on light cycler 480 (Roche Diagnostic, Indianapolis, IN, USA). The specific primers used were as follows: *Tnap*: For CGGATCCTGACCAAAAACC Rev TCATGATGTCCGTGGTCAAT. PCR was also carried out for the housekeeping gene *Gapdh* For AGCTTGTCATCAACGGGAAG, Rev TTTGATGTTAGTGGGGTCTCG and *Hprt* For GGAGCGGTAGCACCTCCT Rev CTGGTTCATCATCGCTAATCAC for kidney samples ; *ActinB1* For CTAAGGCCAACCGTGAAAAG Rev CCAGAGGCATACAGGGACA ; and *RPL32* For GCTGCCATCTGTTTTACGG Rev TGACTGGTCCTGATGAACT for liver samples to normalize the Q-PCR results, using Roche LightCycler 2.0 software (Roche Diagnostics Indianapolis, IN, USA). Results are expressed as 2^−ΔΔCT, where CT is the cycle threshold number, and data are presented as fold increase in mRNA levels.

## Western Blots

Proteins were extracted from kidney (cortex + medulla) and liver tissue using RIPA lysis buffer (Santa Cruz Biotechnology, Heidelberg, Germany) supplemented with a cocktail of protease inhibitors, PMSF and sodium orthovanadate. Equal amounts of denaturing samples were loaded in MiniProtean, TGX, Stain Free Protein Gels (BioRad, Marnes la Coquette, France) and transferred to nitrocellulose membranes. Immunoblotting was performed with the antibody Anti-TNAP (1/1000, ab108337, Abcam, Cambridge, United Kingdom). Proteins were detected using the chemiluminescent substrate (Clarity Western ECL, BioRad, Marnes la Coquette, France) and imaged with ChemiDoc (MP imaging, BioRad, Marnes la Coquette, France). Protein signals were quantified and normalized to the total protein loading signal (Stain Free procedure, BioRad, Marnes la Coquette, France). Western-blots were performed at 3 and 6 months but only the results at 3 months are presented, as results were similar at both stages.

## Alkaline phosphatase activity

Plasma and tissue (kidney, liver) alkaline phosphatase activity was measured by using a fluorometric alkaline phosphatase assay kit (ab83371, Abcam, Cambridge, United Kingdom) following the manufacturer's instructions. Fluorescence (excitation 360nm, emission 440 nm) was measured using an infinite-200 Pro microplate reader
(Tecan, Mannendorf, Switzerland). Alkaline phosphatase activity was calculated as the amount of 4-methylumbelliferone (4-MU) generated during the reaction time and normalized to the concentration of the total amount of protein in the tissue lysates.

In situ TNAP activity was assessed in liver and kidney using a mixture of ready-to-use 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) solution (UP099851, Interchim): BCIP 0.48mmol/l and NBT 0.51mmol/l at pH 9.8 for 5 minutes.

## X-Ray Microtomography and Three-Dimensional Modeling

Aortas from the cardiac aortic valve to the iliac bifurcation were fixed in formaldehyde and embedded in paraffin (n=6 per group). X-ray computed tomography imaging was performed using a Skyscan 1272 (Bruker, Anvers, Belgium) on the imaging platform at Lariboisière Hospital (Paris, France). A resolution scale of 6-µm was obtained. Shadow images were obtained using X-ray energy of 65 kV and 150 mA without filter exposure. The angular step between image acquisitions was 0.5, and each image was averaged after 2 frames. Data were reconstructed using Nrecon software (Bruker, Anvers, Belgium), then exported to a virtual slice stack in 16-bit Tag Image File Format. The Mimics Innovation suite 20.0 (Materialise, Leuven, Belgium) was used for quantitative analysis of vascular calcification volume, and three-dimensional modeling of the aorta and calcifications.

## Statistical Analysis

Data are expressed as means +/- SEM. Data were analyzed using non-parametric tests (Kruskall Wallis and Mann Whitney tests) and Spearman's correlation test, using GraphPad Prism software version 8 (GraphPad Software, Inc., San Diego, CA). The significance level was set at p< 0.05.

## Results

### Murine model of long-term CKD

Repeated injections of aristolochic acid into wild-type animals induced a slight impairment of renal function at 3 months, as expected. Serum creatinine was 19.5±1.7 µmol/l in CKD mice versus 14.4±1.0 µmol/l in control animals (n=5 and 4/group, respectively, p=0.0317; Figure 1A). At 6 months, serum creatinine was slightly but
significantly increased in CKD mice : 21.8±3.1 vs 16.1±0.9 µmol/l in control animals, (n= 6/group, p=0.0216 ; Figure 1B). At 3 and 6 months, CKD had no impact on serum calcium and phosphate levels, which remained similar in CKD and control groups (Figure 1C-F). PPi levels were assessed in plasma and urine at 3 months. In plasma, there was a non-significant downward trend in concentration in CKD vs control mice: 1.4±0.16 µmol/l versus 1.7±0.07 µmol/l, respectively (n=6/group ; p=0.06; Figure 1G). Urinary PPi excretion was significantly reduced by CKD: 0.9±0.2 vs 2.2±0.6 µmol/mmol of creatinine in control mice (n=6/group, p=0.026; Figure 1H).

Masson staining showed the predominance of tubulointerstitial lesions at 3 and 6 months and the progressive development of interstitial fibrosis (Figure 1I,J). Fibrosis was quantified by morphometric analyses based on Sirius red staining analyzed in polarized light. CKD significantly increased renal fibrosis, as expected (n=6/group, p=0.0022 and p=0.0152 at 3 and 6 months respectively; Figure 1K,L).

### CKD alters *TNAP* expression and alkaline phosphatase activity

The expression of TNAP was assessed at 3 and 6 months in CKD and control mice, in kidney and liver. Alkaline phosphatase activity was assessed at 3 months in kidney, liver and plasma.

In kidney, alkaline phosphatase activity (mU/mg of tissue) was very high compared with activity in liver and plasma (Figure 2A,J,O). CKD significantly reduced alkaline phosphatase activity in kidney (p= 0.015, n= 6/group; Figure 2A). CKD reduced significantly *Tnap* expression at the mRNA level at 3 months and there was a non-significant trend toward a lower expression at 6 months (Figure 2B,C). Immunostaining showed that TNAP was still expressed in CKD-affected kidneys but that the pattern of expression was altered : TNAP expression was no longer localized exclusively to the brush border membrane, but was expressed more widely in tubular cells. (Figure 2D,E). Alkaline phosphatase activity *in situ* was reduced by CKD in tubular cells (Figure 2F,G).

In contrast, western-blot experiments showed no decrease in TNAP expression at protein level by CKD and even a trend towards a paradoxical increase in expression (Figure 2H,I).

In the liver, alkaline phosphatase activity was significantly increased by CKD (p= 0.002, n= 6/group; Figure 2J). A non-significant trend towards increased *Tnap* expression at mRNA level was observed at 3 and 6 months (Figure 2K,L). Immunostaining showed very weak TNAP staining in the liver of both control and CKD mice (not shown), and in *in situ* alkaline phosphatase activity was undetectable (not shown). A non-significant trend towards increased protein expression was observed (Figure 2M,N).
Interestingly, alkaline phosphatase activity was significantly increased in plasma by CKD (p=0.002, n=6/group; Figure 2O).

### CKD accelerates vascular calcification in the *Abcc6*⁻/⁻ mouse model

Although CKD increased TNAP expression and alkaline phosphatase activity in liver and blood and decreased PPi levels in urine, induction of mild CKD was not sufficient to induce vascular calcification in wild type mice (Figure 3A,B,E,F). A “two hit” model was developed in parallel, i.e. induction of mild CKD in *Abcc6*⁻/⁻ mice, based on the hypothesis that the pro-calcifying effect of CKD would be unmasked in this predisposed strain. At 6 months, renal function was slightly impaired in *Abcc6*⁻/⁻ mice exposed to aristolochic acid and was similar to the renal function observed in wild-type mice with CKD (Supplemental Figure 1). Compared with wild type mice, *Abcc6*⁻/⁻ mice spontaneously developed moderate calcification in the aorta and renal arteries at 6 months (Figure 3A,B,C,G). Interestingly, the induction of mild CKD was sufficient to significantly increase the burden of calcium phosphate deposits in the mouse model, in both aorta and kidney arteries (Figure 3A-D,H).

As expected, at 3 months, plasma PPi levels were lower in *Abcc6*⁻/⁻ mice than in wild-type animals (p=0.0043, n=6/group), but there was no significant difference in urine PPi concentrations (Figure 4A,B). Induction of CKD caused a dramatic fall in urinary PPi concentrations in both groups (p=0.026, in WT mice and p=0.0087, in *Abcc6*⁻/⁻ mice, n=6/group, Figure 4B) and a non-significant fall in plasma PPi levels in both groups (Figure 4A). Of note, there was an additive effect of *Abcc6*⁻/⁻ genotype and CKD induction on plasma PPi levels. Indeed, plasma PPi was significantly decreased in *Abcc6*⁻/⁻ CKD mice compared to wild-type CKD animals (p=0.0022, n=6/group; Figure 4A). Alkaline phosphatase activity was increased significantly by CKD in the liver and blood of *Abcc6*⁻/⁻ mice, as observed in wild-type mice (p=0.0087 and p=0.0022, respectively, n=6/group; Figure 4C,D).

### Oral PPi supplementation attenuates CKD-accelerated vascular calcification

As PPi deficiency accelerates vascular calcification, *Abcc6*⁻/⁻ mice with or without CKD were exposed for 6 months to oral PPi supplementation. PPi exposure did not significantly affect renal function, serum calcium or serum phosphate levels (Supplemental Figure 1). In *Abcc6*⁻/⁻ mice with preserved renal function (no CKD), oral PPi did not significantly reduce the calcification load in the aorta (92,522±51,163 µm³ vs 195,522±88,899 µm³, p=0.3, n=6/group) but did reduce it in the arteries of the renal cortex (228,488±135,145 µm³ vs 840,389±147,444 µm³,
p=0.0152, n=6/group; Figure 3A,B,G,I). In *Abcc6*⁻/⁻ CKD animals, oral PPi supplementation significantly reduced calcification load in the aorta (268,660±136,978 µm³ vs 846,664±241,319 µm³, p=0.04, n=6/group) and in the arteries of the renal cortex (650,918±185,944 µm³ vs 2538,300±778,567 µm³, p=0.015, n=6/group) (Figure 3A,B,H,J).

As expected *Abcc6*⁻/⁻ mice exposed to PPi had increased excretion of PPi in urine compared with controls in CKD groups (5.1±2.1 vs 0.6±0.2 µmol/mmol of creatinine, p=0.0043, n=6/group; Figure 4B). This increase was not significant in non-CKD groups (4.4±2.4 vs 2.8±0.6 µmol/mmol of creatinine, p=0.81, n=6/group; Figure 4B). No significant increase was detected in the plasma of PPi-supplemented animals (Figure 4A). Alkaline phosphatase activity was also assessed in PPi-exposed animals, confirming the CKD-induced increase in alkaline phosphatase activity in all groups of animals, irrespective of genotype or PPi exposure (Figure 4C,D).

Discussion

Overall, our results confirm that CKD is associated with impaired PPi homeostasis. Although *Abcc6*⁻/⁻ mice are already affected by mild PPi deficiency, CKD led to a further drop in plasma PPi, demonstrating that a mechanism independent of ABCC6 explains the decrease in PPi in CKD. CKD in fact significantly increased alkaline phosphatase activity in plasma and liver, which probably led to reduced plasma PPi levels in wild-type and Abcc6⁻/⁻ mice. There is an apparent paradox with regard to the kidney: alkaline phosphatase activity is extremely high in the kidney in the absence of CKD, and this activity is decreased by CKD. TNAP expression and alkaline phosphatase activity are restricted to the brush border of proximal tubular cells in the absence of CKD. The total amount of TNAP in the kidney was not decreased by CKD, but the enzyme was expressed in the cytosol rather than at the brush border membrane. TNAP is an ecto-enzyme that is biologically active only when expressed at the brush border. In any case, the increase in systemic alkaline phosphatase activity observed in CKD is not due to an increased synthesis by the kidney.

Increased alkaline phosphatase activity is probably the major determinant of vascular calcification in CKD, via decreased PPi and local release of inorganic phosphate. Lomashvli et al. and Villa Bellosta et al. have previously demonstrated that hemodialysis patients have relatively low plasma PPi levels, and that dialytic clearance of serum phosphate increases TNAP activity, limiting the benefits of phosphate removal[13, 14]. More recently, Azpiazu et al. have evidenced that PPi hydrolysis was 51% higher in post-dialysis than in pre-dialysis plasma, and that levamisole, a TNAP inhibitor, reduced the phosphatase activity in patient plasma[11]. Laurain et al. showed that
plasma PPi was severely decreased in hemodialysis patients compared with healthy patients, [15]. Our results are
in line with these studies and demonstrate that PPi deficiency is a major determinant of vascular calcification, even
in a model of mild and long-term CKD without increased plasma phosphate levels.

The most important result, confirming the major role of PPi in vascular calcification, is the demonstration that oral
PPi is sufficient to blunt CKD-induced vascular calcification in murine models.

In a previous study, O'Neill et al. tested the effect of daily intraperitoneal PPi injections on uremic vascular calcification in rats fed an adenine-containing phosphate-rich diet for 28 days to induce uremia[21]. Unfortunately, the incidence of aortic calcification was not altered by PPi but the calcium content of calcified aortas was significantly reduced. Uremic rats were eventually given calcitriol to produce more consistent aortic calcification and were treated with sodium PPi administered intraperitoneally in a large volume of glucose-containing solution to prolong plasma PPi levels : the incidence and amount of calcification were reduced in this model[21]. These seminal studies were major proofs of concept, but they were based on acute and severe models, as well as on intraperitoneal injections of PPi which seem difficult to transpose into clinical practice.

It has recently been shown that oral PPi is not completely degraded in the gut, breaking with an old dogma. Oral PPi supplementation in mice or humans can transiently but effectively increase plasma PPi levels and potentially reduce vascular calcification[22-24]. This treatment or related drugs could therefore be tested in patients with CKD in the near future. The deficiency in PPi also promotes the onset of papillary calcification in Abcc6⁻/⁻ mice [25]. We took the opportunity of this model to test whether PPi treatment could reduce the volume of papillary calcifications on CT scans. Unfortunately, this volume was very low, probably due to the relatively young age of the animals, and we saw no significant benefit from the treatment (data not shown). Extended studies on older animals will be necessary to test the efficacy of PPi on kidney papillary calcifications. In the same way, it would have been of interest to quantify vibrissae calcification as a biomarker of soft tissue calcification in this model.

Our studies have a number of limitations. Firstly, the origin of the increase in alkaline phosphatase activity is not
understood. In fact, we identified that CKD significantly increased alkaline phosphatase activity in plasma and
liver in all three groups of animals (wild-type, Abcc6⁻/⁻ and Abcc6⁻/⁻ exposed to PPi supplementation). A trend
towards increased TNAP synthesis (at mRNA or protein level) in the liver was observed but the results were not
significant. This may be due to a lack of power in our studies. Another hypothesis is that alkaline phosphatase
activity in the liver is due to alkaline phosphatase synthesized in other organs (e.g. bones) or by immune cells, and
not just by hepatocytes. The exact origin of the increased alkaline phosphatase activity observed in CKD merits
further specific studies. Secondly, we used mice affected by a “first hit”, i.e. *Abcc6*⁻/⁻ mice, to have a relevant model of vascular calcification in the media in a “long-term” model. A more acute model, for instance based upon 2,8 dihydroxyadenine precipitation in kidney tubules might have been used to assess the effect of PPi supplementation in more acute and severe conditions.

Thirdly, lack of power and overlap between groups may explain some "negative" results. For example, in *Abcc6*⁻/⁻ mice with preserved renal function, oral PPi did not significantly reduce calcification load in the aorta, despite a 53% reduction in calcification volume.

Finally, plasma PPi levels were lower in all groups of *Abcc6*⁻/⁻ mice, as expected, but we did not observe a significant reduction in urine PPi concentrations in *Abcc6*⁻/⁻ mice. Urine PPi levels were surprisingly high in some individuals, suggesting that unidentified pre-analytical problems may have skewed the results (perhaps contamination of some urine by PPi-containing stool). In contrast, urinary PPi excretion was significantly reduced by CKD. This is probably due to the reduced filtered load of PPi resulting from lower plasma levels and, to a lesser extent, a decrease in glomerular filtration rate.

At last, the use of oral PPi as a potential treatment for patients with CKD needs to be tested with caution, as PPi can be hydrolyzed to orthophosphate in the gut thereby increasing phosphate intake. Ongoing studies testing oral PPi in PXE patients may indicate whether this approach is a therapeutic option[26].

In conclusion, our studies show that PPi deficiency is a major determinant of vascular calcification in a mouse model of CKD since oral PPi is sufficient to blunt CKD-induced vascular calcification, suggesting that PPi or other PPi-mimicking drugs (specific bisphosphonates or others) may be promising drugs to slow the development of medial calcifications affecting CKD patients.

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**Statements and Declarations**

**Funding**

This work was supported by the Agence Nationale de la Recherche (ANR KI-PYRINA AAP CE17 grant) and by the French Society of Nephrology (Société Francophone de Néphrologie Dialyse et Transplantation): “IRCT Dialyse” grant 2020/2021 and Elise Bouderlique Ph.D. funding.

**Acknowledgements**

The authors thank Mrs Agnes Ostertag and Mrs Liliane Louedec for their technical help and animal facilities.

**Competing Interests**

The authors have no relevant financial or non-financial interests to disclose.

**Author Contributions**

E.B., J.K., E.T., J.Z., A.C., I.R., C.D., E.K., E.L. performed experiments, E.B., J.K., E.T., I.R., C.D., JP.H., M.D., G.L., E.L. analyzed the data; E.L., C.D., I.R. secured funding, E.L. and E.B. designed the experiment.
E.L. is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

**Data availability**

The datasets generated during and/or analysed during the current study are not publicly available as only the most relevant data are presented in the manuscript, but are available from the corresponding author on reasonable request.

**Ethics approval**

The study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).

The study was carried out in accordance with national and European Union directives on animal experimentation and ARRIVE guidelines. Ethics committee approval was obtained (Comité d'Éthique en Expérimentation Animale Charles Darwin, France; APAFIS #27030).
**Figure Legends**

**Figure 1: Mouse model of CKD**

Serum creatinine levels in both 3-months (n=4 and 5/group) and 6-months models (n=6/group) (A,B). Serum phosphate and calcium levels at 3 and 6 months (n=5 and 6/group, respectively) (C-F). Plasma and urine PPI levels at 3 months (n=6/group) (G,H). Masson staining showing the predominance of tubulointerstitial lesions at 6 months and the progressive development of interstitial fibrosis in CKD mice versus controls (I,J). Kidney fibrosis quantified by sirius red staining and morphometric analyses at 3-months and 6-months (n=5 and 6/group, respectively) (K,L). Black bar = 100µm. \*p < 0.05.

CKD: Chronic kidney disease

**Figure 2: TNAP expression and alkaline phosphatase activity in kidney, liver and plasma.**

**Kidney.** Alkaline phosphatase (ALP) activity in control and CKD mice (n=6/group) (A). *Tnap* expression at the mRNA level at 3 and 6 months in control and CKD mice (n=6/group) (B,C). TNAP enzyme expression pattern in tubular cells from control and CKD mice at 3 months (D,E). In situ ALP activity in tubular cells from control and CKD mice at 3 months (F,G). TNAP protein expression in the kidney at 3 months (n=6/group, OD normalized/stain-free)

**Liver.** ALP activity in control and CKD mice (n=6/group) (J). TNAP enzyme expression at 3 and 6 months at the mRNA level (n=6/group) (K,L). TNAP protein expression in the liver at 3 months (M,N) (n=6/group, OD normalized/stain-free).

**Plasma.** ALP activity in the plasma at 3 months (O)

Black bar = 100µm. \*p < 0.05. ALP: Alkaline phosphatase; CKD: Chronic kidney disease

**Figure 3: Vascular calcification after induction of a mild CKD for 6 months**

Calcification load quantified through CT-scan reconstruction of the aorta (A, n=6/group) and arteries of the kidney cortex (B, n=6/group). Representative projection of kidney 3D-modeling CT-scan from an *Abcc6*⁻/⁻ CKD mouse (C). Calcifications of the kidney cortex, stained in red, correspond to vascular calcifications, as stated by histological sections. Papillary calcifications (C, in blue) were not vascular and were therefore excluded from the analysis. Representative Yasue-stained calcifications observed in aorta sections (D) from *Abcc6*⁻/⁻ animals exposed to CKD. Representative projections of aortic 3D-modeling CT-scans with calcifications stained in red: E: *Abcc6*⁺/⁺ control; F: *Abcc6*⁺/⁺ CKD; G: *Abcc6*⁻/⁻ control; H: *Abcc6*⁻/⁻ CKD; I: *Abcc6*⁻/⁻ + PPI and J: *Abcc6*⁻/⁻ + PPI + CKD.

PPi: inorganic pyrophosphate; CKD: Chronic kidney disease

Black bar = 20 µm (D) and 10µm (E). \*p < 0.05.

**Figure 4: Plasma PPI concentrations and Alkaline phosphatase (ALP) activity.**

Plasma PPI concentration and urine excretion at 3 months, in control and CKD wild type and *Abcc6*⁻/⁻ mice, with or without supplementation in PPI (A,B, n=6/group).

ALP activity in liver and plasma at 3 months, in control and CKD wild type and *Abcc6*⁻/⁻ mice, with or without supplementation in PPI (C,D, n=6/group).

PPi: inorganic pyrophosphate; ALP: Alkaline phosphatase; CKD: Chronic kidney disease

Black bar = 100µm. \*p < 0.05.
<figure><img src="image_3.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Figure 4</figcaption></figure>