The ABCC6 Transporter: A New Player in Biomineralization

Pseudoxanthoma elasticum (PXE) is an inherited metabolic disease with autosomal recessive inheritance caused by mutations in the ABCC6 gene. Since the first description of the disease in 1896, alleging a disease involving the elastic fibers, the concept evolved with the further discoveries of the pivotal role of ectopic mineralization that is preponderant in the elastin-rich tissues of the skin, eyes and blood vessel walls. After discovery of the causative gene of the disease in 2000, the function of the ABCC6 protein remains elusive. More than 300 mutations have been now reported and the concept of a dermal disease has progressively evolved toward a metabolic disorder resulting from the remote effects caused by lack of a circulating anti-mineralization factor. Very recently, evidence has accumulated that this anti-mineralizing factor is inorganic pyrophosphate (PPi). This leads to decreased PPi/Pi (inorganic phosphate) ratio that results from the lack of extracellular ATP release by hepatocytes and probably renal cells harboring the mutant ABCC6 protein. However, the mechanism by which ABCC6 dysfunction causes diminished ATP release remains an enigma. Studies of other ABC transporters, such as ABCC7 or ABCC1 could help our understanding of what ABCC6 exact function is. Data and a hypothesis on the possible roles of ABCC6 in acquired metabolic diseases are also discussed

Homéostasie du pyrophosphate dans les maladies calcifiantes

Some chronic diseases, notably liver or kidney diseases, are associated with a high prevalence of arterial calcification, the determinants of which are not known. In the most advanced stages of these diseases, characterized by organ fibrosis and then organ failure, liver or kidney transplantation is suggested.Pyrophosphate is a potent circulating physiological inhibitor of calcification. In pathology, pyrophosphate deficiency is associated with arterial calcifications during genetic diseases due to mutations in the genes coding for ABCC6 (adenosine triphosphate ATP-binding cassette, subfamily C, member 6) or ENPP-1 (ectonucleotide pyrophosphatase/phosphodiesterase 1). Both proteins are mainly expressed in the liver and are involved in pyrophosphate synthesis while it is eliminated by urinary and alkaline phosphatase hydrolysis. The influence of organ transplantation on pyrophosphate homeostasis is unknown.The objectives of this thesis are to study the determinants of plasma pyrophosphate concentration and arterial calcifications before and after liver or kidney transplantation.In the first part, a new standardized method for determining the concentration of inorganic pyrophosphate in different biological fluids (culture media, plasma, urine, saliva, joint fluid) was developed. A European patent application has been filed.In the second part, haemodialysis and kidney transplant patients were compared to each other and to controls. Both haemodialysis and transplant patients have a pyrophosphate deficit compared to controls. Urinary pyrophosphate excretion is unchanged. Plasma ENPP1 activity is identical between groups but plasma alkaline phosphatase activity is negatively correlated with plasma pyrophosphate concentration. Pyrophosphate deficiency persists in renal transplant patients despite normal plasma alkaline phosphatase activity two years after transplantation.In the third part, patients with hepatic fibrosis and hepatocellular insufficiency, of varying etiologies, were compared before and 3 months after liver transplantation. Plasma pyrophosphate concentration is low before transplantation, and negatively correlated with the clinical scores of fibrosis or hepatocellular insufficiencies are high. While urinary pyrophosphate elimination remains unchanged, plasma alkaline phosphatase activity is increased. Hepatic expression of ABCC6 and ENPP1 mRNA is significantly decreased in patients with chronic liver pathologies compared to non-cirrhotic controls. Radiological scores of coronary or aortic arterial calcifications are positively correlated with the severity of fibrosis or hepatocellular insufficiency.In conclusion, the elevation of plasma alkaline phosphatase activity before liver or kidney transplantation contributes to pyrophosphate deficiency by increasing its hydrolysis. The complete correction of pyrophosphate deficiency by liver transplantation indicates that the liver is the main source of pyrophosphate. Incomplete correction of pyrophosphate deficiency by renal transplantation suggests a liver-kidney dialogue. Arterial calcifications are not directly correlated with pyrophosphate, suggesting the involvement of other agents that are yet to be determined.Certaines maladies chroniques, notamment hépatiques ou rénales, sont associées à une forte prévalence de calcification artérielle dont les déterminants ne sont pas connus. Aux stades les plus évolués de ces maladies, caractérisées par une fibrose d’organe et une insuffisance fonctionnelle, la transplantation hépatique ou rénale est proposée.Le pyrophosphate est un puissant inhibiteur physiologique circulant de la calcification. En pathologie, un déficit en pyrophosphate est associé aux calcifications artérielles au cours de maladies génétiques secondaires à l’inactivation des gènes codants pour ABCC6 (adénosine triphosphate ATP-binding cassette, sous-famille C, membre 6) ou ENPP-1 (ectonucléotide pyrophosphatase/phosphodiestérase 1). Ces deux protéines sont principalement exprimées dans le foie et elles synthétisent le pyrophosphate qui est éliminé intact par voie urinaire et par hydrolyse grâce aux phosphatases alcalines. L’influence de la transplantation d’organe sur l’homéostasie du pyrophosphate est inconnue.Les objectifs de la thèse sont d’étudier les déterminants de la concentration plasmatique en pyrophosphate et des calcifications artérielles avant et après transplantation hépatique ou rénale.Dans une première partie, une nouvelle méthode de dosage standardisée du pyrophosphate dans le plasma a été mise au point pour différents fluides biologiques (milieux de culture, plasma, urine, salive, liquide articulaire). Une demande de brevet européen a été déposée.Dans une deuxième partie, des patients hémodialysés et transplantés rénaux ont été comparés entre eux et à des témoins. Les patients hémodialysés et les patients transplantés ont un déficit en pyrophosphate par rapport aux témoins. L ‘élimination urinaire de pyrophosphate est inchangée. L’activité plasmatique d’ENPP1 est identique entre les groupes mais l’activité plasmatique des phosphatases alcalines est négativement corrélée à la concentration plasmatique de pyrophosphate. Le déficit en pyrophosphate persiste chez les patients transplantés rénaux malgré une activité plasmatique des phosphatases alcalines normale à distance de la transplantation.Dans une troisième partie, des patients présentant une fibrose hépatique et une insuffisance hépatocellulaire, d’étiologies variables, ont été comparés entre eux avant et 3 mois après transplantation hépatique. La concentration plasmatique de pyrophosphate est basse avant transplantation, d’autant plus que les scores cliniques de fibrose ou d’insuffisance hépatocellulaire sont élevés. L ‘élimination urinaire de pyrophosphate est inchangée. L’activité plasmatique des phosphatases alcalines est augmentée. L’expression hépatique des ARNms d’ABCC6 et d’ENPP1 est significativement abaissée par rapport à celle de témoins non cirrhotiques. Les scores radiologiques de calcifications artérielles coronaires ou aortiques sont positivement corrélés à la sévérité de la fibrose ou de l’insuffisance hépatocellulaire.En conclusion, l’élévation de l’activité plasmatique des phosphatases alcalines avant transplantation hépatique ou rénale participe au déficit en pyrophosphate en augmentant son hydrolyse. La correction complète du déficit en pyrophosphate par la transplantation hépatique indique que le foie est la principale source de pyrophosphate. Le caractère incomplet de la correction du déficit en pyrophosphate par la transplantation rénale est évocateur d’un dialogue foie-rein. Les calcifications artérielles ne sont pas directement corrélées au pyrophosphate, suggérant l’intervention de d’autres agents qu’ils restent à caractériser

Pyrophosphate homeostasis during calcifying diseases

Certaines maladies chroniques, notamment hépatiques ou rénales, sont associées à une forte prévalence de calcification artérielle dont les déterminants ne sont pas connus. Aux stades les plus évolués de ces maladies, caractérisées par une fibrose d’organe et une insuffisance fonctionnelle, la transplantation hépatique ou rénale est proposée.Le pyrophosphate est un puissant inhibiteur physiologique circulant de la calcification. En pathologie, un déficit en pyrophosphate est associé aux calcifications artérielles au cours de maladies génétiques secondaires à l’inactivation des gènes codants pour ABCC6 (adénosine triphosphate ATP-binding cassette, sous-famille C, membre 6) ou ENPP-1 (ectonucléotide pyrophosphatase/phosphodiestérase 1). Ces deux protéines sont principalement exprimées dans le foie et elles synthétisent le pyrophosphate qui est éliminé intact par voie urinaire et par hydrolyse grâce aux phosphatases alcalines. L’influence de la transplantation d’organe sur l’homéostasie du pyrophosphate est inconnue.Les objectifs de la thèse sont d’étudier les déterminants de la concentration plasmatique en pyrophosphate et des calcifications artérielles avant et après transplantation hépatique ou rénale.Dans une première partie, une nouvelle méthode de dosage standardisée du pyrophosphate dans le plasma a été mise au point pour différents fluides biologiques (milieux de culture, plasma, urine, salive, liquide articulaire). Une demande de brevet européen a été déposée.Dans une deuxième partie, des patients hémodialysés et transplantés rénaux ont été comparés entre eux et à des témoins. Les patients hémodialysés et les patients transplantés ont un déficit en pyrophosphate par rapport aux témoins. L ‘élimination urinaire de pyrophosphate est inchangée. L’activité plasmatique d’ENPP1 est identique entre les groupes mais l’activité plasmatique des phosphatases alcalines est négativement corrélée à la concentration plasmatique de pyrophosphate. Le déficit en pyrophosphate persiste chez les patients transplantés rénaux malgré une activité plasmatique des phosphatases alcalines normale à distance de la transplantation.Dans une troisième partie, des patients présentant une fibrose hépatique et une insuffisance hépatocellulaire, d’étiologies variables, ont été comparés entre eux avant et 3 mois après transplantation hépatique. La concentration plasmatique de pyrophosphate est basse avant transplantation, d’autant plus que les scores cliniques de fibrose ou d’insuffisance hépatocellulaire sont élevés. L ‘élimination urinaire de pyrophosphate est inchangée. L’activité plasmatique des phosphatases alcalines est augmentée. L’expression hépatique des ARNms d’ABCC6 et d’ENPP1 est significativement abaissée par rapport à celle de témoins non cirrhotiques. Les scores radiologiques de calcifications artérielles coronaires ou aortiques sont positivement corrélés à la sévérité de la fibrose ou de l’insuffisance hépatocellulaire.En conclusion, l’élévation de l’activité plasmatique des phosphatases alcalines avant transplantation hépatique ou rénale participe au déficit en pyrophosphate en augmentant son hydrolyse. La correction complète du déficit en pyrophosphate par la transplantation hépatique indique que le foie est la principale source de pyrophosphate. Le caractère incomplet de la correction du déficit en pyrophosphate par la transplantation rénale est évocateur d’un dialogue foie-rein. Les calcifications artérielles ne sont pas directement corrélées au pyrophosphate, suggérant l’intervention de d’autres agents qu’ils restent à caractériser.Some chronic diseases, notably liver or kidney diseases, are associated with a high prevalence of arterial calcification, the determinants of which are not known. In the most advanced stages of these diseases, characterized by organ fibrosis and then organ failure, liver or kidney transplantation is suggested.Pyrophosphate is a potent circulating physiological inhibitor of calcification. In pathology, pyrophosphate deficiency is associated with arterial calcifications during genetic diseases due to mutations in the genes coding for ABCC6 (adenosine triphosphate ATP-binding cassette, subfamily C, member 6) or ENPP-1 (ectonucleotide pyrophosphatase/phosphodiesterase 1). Both proteins are mainly expressed in the liver and are involved in pyrophosphate synthesis while it is eliminated by urinary and alkaline phosphatase hydrolysis. The influence of organ transplantation on pyrophosphate homeostasis is unknown.The objectives of this thesis are to study the determinants of plasma pyrophosphate concentration and arterial calcifications before and after liver or kidney transplantation.In the first part, a new standardized method for determining the concentration of inorganic pyrophosphate in different biological fluids (culture media, plasma, urine, saliva, joint fluid) was developed. A European patent application has been filed.In the second part, haemodialysis and kidney transplant patients were compared to each other and to controls. Both haemodialysis and transplant patients have a pyrophosphate deficit compared to controls. Urinary pyrophosphate excretion is unchanged. Plasma ENPP1 activity is identical between groups but plasma alkaline phosphatase activity is negatively correlated with plasma pyrophosphate concentration. Pyrophosphate deficiency persists in renal transplant patients despite normal plasma alkaline phosphatase activity two years after transplantation.In the third part, patients with hepatic fibrosis and hepatocellular insufficiency, of varying etiologies, were compared before and 3 months after liver transplantation. Plasma pyrophosphate concentration is low before transplantation, and negatively correlated with the clinical scores of fibrosis or hepatocellular insufficiencies are high. While urinary pyrophosphate elimination remains unchanged, plasma alkaline phosphatase activity is increased. Hepatic expression of ABCC6 and ENPP1 mRNA is significantly decreased in patients with chronic liver pathologies compared to non-cirrhotic controls. Radiological scores of coronary or aortic arterial calcifications are positively correlated with the severity of fibrosis or hepatocellular insufficiency.In conclusion, the elevation of plasma alkaline phosphatase activity before liver or kidney transplantation contributes to pyrophosphate deficiency by increasing its hydrolysis. The complete correction of pyrophosphate deficiency by liver transplantation indicates that the liver is the main source of pyrophosphate. Incomplete correction of pyrophosphate deficiency by renal transplantation suggests a liver-kidney dialogue. Arterial calcifications are not directly correlated with pyrophosphate, suggesting the involvement of other agents that are yet to be determined

A Plasma Pyrophosphate Cutoff Value for Diagnosing Pseudoxanthoma Elasticum

Pseudoxanthoma elasticum (PXE) is a rare inherited systemic disease responsible for a juvenile peripheral arterial calcification disease. The clinical diagnosis of PXE is only based on a complex multi-organ phenotypic score and/or genetical analysis. Reduced plasma inorganic pyrophosphate concentration [PPi]p has been linked to PXE. In this study, we used a novel and accurate method to measure [PPi]p in one of the largest cohorts of PXE patients, and we reported the valuable contribution of a cutoff value to PXE diagnosis. Plasma samples and clinical records from two French reference centers for PXE (PXE Consultation Center, Angers, and FAVA-MULTI South Competent Center, Nice) were assessed. Plasma PPi were measured in 153 PXE and 46 non-PXE patients. The PPi concentrations in the plasma samples were determined by a new method combining enzymatic and ion chromatography approaches. The best match between the sensitivity and specificity (Youden index) for diagnosing PXE was determined by ROC analysis. [PPi]p were lower in PXE patients (0.92 ± 0.30 µmol/L) than in non-PXE patients (1.61 ± 0.33 µmol/L, p < 0.0001), corresponding to a mean reduction of 43 ± 19% (SD). The PPi cutoff value for diagnosing PXE in all patients was 1.2 µmol/L, with a sensitivity of 83.3% and a specificity of 91.1% (AUC = 0.93), without sex differences. In patients aged <50 years (i.e., the age period for PXE diagnosis), the cutoff PPi was 1.2 µmol/L (sensitivity, specificity, and AUC of 93%, 96%, and 0.97, respectively). The [PPi]p shows high accuracy for diagnosing PXE; thus, quantifying plasma PPi represents the first blood assay for diagnosing PXE

Relationships between Plasma Pyrophosphate, Vascular Calcification and Clinical Severity in Patients Affected by Pseudoxanthoma Elasticum

International audiencePseudoxanthoma elasticum (PXE; OMIM 264800) is an autosomal recessive metabolic disorder characterized by progressive calcification in the skin, the Bruch’s membrane, and the vasculature. Calcification in PXE results from a low level of circulating pyrophosphate (PPi) caused by ABCC6 deficiency. In this study, we used a cohort of 107 PXE patients to determine the pathophysiological relationship between plasma PPi, coronary calcification (CAC), lower limbs arterial calcification (LLAC), and disease severity. Overall, our data showed a deficit in plasma PPi in PXE patients compared to controls. Remarkably, affected females showed higher PPi levels than males, but a lower LLAC. There was a strong correlation between age and PPi in PXE patients (r = 0.423, p < 0.0001) but not in controls (r = 0.059, p = 0.828). A weak correlation was found between PPi and CAC (r = 0.266, p < 0.02); however, there was no statistically significant connection with LLAC (r = 0.068, p = 0.518) or a severity score (r = 0.077, p = 0.429). Surprisingly, we found no significant correlation between plasma alkaline phosphatase activity and PPi (r = 0.113, p = 0.252) or between a 10-year cardiovascular risk score and all other variables. Multivariate analysis confirmed that LLAC and CAC were strongly dependent on age, but not on PPi. Our data showed that arterial calcification is only weakly linked to circulating PPi levels and that time (i.e., age) appears to be the major determinant of disease severity and calcification in PXE. These data are important to better understand the natural history of this disease but also for the follow-up and management of patients, and the design of future clinical trials. Our results also show that PPi is not a good biomarker for the evaluation of disease severity and progression

New Strategies for Volume Control in Patients with Diabetes Mellitus, a Narrative Review

Sodium is reabsorbed all along the renal tubules. The positive impacts of sodium-glucose cotransporter-2 inhibitors (SGLT2i), angiotensin receptor neprilysin inhibitor (ARNI) and mineralocorticoid receptor antagonists (MRA) on hard renal and/or cardiac endpoints calls for the role of diuretics in nephroprotection and cardioprotection in patients with diabetes mellitus to be reviewed. Here, we review: (a) the mechanisms of action of the available natriuretics; (b) the physiological adaptations to chronic loop diuretic usage that lead to increased sodium reabsorption in the proximal and distal convoluted tubules; (c) the physiology of sodium retention in patients with diabetes mellitus; and (d) the mechanisms of aldosterone breakthrough. We show the rationale for combined diuretics to target not only the loop of Henle, but also the proximal and distal convoluted tubules. Indeed, higher residual proteinuria in patients treated with renin-angiotensin-aldosterone system (RAAS) blockers portends poorer renal and cardiovascular outcomes. Diuretics are known to optimize the reduction of proteinuria, in addition to RAAS blockers, but may favor aldosterone breakthrough in the absence of MRA. The aim of our study is to support a combined diuretics strategy to improve the management of patients with diabetes mellitus and chronic kidney disease or heart failure

Regulation of Monocytes/Macrophages by the Renin–Angiotensin System in Diabetic Nephropathy: State of the Art and Results of a Pilot Study

Diabetic nephropathy (DN) is characterized by albuminuria, loss of renal function, renal fibrosis and infiltration of macrophages originating from peripheral monocytes inside kidneys. DN is also associated with intrarenal overactivation of the renin–angiotensin system (RAS), an enzymatic cascade which is expressed and controlled at the cell and/or tissue levels. All members of the RAS are present in the kidneys and most of them are also expressed in monocytes/macrophages. This review focuses on the control of monocyte recruitment and the modulation of macrophage polarization by the RAS in the context of DN. The local RAS favors the adhesion of monocytes on renal endothelial cells and increases the production of monocyte chemotactic protein-1 and of osteopontin in tubular cells, driving monocytes into the kidneys. There, proinflammatory cytokines and the RAS promote the differentiation of macrophages into the M1 proinflammatory phenotype, largely contributing to renal lesions of DN. Finally, resolution of the inflammatory process is associated with a phenotype switch of macrophages into the M2 anti-inflammatory subset, which protects against DN. The pharmacologic interruption of the RAS reduces albuminuria, improves the trajectory of the renal function, decreases macrophage infiltration in the kidneys and promotes the switch of the macrophage phenotype from M1 to M2

The ABCC6 Transporter: A New Player in Biomineralization

Pseudoxanthoma elasticum (PXE) is an inherited metabolic disease with autosomal recessive inheritance caused by mutations in the ABCC6 gene. Since the first description of the disease in 1896, alleging a disease involving the elastic fibers, the concept evolved with the further discoveries of the pivotal role of ectopic mineralization that is preponderant in the elastin-rich tissues of the skin, eyes and blood vessel walls. After discovery of the causative gene of the disease in 2000, the function of the ABCC6 protein remains elusive. More than 300 mutations have been now reported and the concept of a dermal disease has progressively evolved toward a metabolic disorder resulting from the remote effects caused by lack of a circulating anti-mineralization factor. Very recently, evidence has accumulated that this anti-mineralizing factor is inorganic pyrophosphate (PPi). This leads to decreased PPi/Pi (inorganic phosphate) ratio that results from the lack of extracellular ATP release by hepatocytes and probably renal cells harboring the mutant ABCC6 protein. However, the mechanism by which ABCC6 dysfunction causes diminished ATP release remains an enigma. Studies of other ABC transporters, such as ABCC7 or ABCC1 could help our understanding of what ABCC6 exact function is. Data and a hypothesis on the possible roles of ABCC6 in acquired metabolic diseases are also discussed

Arterial Calcifications in Patients with Liver Cirrhosis Are Linked to Hepatic Deficiency of Pyrophosphate Production Restored by Liver Transplantation

International audienceLiver fibrosis is associated with arterial calcification (AC). Since the liver is a source of inorganic pyrophosphate (PPi), an anti-calcifying compound, we investigated the relationship between plasma PPi ([PPi]pl), liver fibrosis, liver function, AC, and the hepatic expression of genes regulating PPi homeostasis. To that aim, we compared [PPi]pl before liver transplantation (LT) and 3 months after LT. We also assessed the expression of four key regulators of PPi in liver tissues and established correlations between AC, and scores of liver fibrosis and liver failure in these patients. LT candidates with various liver diseases were included. AC scores were assessed in coronary arteries, abdominal aorta, and aortic valves. Liver fibrosis was evaluated on liver biopsies and from non-invasive tests (FIB-4 and APRI scores). Liver functions were assessed by measuring serum albumin, ALBI, MELD, and Pugh–Child scores. An enzymatic assay was used to dose [PPi]pl. A group of patients without liver alterations from a previous cohort provided a control group. Gene expression assays were performed with mRNA extracted from liver biopsies and compared between LT recipients and the control individuals. [PPi]pl negatively correlated with APRI (r = −0.57, p = 0.001, n = 29) and FIB-4 (r = −0.47, p = 0.006, n = 29) but not with interstitial fibrosis index from liver biopsies (r = 0.07, p = 0.40, n = 16). Serum albumin positively correlated with [PPi]pl (r = 0.71; p < 0.0001, n = 20). ALBI, MELD, and Pugh–Child scores correlated negatively with [PPi]pl (r = −0.60, p = 0.0005; r = −0.56, p = 0.002; r = −0.41, p = 0.02, respectively, with n = 20). Liver fibrosis assessed on liver biopsies by FIB-4 and by APRI positively correlated with coronary AC (r = 0.51, p = 0.02, n = 16; r = 0.58, p = 0.009, n = 20; r = 0.41, p = 0.04, n = 20, respectively) and with abdominal aorta AC (r = 0.50, p = 0.02, n = 16; r = 0.67, p = 0.002, n = 20; r = 0.61, p = 0.04, n = 20, respectively). FIB-4 also positively correlated with aortic valve calcification (r = 0.40, p = 0.046, n = 20). The key regulator genes of PPi production in liver were lower in patients undergoing liver transplantation as compared to controls. Three months after surgery, serum albumin levels were restored to physiological levels (40 [37–44] vs. 35 [30–40], p = 0.009) and [PPi]pl was normalized (1.40 [1.07–1.86] vs. 0.68 [0.53–0.80] µmol/L, p = 0.0005, n = 12). Liver failure and/or fibrosis correlated with AC in several arterial beds and were associated with low plasma PPi and dysregulation of key proteins involved in PPi homeostasis. Liver transplantation normalized these parameters