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Sclerostin is a negative regulator of the Wnt/β-catenin signaling and is, therefore, an important inhibitor of bone formation and turnover. Since ectopic vascular calcification develops in a similar way to bone formation, one might reasonably attribute a role to sclerostin in this pathological process. Ectopic calcification, especially vascular calcification, importantly contributes to mortality in elderly and patients with diabetes, osteoporosis, chronic kidney disease (CKD) and hypertension. The central players in this ectopic calcification process are the vascular smooth muscle cells that undergo dedifferentiation and thereby acquire characteristics of bone-like cells. Therefore, we hypothesize that depletion/de-activation of the Wnt/β-catenin signaling inhibitor sclerostin, may promote the development of ectopic calcifications through stimulation of bone-anabolic effects at the level of the arteries. We investigated the role of sclerostin (encoded by the Sost-gene) during vascular calcification by using either Sost-/- mice or anti-sclerostin antibody. Sost-/- and wild-type mice (C57BL/6J background) were administered an adenine-containing diet to promote the development of CKD-induced vascular calcification. Calcifications developed more extensively in the cardiac vessels of adenine-exposed Sost-/- mice, compared to adenine-exposed WT mice. This could be concluded from the cardiac calcium content as well as from cardiac tissue sections on which calcifications were visualized histochemically. In a second experiment, DBA/2J mice were administered a warfarin-containing diet to induce vascular calcifications in the absence of CKD. Here, warfarin-exposure led to significantly increased aortic and renal tissue calcium content. Calcifications, which were present in the aortic medial layer and renal vessels, were significantly more pronounced when warfarin-treatment was combined with anti-sclerostin antibody treatment. This study demonstrates a protective effect of sclerostin during vascular calcification.

Arterial media calcification refers to the pathological deposition of calcium phosphate crystals in the arterial wall. This pathology is a common and life-threatening complication in chronic kidney disease, diabetes and osteoporosis patients. Recently, we reported that the use of a TNAP inhibitor, SBI-425, attenuated arterial media calcification in a warfarin rat model. Employing a high-dimensionality unbiased proteomic approach, we also investigated the molecular signaling events associated with blocking arterial calcification through SBI-425 dosing. The remedial actions of SBI-425 were strongly associated with (i) a significant downregulation of inflammatory (acute phase response signaling) and steroid/glucose nuclear receptor signaling (LXR/RXR signaling) pathways and (ii) an upregulation of mitochondrial metabolic pathways (TCA cycle II and Fatty Acid β-oxidation I). Interestingly, we previously demonstrated that uremic toxin-induced arterial calcification contributes to the activation of the acute phase response signaling pathway. Therefore, both studies suggest a strong link between acute phase response signaling and arterial calcification across different conditions. The identification of therapeutic targets in these molecular signaling pathways may pave the way to novel therapies against the development of arterial media calcification.

Arterial media calcification is an active cell process. This encompasses osteochondrogenic transdifferentiation of vascular smooth muscle cells followed by the deposition of calcium-phosphate crystals. Increasing evidence suggests a significant role for endothelial cells (ECs) in the development of arterial media calcification. This manuscript explores a role for endothelial dysfunction in the disease progression of arterial media calcification. Male rats were randomly assigned to four different groups. The first group received standard chow. The second group was given L-NAME (≈50 mg kg−1 · d−1), to induce endothelial dysfunction, in addition to standard chow. The third group and fourth group received a warfarin-supplemented diet to induce mild calcification and the latter group was co-administered L-NAME. Prior to sacrifice, non-invasive measurement of aortic distensibility was performed. Animals were sacrificed after 6 weeks. Arterial media calcification was quantified by measuring aortic calcium and visualized on paraffin-embedded slices by the Von Kossa method. Arterial stiffness and aortic reactivity was assessed on isolated carotid segments using specialized organ chamber setups. Warfarin administration induced mineralization. Simultaneous administration of warfarin and L-NAME aggravated the arterial media calcification process. Through organ chamber experiments an increased vessel tonus was found, which could be linked to reduced basal NO availability, in arteries of warfarin-treated animals. Furthermore, increased calcification because of L-NAME administration was related to a further compromised endothelial function (next to deteriorated basal NO release also deteriorated stimulated NO release). Our findings suggest early EC changes to impact the disease progression of arterial media calcification.

Arterial medial calcification (AMC) is the deposition of calcium phosphate in the arteries. AMC is widely thought to share similarities with physiological bone formation; however, emerging evidence suggests several key differences between these processes. N-acetylcysteine (NAC) displays antioxidant properties and can generate hydrogen sulphide (H2S) and glutathione (GSH) from its deacetylation to l-cysteine. This study found that NAC exerts divergent effects in vitro, increasing osteoblast differentiation and bone formation by up to 5.5-fold but reducing vascular smooth muscle cell (VSMC) calcification and cell death by up to 80%. In vivo, NAC reduced AMC in a site-specific manner by 25% but had no effect on the bone. The actions of l-cysteine and H2S mimicked those of NAC; however, the effects of H2S were much less efficacious than NAC and l-cysteine. Pharmacological inhibition of H2S-generating enzymes did not alter the actions of NAC or l-cysteine; endogenous production of H2S was also unaffected. In contrast, NAC and l-cysteine increased GSH levels in calcifying VSMCs and osteoblasts by up to 3-fold. This suggests that the beneficial actions of NAC are likely to be mediated via the breakdown of l-cysteine and the subsequent GSH generation. Together, these data show that while the molecular mechanisms driving the actions of NAC appear similar, the downstream effects on cell function differ significantly between osteoblasts and calcifying VSMCs. The ability of NAC to exert these differential actions further supports the notion that there are differences between the development of pathological AMC and physiological bone formation. NAC could represent a therapeutic option for treating AMC without exerting negative effects on bone.

Current treatment strategies for chronic kidney disease (CKD) mainly focus on controlling risk factors. Metformin, a first-line drug for type-2 diabetes, exerts beneficial pleiotropic actions beyond its prescribed use and incipient data has revealed protective effects against the development of kidney impairment. This study evaluated the therapeutic efficacy of metformin and canagliflozin, a sodium-glucose cotransporter-2 (SGLT2) inhibitor recently approved by the United States Food and Drug Administration to treat diabetic nephropathy, in slowing the progression of established non-diabetic CKD. Rats with adenine-induced CKD were assigned to different treatment groups to receive either 200 mg/kg metformin, four or five weeks after the start of the adenine diet (established mild-moderate CKD), or 25 mg/kg canagliflozin four weeks after the start of the diet, by daily oral gavage administered during four weeks. Each treatment group was compared to a vehicle group. Chronic adenine dosing resulted in severe CKD in vehicle-treated rats as indicated by a marked rise in serum creatinine levels, a marked decrease in creatinine clearance, and a disturbed mineral metabolism. Metformin, but not canagliflozin, halted functional kidney decline. Additionally, kidneys of metformin-treated animals showed less interstitial area and inflammation as compared to the vehicle group. Proteomic analyses revealed that metformin’s kidney-protective effect was associated with the activation of the Hippo signaling pathway, a highly conserved multiprotein kinase cascade that controls tissue development, organ size, cell proliferation, and apoptosis. Thus, metformin demonstrated therapeutic efficacy by halting the progression of established CKD in a rat model.

Arterial calcification, the deposition of calcium-phosphate crystals in the extracellular matrix, resembles physiological bone mineralization. It is well-known that extracellular nucleotides regulate bone homeostasis raising an emerging interest in the role of these molecules on arterial calcification. The purinergic independent pathway involves the enzymes ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs), ecto-nucleoside triphosphate diphosphohydrolases (NTPDases), 5′-nucleotidase and alkaline phosphatase. These regulate the production and breakdown of the calcification inhibitor—pyrophosphate and the calcification stimulator—inorganic phosphate, from extracellular nucleotides. Maintaining ecto-nucleotidase activities in a well-defined range is indispensable as enzymatic hyper- and hypo-expression has been linked to arterial calcification. The purinergic signaling dependent pathway focusses on the activation of purinergic receptors (P1, P2X and P2Y) by extracellular nucleotides. These receptors influence arterial calcification by interfering with the key molecular mechanisms underlying this pathology, including the osteogenic switch and apoptosis of vascular cells and possibly, by favoring the phenotypic switch of vascular cells towards an adipogenic phenotype, a recent, novel hypothesis explaining the systemic prevention of arterial calcification. Selective compounds influencing the activity of ecto-nucleotidases and purinergic receptors, have recently been developed to treat arterial calcification. However, adverse side-effects on bone mineralization are possible as these compounds reasonably could interfere with physiological bone mineralization.

Background and Aims Vascular media calcification is frequently seen in elderly and patients with chronic kidney disease (CKD), diabetes and osteoporosis. Pyrophosphate is a well-known calcification inhibitor that binds to nascent hydroxyapatite crystals and prevents further incorporation of inorganic phosphate into these crystals. However, the enzyme tissue-nonspecific alkaline phosphatase (TNAP), which is highly expressed in calcified arteries, degrades extracellular pyrophosphate into phosphate ions, by which pyrophosphate loses its ability to block vascular calcification. Here, we aimed to evaluate whether a TNAP inhibitor is able to prevent the development of arterial calcification in a rat model of warfarin-induced vascular calcification. Method To induce vascular calcification, rats received a diet containing 0.30% warfarin and 0.15% vitamin K1 throughout the entire study and were subjected to the following daily treatments: (i) vehicle (n=10) or (ii) 10 mg/kg/day TNAP-inhibitor (n=10) administered via an intraperitoneal catheter from start of the study until sacrifice at week 7. Calcium, phosphorus and parathyroid hormone (PTH) levels were determined in serum samples as these are important determinants of vascular calcification. As TNAP is also expressed in the liver, serum alanine aminotransferase (ALT) and aspartate (AST) levels were analyzed. At sacrifice, vascular calcification was evaluated by measurement of the total calcium content in the arteries and quantification of the area % calcification on Von Kossa stained sections of the aorta. The mRNA expression of osteo/chondrogenic marker genes (runx2, TNAP, SOX9, collagen 1 and collagen 2) was analyzed in the aorta by qPCR to verify whether vascular smooth muscle cells underwent reprogramming towards bone-like cells. Bone histomorphometry was performed on the left tibia to measure static and dynamic bone parameters as TNAP also regulates physiological bone mineralization. Results No differences in serum calcium, phosphorus and PTH levels was observed between both study groups. Warfarin exposure resulted in distinct calcification in the aorta and peripheral arteries. Daily dosing with the TNAP inhibitor (10 mg/kg/day) for 7 weeks significantly reduced vascular calcification as indicated by a significant decrease in calcium content in the aorta (vehicle 3.84±0.64 mg calcium/g wet tissue vs TNAP inhibitor 0.70±0.23 mg calcium/g wet tissue) and peripheral arteries and a distinct reduction in area % calcification on Von Kossa stained aortic sections as compared to vehicle condition. The inhibitory effects of SBI-425 on vascular calcification were without altering serum liver markers ALT and AST levels. Furthermore, TNAP-inhibitor SBI-425 did not modulate the mRNA expression of osteo/chondrogenic marker genes runx2, TNAP, SOX9, collagen 1 and 2. Dosing with SBI-425 resulted in decreased bone formation rate and mineral apposition rate, and increased osteoid maturation time and this without significant changes in osteoclast- and eroded perimeter. Conclusion Dosing with TNAP inhibitor SBI-425 significantly reduced the calcification in the aorta and peripheral arteries of a rat model of warfarin-induced vascular calcification and this without affecting liver function. However, suppression of TNAP activity should be limited in order to maintain adequate physiological bone mineralization.

Arterial media calcification (AMC) is predominantly regulated by vascular smooth muscle cells (VSMCs), which transdifferentiate into pro-calcifying cells. In contrast, there is little evidence for endothelial cells playing a role in the disease. The current study investigates cellular functioning and molecular pathways underlying AMC, respectively by, an ex vivo isometric organ bath set-up to explore the interaction between VSMCs and ECs and quantitative proteomics followed by functional pathway interpretation. AMC development, which was induced in mice by dietary warfarin administration, was proved by positive Von Kossa staining and a significantly increased calcium content in the aorta compared to that of control mice. The ex vivo organ bath set-up showed calcified aortic segments to be significantly more sensitive to phenylephrine induced contraction, compared to control segments. This, together with the fact that calcified segments as compared to control segments, showed a significantly smaller contraction in the absence of extracellular calcium, argues for a reduced basal NO production in the calcified segments. Moreover, proteomic data revealed a reduced eNOS activation to be part of the vascular calcification process. In summary, this study identifies a poor endothelial function, next to classic pro-calcifying stimuli, as a possible initiator of arterial calcification.

Introduction Sucroferric oxyhydroxide (PA21) is an efficacious, well-tolerated iron-based phosphate binder and a promising alternative to existing compounds. We compared the effects of PA21 with those of a conventional phosphate binder on renal function, mineral homeostasis and vascular calcification in a chronic kidney disease–mineral and bone disorder (CKD-MBD) rat model. Methods To induce stable renal failure, rats were administered a 0.25% adenine diet for 8 weeks. Concomitantly, rats were treated with vehicle, 2.5 g/kg/day PA21, 5.0 g/kg/day PA21 or 3.0 g/kg/day calcium carbonate (CaCO3). Renal function and calcium/phosphorus/iron metabolism were evaluated during the study course. Renal fibrosis, inflammation, vascular calcifications and bone histomorphometry were quantified. Results Rats treated with 2.5 or 5.0 g/kg/day PA21 showed significantly lower serum creatinine and phosphorus and higher ionized calcium levels after 8 weeks of treatment compared with vehicle-treated rats. The better preserved renal function with PA21 went along with less severe anaemia, which was not observed with CaCO3. Both PA21 doses, in contrast to CaCO3, prevented a dramatic increase in fibroblast growth factor (FGF)-23 and significantly reduced the vascular calcium content while both compounds ameliorated CKD-related hyperparathyroid bone. Conclusions PA21 treatment prevented an increase in serum FGF-23 and had, aside from its phosphate-lowering capacity, a beneficial impact on renal function decline (as assessed by the renal creatinine clearance) and related disorders. The protective effect of this iron-based phosphate binder on the kidney in rats, together with its low pill burden in humans, led us to investigate its use in patients with impaired renal function not yet on dialysis.

Sclerostin is a well-known inhibitor of bone formation that acts on Wnt/&beta, catenin signaling. This manuscript considers the possible role of sclerostin in vascular calcification, a process that shares many similarities with physiological bone formation. Rats were exposed to a warfarin-containing diet to induce vascular calcification. Vascular smooth muscle cell transdifferentiation, vascular calcification grade, and bone histomorphometry were examined. The presence and/or production of sclerostin was investigated in serum, aorta, and bone. Calcified human aortas were investigated to substantiate clinical relevance. Warfarin-exposed rats developed vascular calcifications in a time-dependent manner which went along with a progressive increase in serum sclerostin levels. Both osteogenic and adipogenic pathways were upregulated in calcifying vascular smooth muscle cells, as well as sclerostin mRNA and protein levels. Evidence for the local vascular action of sclerostin was found both in human and rat calcified aortas. Warfarin exposure led to a mildly decreased bone and mineralized areas. Osseous sclerostin production and bone turnover did not change significantly. This study showed local production of sclerostin in calcified vessels, which may indicate a negative feedback mechanism to prevent further calcification. Furthermore, increased levels of serum sclerostin, probably originating from excessive local production in calcified vessels, may contribute to the linkage between vascular pathology and impaired bone mineralization.

BACKGROUND: Protein-bound uremic toxins indoxyl sulfate (IS) and p-cresyl sulfate (PCS) have been associated with cardiovascular morbidity and mortality in patients with CKD. However, direct evidence for a role of these toxins in CKD-related vascular calcification has not been reported. METHODS: To study early and late vascular alterations by toxin exposure, we exposed CKD rats to vehicle, IS (150 mg/kg per day), or PCS (150 mg/kg per day) for either 4 days (short-term exposure) or 7 weeks (long-term exposure). We also performed unbiased proteomic analyses of arterial samples coupled to functional bioinformatic annotation analyses to investigate molecular signaling events associated with toxin-mediated arterial calcification. RESULTS: Long-term exposure to either toxin at serum levels similar to those experienced by patients with CKD significantly increased calcification in the aorta and peripheral arteries. Our analyses revealed an association between calcification events, acute-phase response signaling, and coagulation and glucometabolic signaling pathways, whereas escape from toxin-induced calcification was linked with liver X receptors and farnesoid X/liver X receptor signaling pathways. Additional metabolic linkage to these pathways revealed that IS and PCS exposure engendered a prodiabetic state evidenced by elevated resting glucose and reduced GLUT1 expression. Short-term exposure to IS and PCS (before calcification had been established) showed activation of inflammation and coagulation signaling pathways in the aorta, demonstrating that these signaling pathways are causally implicated in toxin-induced arterial calcification. CONCLUSIONS: In CKD, both IS and PCS directly promote vascular calcification via activation of inflammation and coagulation pathways and were strongly associated with impaired glucose homeostasis. ispartof: JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY vol:30 issue:5 pages:751-766 ispartof: location:United States status: published

Arterial media calcification is frequently seen in elderly and patients with chronic kidney disease (CKD), diabetes and osteoporosis. Pyrophosphate is a well-known calcification inhibitor that binds to nascent hydroxyapatite crystals and prevents further incorporation of inorganic phosphate into these crystals. However, the enzyme tissue-nonspecific alkaline phosphatase (TNAP), which is expressed in calcified arteries, degrades extracellular pyrophosphate into phosphate ions, by which pyrophosphate loses its ability to block vascular calcification. Here, we aimed to evaluate whether pharmacological TNAP inhibition is able to prevent the development of arterial calcification in a rat model of warfarin-induced vascular calcification. To investigate the effect of the pharmacological TNAP inhibitor SBI-425 on vascular calcification and bone metabolism, a 0.30% warfarin rat model was used. Warfarin exposure resulted in distinct calcification in the aorta and peripheral arteries. Daily administration of the TNAP inhibitor SBI-425 (10 mg/kg/day) for 7 weeks significantly reduced vascular calcification as indicated by a significant decrease in calcium content in the aorta (vehicle 3.84 ± 0.64 mg calcium/g wet tissue vs TNAP inhibitor 0.70 ± 0.23 mg calcium/g wet tissue) and peripheral arteries and a distinct reduction in area % calcification on Von Kossa stained aortic sections as compared to vehicle. Administration of SBI-425 resulted in decreased bone formation rate and mineral apposition rate, and increased osteoid maturation time and this without significant changes in osteoclast- and eroded perimeter. Administration of TNAP inhibitor SBI-425 significantly reduced the calcification in the aorta and peripheral arteries of a rat model of warfarin-induced vascular calcification. However, suppression of TNAP activity should be limited in order to maintain adequate physiological bone mineralization.

The use of geo-engineering materials to manage phosphorus in lakes has increased in recent years with aluminium and lanthanum based materials being most commonly applied. Hence the potential impact of the use of these compounds on human health is receiving growing interest. This review seeks to understand, evaluate and compare potential unintended consequences on human health and ecotoxicological risks associated with the use of lanthanum- and aluminium-based materials to modify chemical and ecological conditions in water bodies. In addition to their therapeutic use for the reduction of intestinal phosphate absorption in patients with impaired renal function, the phosphate binding capacity of aluminium and lanthanum also led to the development of materials used for water treatment. Although lanthanum and aluminium share physicochemical similarities and have many common applications, their uptake and kinetics within the human body and living organisms importantly differ from each other which is reflected in a different toxicity profile. Whilst a causal role in the development of neurological pathologies, skeletal lesions, hematopoietic disorders and respiratory effects has unequivocally been demonstrated with increased exposure to aluminium, studies until now have failed to find such a clear association after exposure to lanthanum although caution is warranted. Our review indicates that lanthanum and aluminium have a distinctly different profile with respect to their potential effects on human health. Regular monitoring of both aluminium and lanthanum concentrations in lanthanum-/aluminium-treated water by the responsible authorities is recommended to avoid acute accidental or chronic low level accumulation.

Because current rat models used to study chronic kidney disease (CKD)-related vascular calcification show consistent but excessive vascular calcification and chaotic, immeasurable, bone mineralization due to excessive bone turnover, they are not suited to study the bone-vascular axis in one and the same animal. Because vascular calcification and bone mineralization are closely related to each other, an animal model in which both pathologies can be studied concomitantly is highly needed. CKD-related vascular calcification in rats was induced by a 0.25% adenine/low vitamin K diet. To follow vascular calcification and bone pathology over time, rats were killed at weeks 4, 8, 10, 11, and 12. Both static and dynamic bone parameters were measured. Vascular calcification was quantified by histomorphometry and measurement of the arterial calcium content. Stable, severe CKD was induced along with hyperphosphatemia, hypocalcemia as well as increased serum PTH and FGF23. Calcification in the aorta and peripheral arteries was present from week 8 of CKD onward. Four and 8 weeks after CKD, static and dynamic bone parameters were measurable in all animals, thereby presenting typical features of hyperparathyroid bone disease. Multiple regression analysis showed that the eroded perimeter and mineral apposition rate in the bone were strong predictors for aortic calcification. This rat model presents a stable CKD, moderate vascular calcification, and quantifiable bone pathology after 8 weeks of CKD and is the first model that lends itself to study these main complications simultaneously in CKD in mechanistic and intervention studies.

Canonical Wnt signaling activity contributes to physiological and adaptive bone mineralization and is an essential player in bone remodeling. Sclerostin is a prototypic soluble canonical Wnt signaling pathway inhibitor that is produced in osteocytes and blocks osteoblast differentiation and function. Therefore, sclerostin is a potent inhibitor of bone formation and mineralization. Accordingly, rodent sclerostin-deficiency models exhibit a strong bone phenotype. Moreover, blocking sclerostin represents a promising treatment perspective against osteoporosis. Beyond the bone field novel data definitely associate Wnt signaling in general and sclerostin in particular with ectopic extraosseous mineralization processes, as is evident in cardiovascular calcification or calciphylaxis. Uremia is characterized by parallel occurrence of disordered bone mineralization and accelerated cardiovascular calcification (chronic kidney disease mineral and bone disorder, CKD-MBD), linking skeletal and cardiovascular diseasethe so-called bone-vascular calcification paradox. In consequence, sclerostin may qualify as an emerging player in CKD-MBD. We present a stepwise review approach regarding the rapidly evolving field sclerostin participation in CKD-MBD. Starting from data originating in the classical bone field we look separately at three major areas of CKD-MBD: disturbed mineral metabolism, renal osteodystrophy, and uremic cardiovascular disease. Our review is intended to help the nephrologist revise the potential importance of sclerostin in CKD by focusing on how sclerostin research is gradually evolving from the classical osteoporosis niche into the area of CKD-MBD. In particular, we integrate the limited amount of available data in the context of pediatric nephrology.

Chronic kidney disease (CKD) causes dysregulation of mineral metabolism, vascular calcification and renal osteodystrophy, an entity called CKDMineral and Bone Disorder (CKD-MBD). Here we determine whether metformin, an anti-diabetic drug, exerts favorable effects on progressive, severe CKD and concomitant mineral metabolism disturbances. Rats with CKD-MBD, induced by a 0.25% adenine diet for eight weeks, were treated with 200 mg/kg/day metformin or vehicle from one week after CKD induction onward. Severe, stable CKD along with marked hyperphosphatemia and hypocalcemia developed in these rats which led to arterial calcification and high bone turnover disease. Metformin protected from development toward severe CKD. Metformin-treated rats did not develop hyperphosphatemia or hypocalcemia and this prevented the development of vascular calcification and inhibited the progression toward high bone turnover disease. Kidneys of the metformin group showed significantly less cellular infiltration, fibrosis and inflammation. To study a possible direct effect of metformin on the development of vascular calcification, independent of its effect on renal function, metformin (200 mg/kg/day) or vehicle was dosed for ten weeks to rats with warfarin-induced vascular calcification. The drug did not reduce aorta or small vessel calcification in this animal model. Thus, metformin protected against the development of severe CKD and preserved calcium phosphorus homeostasis. As a result of its beneficial impact on renal function, associated comorbidities such as vascular calcification and high bone turnover disease were also prevented.

Calcium-based phosphate binders are used to control hyperphosphatemia; however, they promote hypercalcemia and may accelerate aortic calcification. Here we compared the effect of a phosphate binder containing calcium acetate and magnesium carbonate (CaMg) to that of sevelamer carbonate on the development of medial calcification in rats with chronic renal failure induced by an adenine diet for 4 weeks. After 1 week, rats with chronic renal failure were treated with vehicle, 375 or 750 mg/kg CaMg, or 750 mg/kg sevelamer by daily gavage for 5 weeks. Renal function was significantly impaired in all groups. Vehicle-treated rats with chronic renal failure developed severe hyperphosphatemia, but this was controlled in treated groups, particularly by CaMg. Neither CaMg nor sevelamer increased serum calcium ion levels. Induction of chronic renal failure significantly increased serum PTH, dose-dependently prevented by CaMg but not sevelamer. The aortic calcium content was significantly reduced by CaMg but not by sevelamer. The percent calcified area of the aorta was significantly lower than vehicle-treated animals for all three groups. The presence of aortic calcification was associated with increased sox9, bmp-2, and matrix gla protein expression, but this did not differ in the treatment groups. Calcium content in the carotid artery was lower with sevelamer than with CaMg but that in the femoral artery did not differ between groups. Thus, treatment with either CaMg or sevelamer effectively controlled serum phosphate levels in CRF rats and reduced aortic calcification.

Background: The cardiorenal syndrome (CRS) is a major health problem in our aging population. The term was introduced to cover disorders of the kidneys and heart, whereby dysfunction of one organ may induce dysfunction of the other. As the natural history of the CRS is mostly slow, hence difficult to explore in clinical trials, adequate animal models combining cardiovascular and renal disease are required. Therefore, we developed and characterized a usable model for CRS type 4, i.e. chronic kidney disease (CKD) causing cardiac dysfunction. Methods: CKD was induced in rats by supplementing the diet with adenine. During 8 weeks, several aspects of CRS were studied: CKD, mineral-bone disorder (MBD), cardiovascular disease, and (iron-deficiency) anemia. Hereto, the following parameters were monitored: serum creatinine, calcium, phosphate, FGF23, dynamic bone parameters, aortic Ca deposits, heart weight, serum NT-proANP, Hct, Hb, reticulocytes, spleen iron, and serum hepcidin. Results: Animals developed a severe CKD together with a disturbed mineral balance as reflected by the increased serum creatinine and phosphorus levels and decreased serum calcium levels; and in association herewith aberrations in hormonal levels of FGF-23. In turn, the well-known and highly undesirable complications of CKD, i.e. high turnover bone disease and pathological vessel calcification were induced. Furthermore (iron-deficiency) anemia developed quickly. Conclusion: The animal model described in this article in many aspects mimics the human situation of the CRS type 4 and will be useful to concomitantly evaluate the effects of new treatment strategies on the various aspects of CRS.

Processes similar to endochondral or intramembranous bone formation occur in the vascular wall. Bone and cartilage tissue as well as osteoblast- and chondrocyte-like cells are present in calcified arteries. As in bone formation, apoptosis and matrix vesicles play an important role in the initiation of vascular calcification. Recent evidence indicates that nanocrystals initially formed in the vessel wall may actively be involved in the progression of the calcification process. This review focuses on the cellular and structural similarities between bone formation and vascular calcification and discusses the initial events in this pathological mineralization process.

Iron deficiency is frequently seen in patients with end-stage renal disease, particularly in those treated by dialysis, this is because of an impairment in gastrointestinal absorption and ongoing blood losses or alternatively, due to an impaired capacity to mobilize iron from its stores, called functional iron deficiency. Therefore, these patients may require intravenous iron to sustain adequate treatment with erythropoietin-stimulating agents. Aside from this, they are also prone to vascular calcification, which has been reported a major contributing factor in the development of cardiovascular disease and the increased mortality associated herewith. Several factors and mechanisms underlying the development of vascular calcification in chronic kidney diseased patients have been put forward during recent years. In view of the ability of iron to exert direct toxic effects and to induce oxidative stress on the one hand versus its essential role in various cellular processes on the other hand, the possible role of iron in the development of vascular calcification should be considered.

Background: Increased bone loss has been associated with the development of vascular calcification in patients with chronic renal failure (CRF). In this study, the effect of impaired bone metabolism on aortic calcifications was investigated in uremic rats with or without ovariectomy. Methods: CRF was induced by administration of a 0.75% adenine/2.5% protein diet for 4 weeks. In one group, osteoporosis was induced by ovariectomy (CRF-OVX), while the other group underwent a sham-operation instead (CRF). A third group consisted of ovariectomized rats with normal renal function (OVX). At regular time intervals throughout the study, bone status and aortic calcifications were evaluated by in vivo micro-CT. At sacrifice after 6 weeks of CRF, bone histomorphometry was performed and vascular calcification was assessed by bulk calcium analysis and Von Kossa staining. Results: Renal function was significantly impaired in the CRF-OVX and CRF groups. Trabecular bone loss was seen in all groups. In the CRF-OVX and CRF groups, trabecular bone density was restored after adenine withdrawal, which coincided with cortical bone loss and the development of medial calcifications in the aorta. No significant differences with regard to the degree of aortic calcifications were seen between the two CRF groups. Neither cortical bone loss nor calcifications were seen in the OVX group. Cortical bone loss significantly correlated with the severity of vascular calcification in the CRF-OVX and CRF groups, but no associations with trabecular bone changes were found. Conclusions: Cortical rather than trabecular bone loss is associated with the process of calcification in rats with adenine- induced CRF.

Over the past decades metformin has been the optimal first-line treatment for type 2 diabetes mellitus (T2DM). Only in the last few years, it has become increasingly clear that metformin exerts benign pleiotropic actions beyond its prescribed use and ongoing investigations focus on a putative beneficial impact of metformin on the kidney. Both acute kidney injury (AKI) and chronic kidney disease (CKD), two major renal health issues, often result in the need for renal replacement therapy (dialysis or transplantation) with a high socio-economic impact for the patients. Unfortunately, to date, effective treatment directly targeting the kidney is lacking. Metformin has been shown to exert beneficial effects on the kidney in various clinical trials and experimental studies performed in divergent rodent models representing different types of renal diseases going from AKI to CKD. Despite growing evidence on metformin as a candidate drug for renal diseases, in-depth research is imperative to unravel the molecular signaling pathways responsible for metformins renoprotective actions. This review will discuss the current state-of-the-art literature on clinical and preclinical data, and put forward potential cellular mechanisms and molecular pathways by which metformin ameliorates AKI/CKD. View Full-Text Keywords: metformin; type 2 diabetes mellitus; acute kidney injury; chronic kidney disease; lactic acidosis; renoprotection; AMP-activated protein kinase pathway

Objective— To investigate cell biological changes in calcified aortas of rats that experienced chronic renal failure. Methods and Results— Vascular smooth muscle cells have the potential to transdifferentiate to either chondrocytes or osteoblasts, depending on the molecular pathways that are stimulated. Uremia-related medial calcification was induced by feeding rats an adenine low-protein diet for 4 weeks. Aortic calcification was evaluated biochemically and histochemically and with in vivo micro–computed tomographic scanning. Immunohistochemistry and RT-PCR were applied to analyze the time-dependent aortic expression of molecules involved in the segregation between the chondrocyte versus osteoblast differentiation pathway. After 4 weeks, 85% of the uremic rats had developed distinct aortic medial calcification, which increased to severely calcified lesions during further follow-up. The calcification process was accompanied by a significant time-dependent increase in the expression of the chondrocyte-specific markers sex determining region Y-box 9 (sox9), collagen II, and aggrecan and a nonsignificant trend toward enhanced core binding factor alpha 1 (cbfa1), and collagen I. The expression of the osteoblast marker osterix and both lipoprotein receptor–related protein 6 and β-catenin, molecules of the wingless-type MMTV integration site family member (Wnt)/β-catenin pathway induced during osteoblast differentiation, was suppressed. Conclusion— In the aorta of uremic rats, medial smooth muscle cells acquire a chondrocyte rather than osteoblast phenotype during the calcification process.

Vascular calcification significantly contributes to mortality in chronic kidney disease (CKD) patients. Sevelamer and pyrophosphate (PPi) have proven to be effective in preventing vascular calcification, the former by controlling intestinal phosphate absorption, the latter by directly interfering with the hydroxyapatite crystal formation. Since most patients present with established vascular calcification, it is important to evaluate whether these compounds may also halt or reverse the progression of preexisting vascular calcification. CKD and vascular calcification were induced in male Wistar rats by a 0.75 % adenine low protein diet for 4 weeks. Treatment with PPi (30 or 120 µmol/kg/day), sevelamer carbonate (1500 mg/kg/day) or vehicle was started at the time point at which vascular calcification was present and continued for 3 weeks. Hyperphosphatemia and vascular calcification developed prior to treatment. A significant progression of aortic calcification in vehicle-treated rats with CKD was observed over the final 3-week period. Sevelamer treatment significantly reduced further progression of aortic calcification as compared to the vehicle control. No such an effect was seen for either PPi dose. Sevelamer but not PPi treatment resulted in an increase in both osteoblast and osteoid perimeter. Our study shows that sevelamer was able to reduce the progression of moderate to severe preexisting aortic calcification in a CKD rat model. Higher doses of PPi may be required to induce a similar reduction of severe established arterial calcification in this CKD model.

In the present study, we characterized and compared the mineral phase deposited in the aortic wall of two different frequently used chronic renal failure rat models of vascular calcification. Vascular calcification was induced in rats by either a 4-week adenine treatment followed by a 10-week high-phosphate diet or 5/6 nephrectomy followed by 6 weeks of 0.25 μ g/kg/day calcitriol treatment and a high-phosphate diet. Multi-element mapping for calcium and phosphate together with mineral identification was performed on several regions of aortic sections by means of synchrotron X-ray- μ -fluorescence and diffraction. Bulk calcium and magnesium content of the aorta was assessed using flame atomic absorption spectrometry. Based on the diffraction data the Von Kossa-positive precipitate in the aortic regions ( N =38) could be classified into three groups: (1) amorphous precipitate (absence of any diffraction peak pattern, N =12); (2) apatite ( N =16); (3) a combination of apatite and magnesium-containing whitlockite ( N =10). The occurrence of these precipitates differed significantly between the two models. Furthermore, the combination of apatite and whitlockite was exclusively found in the calcitriol-treated animals. These data indicate that in adenine/phosphate-induced uremia-related vascular calcification, apatite is the main component of the mineral phase. The presence of magnesium-containing whitlockite found in addition to apatite in the vitamin D-treated rats, has to be seen in view of the well-known vitamin D-stimulated gastrointestinal absorption of magnesium.

Because current rat models used to study chronic kidney disease (CKD)-related vascular calcification show consistent but excessive vascular calcification and chaotic, immeasurable, bone mineralization due to excessive bone turnover, they are not suited to study the bone-vascular axis in one and the same animal. Because vascular calcification and bone mineralization are closely related to each other, an animal model in which both pathologies can be studied concomitantly is highly needed. CKD-related vascular calcification in rats was induced by a 0.25% adenine/low vitamin K diet. To follow vascular calcification and bone pathology over time, rats were killed at weeks 4, 8, 10, 11, and 12. Both static and dynamic bone parameters were measured. Vascular calcification was quantified by histomorphometry and measurement of the arterial calcium content. Stable, severe CKD was induced along with hyperphosphatemia, hypocalcemia as well as increased serum PTH and FGF23. Calcification in the aorta and peripheral arteries was present from week 8 of CKD onward. Four and 8 weeks after CKD, static and dynamic bone parameters were measurable in all animals, thereby presenting typical features of hyperparathyroid bone disease. Multiple regression analysis showed that the eroded perimeter and mineral apposition rate in the bone were strong predictors for aortic calcification. This rat model presents a stable CKD, moderate vascular calcification, and quantifiable bone pathology after 8 weeks of CKD and is the first model that lends itself to study these main complications simultaneously in CKD in mechanistic and intervention studies.

There is increasing interest in the colonic microbiota as a relevant source of uremic retention solutes accumulating in CKD. Renal disease can also profoundly affect the colonic microenvironment and has been associated with a distinct colonic microbial composition. However, the influence of CKD on the colonic microbial metabolism is largely unknown. Therefore, we studied fecal metabolite profiles of hemodialysis patients and healthy controls using a gas chromatography-mass spectrometry method. We observed a clear discrimination between both groups, with 81 fecal volatile organic compounds detected at significantly different levels in hemodialysis patients and healthy controls. To further explore the differential impact of renal function loss per se versus the effect of dietary and other CKD-related factors, we also compared fecal metabolite profiles between patients on hemodialysis and household contacts on the same diet, which revealed a close resemblance. In contrast, significant differences were noted between the fecal samples of rats 6 weeks after 5/6th nephrectomy and those of sham-operated rats, still suggesting an independent influence of renal function loss. Thus, CKD associates with a distinct colonic microbial metabolism, although the effect of renal function loss per se in humans may be inferior to the effects of dietary and other CKD-related factors. The potential beneficial effect of therapeutics targeting colonic microbiota in patients with CKD remains to be examined. ispartof: Journal of the American Society of Nephrology vol:27 issue:5 pages:1389-1399 ispartof: location:United States status: published

Although it is known that bisphosphonates prevent medial vascular calcification in vivo, their mechanism of action remains unknown and, in particular, whether they act directly on the blood vessels or indirectly through inhibition of bone resorption. To determine this, we studied the effects of two bisphosphonates on calcification of rat aortas in vitro and on in vivo aortic calcification and bone metabolism in rats with renal failure. We produced vascular calcification in rats with adenine-induced renal failure fed a high-phosphate diet. Daily treatment with either etidronate or pamidronate prevented aortic calcification, with the latter being 100-fold more potent. Both aortic calcification and bone formation were reduced in parallel; however, bone resorption was not significantly affected. In all uremic rats, aortic calcium content correlated with bone formation but not with bone resorption. Bisphosphonates also inhibited calcification of rat aortas in culture and arrested further calcification of precalcified vessels but did not reverse their calcification. Expression of osteogenic factors or calcification inhibitors was not altered by etidronate in vitro. Hence, these studies show that bisphosphonates can directly inhibit uremic vascular calcification independent of bone resorption. The correlation between inhibition of aortic calcification and bone mineralization is consistent with a common mechanism such as the prevention of hydroxyapatite formation and suggests that bisphosphonates may not be able to prevent vascular calcification without inhibiting bone formation in uremic rats.

The present study investigated to what extent normalization of bone turnover goes along with a reduction of high-dose calcitriol-induced vascular calcifications in uremic rats. Five groups of male SpragueDawley rats were studied: sham-operated controls (n = 7), subtotally nephrectomized (SNX) uremic (CRF) animals (n = 12), CRF + calcitriol (vitD) (0.25 μg/kg/day) (n = 12), CRF + vitD + cinacalcet (CIN) (10 mg/kg/day) (n = 12), and CRF + vitD + parathyroidectomy (PTX) (n = 12). Treatment started 2 weeks after SNX and continued for the next 14 weeks. High-dose calcitriol treatment in hyperparathyroid rats went along with the development of distinct vascular calcification, which was significantly reduced by >50 %, in both CIN-treated and PTX animals. Compared to control animals and those of the CRF group, calcitriol treatment either in combination with CIN or PTX or not was associated with a significant increase in bone area comprising ±50 % of the total tissue area. However, whereas excessive woven bone accompanied by a dramatically increased osteoid width/area was seen in the CRF + vitD group, CIN treatment and PTX resulted in significantly reduced serum PTH level, which was accompanied by a distinct reduction of both the bone formation rate and the amount of osteoid. These data indicate that less efficient calcium and phosphorus incorporation in bone inherent to the severe hyperparathyroidism in vitamin D-treated uremic rats goes along with excessive vascular calcification, a process which is partially reversed by CIN treatment in combination with a more efficacious bone mineralization, thus restricting the availability of calcium and phosphate for being deposited in the vessel wall.

Accelerated atherosclerotic plaque calcification and extensive medial calcifications are common and highly detrimental complications of chronic kidney disease. Valid murine models have been developed to investigate both pathologically distinguishable complications, which allow for better insight into the cellular mechanisms underlying these vascular pathologies and evaluation of compounds that might prevent or retard the onset or progression of vascular calcification. This review describes various experimental models that have been used for the study of arterial intimal and/or medial calcification and discusses the extent to which this experimental research has contributed to our current understanding of vascular calcification, particularly in the setting of chronic renal failure.

Background. Hyperphosphataemia is a risk factor for arterial calcification contributing to the high cardiovascular mortality in patients with chronic kidney disease. Calcium-based phosphate binders can induce hypercalcaemia and are associated with progression of vascular calcification. Therefore, the effect of lanthanum carbonate, a non-calcium phosphate binder, on the development of vascular calcification was investigated in uraemic rats. Methods. Chronic renal failure (CRF) was induced by feeding rats an adenine-enriched diet for 4 weeks. After 2 weeks, 1% or 2% lanthanum carbonate was added to the diet for 6 weeks. Calcification in the aorta, carotid and femoral arteries was evaluated histomorphometrically, biochemically and by ex vivo micro-CT. Chondro-/osteogenic conversion of vascular smooth muscle cells was also analysed in the rat aorta. Results. Treatment with 1% lanthanum carbonate (1% La) did not reduce vascular calcification, but in the 2% lanthanum carbonate (2% La) group vascular calcium content and area% Von Kossa positivity were decreased compared with control CRF rats. The aortic calcified volume measured with ex vivo micro-CT was significantly reduced in rats treated with 2% La. Although calcification was inhibited by treatment with 2% La, the chondrocyte transcription factor sox-9 was abundantly expressed in the aorta. Conclusion. Treatment of CRF rats with 2% La reduces the development of vascular calcification by adequate phosphate binding resulting in a decreased supply of phosphate as a substrate for vascular calcification.

In the present paper, vascular calcifications due to chronic renal failure in rats are studied by X-ray microtomography (micro-CT). Although micro-CT is traditionally used as an imaging technique, a quantitative analysis of data obtained by in vivo and ex vivo micro-CT is described and discussed. By comparison with traditional destructive methods, such as histomorphometry and atomic absorption, the detection limits for calcium were determined in living rats and in extracted aortas. micro-CT proved to be an effective non-invasive imaging technique allowing non-destructive quantification of ectopic calcifications.

Arterial media calcification is often considered a cell-regulated process resembling intramembranous bone formation, implying a conversion of vascular tissue into a bone-like structure without a cartilage intermediate. In this study, we examined the association of chondrocyte-specific marker expression with media calcification in arterial samples derived from rats with chronic renal failure (CRF) and from human transplant donors. CRF was induced in rats with a diet supplemented with adenine. Vascular calcification was evaluated histomorphometrically on Von Kossa-stained sections and the expression of the chondrocyte markers sox9 and collagen II with the osteogenic marker core-binding factor alpha1 (cbfa1) was determined immunohistochemically. Media calcification was detected in more than half of the rats with CRF. In over half of the rats with severe media calcification, a typical cartilage matrix was found by morphology. All of the animals with severe calcification showed the presence of chondrocyte-like cells expressing the markers sox9, collagen II, and cbfa1. Human aorta specimens showing mild to moderate media calcification also showed sox9, collagen II, and cbfa1 expression. The presence of chondrocytes in association with calcification of the media in aortas of rats with CRF mimics endochondral bone formation. The relevance of this association is further demonstrated by the chondrogenic conversion of medial smooth muscle cells in the human aorta.

Objective— Chronic renal failure (CRF) is associated with a 10- to 20-fold increase in cardiovascular risk. Vascular calcification is a prominent feature of cardiovascular disease in patients with end-stage renal failure and contributes to the excess mortality in this population. In this study, we explored in vivo X-ray microtomography (micro-CT) as a tool to detect and follow-up vascular calcifications in the aorta of living rats with adenine-induced CRF. Methods and Results— With in vivo micro-CT, calcification of the aorta in uremic rats was clearly discernible on transversal virtual cross-sections. Micro-CT findings correlated well with tissue calcium content and histology. Repetitive scans in animals with light, moderate, and severe vascular calcification showed good reproducibility with minimal interference of motion artifacts. Moreover, both calcified volume and area could be quantified with this method. Conclusions— In vivo micro-CT scanning is a sensitive method to detect vascular calcifications in CRF rats, allowing follow-up and quantification of the development, and potential reversal during treatment, of vascular calcifications in living animals.

Short stature is an important clinical problem in children with chronic kidney disease. Calcitriol is used as standard therapy to control secondary hyperparathyroidism, but its effect on linear growth remains controversial. Sanchez and He report multiple effects of calcitriol on chondrocyte proliferation and maturation that might help to clarify this controversy.