Introduction Kidneys have a limited capacity for self-repair, and injury frequently progresses to chronic kidney disease (CKD). Remarkably, successful recovery is observed in models of unilateral acute kidney injury (AKI) upon contralateral nephrectomy. Here, we aim to better understand the cellular and molecular mechanisms underlying this enhanced recovery. Methods Six rodent studies were performed, including optimization and time-course experiments in Wistar rats and C57BL/6J mice to define left ischemia conditions, right nephrectomy delay times, and functional outcome. Kidneys were analyzed by quantitative histomorphometry (pathomics) on whole slide sections, immunostaining, and qPCR. Bulk RNA sequencing was conducted in two independent mouse time-course studies to discern repair and injury trajectories. Clonal expansion of tubular progenitor cells was assessed by lineage tracing in PAX2/Confetti mice. Tubular epithelial cell proliferative dynamics and polyploidization were analyzed using live cell cycle and DNA content profiling in PAX8/FUCCI2aR mice. Results Nephrectomy at day three after AKI induced full functional recovery in rats and mice, whereas longer delays (10 and 20 days) failed to prevent CKD progression. Early nephrectomy reduced tubular atrophy, fibrosis, and inflammation. Pathomics revealed distinct tubular morphological trajectories distinguishing between atrophy and repair, as adaptive signatures. Transcriptomic analyses with orthogonal validation demonstrated attenuation and reshaping of immune cell signatures and inflammatory pathways. Additionally, specific gene sets unique to nephrectomy-induced repair were identified. Lineage tracing showed that nephrectomy enhanced clonal expansion of tubular progenitor cells, beyond the levels of spontaneous repair. Cell cycle and DNA-content analysis of tubular cells demonstrated a strong polyploid response immediately after the ischemic insult, while early nephrectomy attenuated persistent tubular cell polyploidization, a contributor to CKD. Conclusions Early nephrectomy in experimental AKI triggers efficient repair by modulating immune responses, promoting tubular regeneration, inducing pro-repair transcriptional reprogramming, and counteracting progression to CKD through attenuation of sustained polyploidization.

Background:Serum total alkaline phosphatase (ALP) is a robust predictor of all-cause mortality in both the general population and in patients with chronic kidney disease (CKD). Individual ALP isozymes and isoforms exert specific physiological functions and may therefore inform separately on different disease processes and risks related to these. Serum ALP consists predominantly of bone and liver isoforms, with a smaller contribution from the intestinal isoenzyme. We aimed to characterize the impact of CKD on serum ALP fractions and to identify clinical and biochemical correlates. Methods:Serum ALP liver, bone and intestinal fractions were analysed by electrophoresis in 523 individuals across stages of CKD (stage G1-2: n = 90; G3: n = 100; G4-5: n = 139; G5D: n = 194) and in 21 kidney-healthy controls. Associations with demographics and parameters of mineral metabolism and inflammation were examined. In haemodialysis patients, we further explored the association between ALP isozymes and all-cause mortality. Results:Total ALP levels increased significantly across stages of CKD, with liver, bone and intestinal fractions all contributing. In patients with CKD G5D, circulating levels of all three fractions were more than two-fold higher than in controls. Bone ALP associated with PTH while liver ALP associated with C-reactive protein, both independent of demographics and kidney function. Higher liver ALP levels associated with increased mortality risk, independent of traditional risk factors and inflammation. Conclusions:In patients with CKD, elevations in liver, bone and intestinal ALP fractions reflect distinct biological pathways and may differentially inform on risk related to different disease processes. While bone ALP reflects bone metabolism, liver ALP is linked with inflammation. The elevation in CKD and the independent association of liver ALP fraction with mortality in patients treated with haemodialysis calls for additional clinical and mechanistic studies.

BACKGROUND AND HYPOTHESIS:Hyperoxaluria is an important risk factor for kidney stone formation. Currently available treatment options for idiopathic hyperoxaluria have limited efficacy and are difficult to maintain in the long-term. Pre-clinical data suggested that Lanthanum Carbonate (LaCa) might efficiently bind oxalate. In this multicenter, open-label, dose-response study we assessed the efficacy and safety of LaCa in the reduction of urinary oxalate excretion in stone formers with idiopathic hyperoxaluria. METHODS:Stone formers with urinary oxalate excretion > 45 mg/24h were treated with LaCa at a dose of 750 mg daily during a first 14-day treatment period, followed by a second 14-day treatment period with 1500 mg LaCa daily. The study was powered for the primary efficacy endpoint of detecting a ≥20% urinary oxalate excretion reduction after the first treatment period (AFTP). Secondary endpoints were the incremental reduction of urinary oxalate excretion with LaCa dose increase to 1500 mg during the second treatment period (ASTP) and the effect on serum phosphate and urinary phosphate excretion. RESULTS:Forty-four patients started the study, 42 completed the two months follow-up. LaCa treatment with both the 750 and 1500 mg dose had no effect on urinary oxalate excretion (52.0 mg/24h at baseline [BL], 49.1 mg/24h AFTP [p=0.25], 52.6 mg/24h ASTP [p=0.01 compared to AFTP, p=0.86 compared to BL]). Urinary phosphate/creatinine ratio and fractional excretion (FE) of phosphate decreased significantly after treatment with 1500 mg LaCa (17.1 mmol/g at BL, 15.2 mmol/g ASTP, p=0.003 for urinary phosphate/creatinine ratio and 16.6% at BL, 14.7% ASTP, p=0.002 for FE phosphate), while LaCa had no effect on serum phosphate (0.97 mmol/L at BL, 0.95 mmol/L ASTP, p=0.29). CONCLUSION:Short-term treatment with LaCa at doses up to 1500 mg did not reduce urinary oxalate excretion in stone formers with idiopathic hyperoxaluria. Urinary phosphate excretion decreased without effect on serum phosphate.

Histomorphometric analysis of an iliac bone biopsy remains the gold standard for the diagnosis of renal osteodystrophy (ROD), which comprises various histological lesions induced by chronic kidney disease (CKD). ROD belongs to the framework of CKD-associated osteoporosis. The use of bone biopsy in the routine management of CKD-associated osteoporosis has decreased over the past decades for various reasons, including diminishing expertise in performing the procedure, and major variability in processing bone samples and reporting of results. In this context, the European Renal Osteodystrophy group, a part of the CKD-mineral and bone disorder working group of the European Renal Association launched an initiative to evaluate various issues related to bone histomorphometry in the context of ROD. To this effect, 28 experts from 14 European countries engaged in rounds of discussions to reach a consensus related to the bone biopsy procedure, sample handling, and reading and reporting findings. Key conclusions include a recommendation that all practitioners in this field move towards reporting diagnostic findings by the turnover, mineralization, and volume (TMV) classification and that external quality control is prioritized to ensure validity and reproducibility of results. The consensus group recognises that the lack of an accepted normative reference for bone histomorphometry is a barrier towards uniform diagnostic definitions and recommends further collaborative efforts in this area. Until these issues are solved, transparent reporting on the choice of reference and diagnostic definitions applied should be adhered to, both in clinical reports and research settings.

Arterial media calcification or pathological deposition of calcium-phosphate crystals in the vessel wall contributes significantly to the high mortality rate observed in patients with CKD. Extracellular nucleotides (ie, ATP or UTP) regulate the arterial calcification process by interacting with (1) purinergic receptors and (2) breakdown via ecto-nucleotidases, such as ectonucleotide pyrophosphatase/phosphodiesterase NPP1 or NPP3, affecting the local levels of calcification inhibitor, pyrophosphate, and stimulator inorganic phosphate (PPi/Pi ratio). Also, it has been shown that ATP analogs (ie, beta,gamma-methylene-ATP [beta,gamma-meATP]) inhibit vascular smooth muscle cell calcification in vitro. In the first experiment, daily dosing of beta,gamma-meATP (2 mg/kg) was investigated in rats fed a warfarin diet to trigger the development of non-CKD-related arterial medial calcifications. This study showed that beta,gamma-meATP significantly lowered the calcium scores in the aorta and peripheral vessels in warfarin-exposed rats. In a second experiment, daily dosing of 4 mg/kg beta,gamma-meATP and its metabolite medronic acid (MDP) was analyzed in rats fed an adenine diet to promote the development of CKD-related arterial medial calcification. Administration of beta,gamma-meATP and MDP did not significantly decrease aortic calcification scores in this model. Moreover, both compounds induced deleterious effects on physiological bone mineralization, causing an imminent risk for worsening the already compromised bone status in CKD. Due to this, it was not possible to raise the dosage of both compounds to tackle CKD-related arterial calcification. Again, this points out the difficult task of targeting solely ectopic calcifications without negatively affecting physiological bone mineralization. On the other hand, aortic mRNA expression of Enpp1 and Enpp3 was significantly and positively associated with aortic calcification scores, suggesting that normalizing the aortic NPP1/3 activity to control values might be a possible target to treat (CKD-induced) arterial media calcifications. The deposition of calcium-phosphate crystals in the arteries, also called arterial calcification, has a serious impact on the mortality in patients with chronic renal failure. We have investigated the therapeutic efficiency and safety of the drug beta,gamma-meATP on arterial calcification in rats. In a first experiment, rats were exposed to high, toxic dosages of warfarin, an anticoagulant, to induce arterial calcification without affecting the kidney function. The drug beta,gamma-meATP was able to prevent the development of mild arterial calcifications in rats exposed to warfarin. In a second experiment, rats were fed an adenine diet to trigger chronic renal failure, which leads to the development of severe arterial calcifications. Unfortunately, the drug beta,gamma-meATP and its metabolite medronic acid did not significantly reduce the presence of severe arterial calcifications in rats with chronic renal failure. Moreover, these compounds induced side-effects at the level of the bones, causing an imminent risk for worsening the already compromised bone status in chronic renal failure. Due to this, it was not possible to raise the dosage of both compounds to tackle arterial calcification in a chronic kidney failure setting. Again, this points out the difficult task of targeting solely arterial calcifications without negatively affecting physiological bone mineralization.

Abstract Background and Aims Diabetes mellitus is common as both cause and comorbidity of patients with chronic kidney disease (CKD). Both diabetes and CKD are states of increased fracture risk compared to healthy controls, with multiple potential contributing mechanisms. In type 2 diabetes, hyperglycemia contributes to the accumulation of advanced glycation end-products and osteocyte dysfunction, and it may play a pathogenic role in bone fragility. In this study, we wanted to examine the association between glycemic status and bone phenotype in patients with end-stage kidney disease (ESKD). Method This is a cross-sectional cohort study of 626 patients with CKD G5-5D referred for kidney transplantation between April 2006 and December 2013. Patients with type 1 diabetes were excluded. Patients were divided into quartiles based on HbA1c. Laboratory parameters of bone mineral metabolism (including biointact parathyroid hormone (PTH), fibroblast growth factor-23 (FGF23), and sclerostin), and bone turnover markers (bone alkaline phosphatase (BALP), trimeric pro-collagen type 1 N-terminal pro-peptide (intact P1NP), and tartrate-resistant acid phosphatase 5b (TRAP5b)) were measured. Bone mineral density (BMD) assessed by dual-energy X-ray absorptiometry (DEXA) (n = 537), bone histomorphometry (n = 180), and bone trabecular score (TBS) (n = 192) were available for subsets of patients. Results The median HbA1c were 4.8% in Q1, 5.1% in Q2, 5.4% in Q3, and 5.9% in Q4. Age and BMI differed across quartiles, with the highest age and BMI in Q4. Differences in bone turnover markers were found, with decreasing bone formation (BALP and intact P1NP) and decreasing bone resorption (TRAP5b) with ascending quartiles (Fig. 1). No differences across quartiles were found for FGF23, or circulating levels of sclerostin; however a weak, positive correlation between HbA1c and sclerostin was noted (rho = 0.08, p = 0.047). Overall, BMD at lumbar spine, femoral neck, and total hip increased across quartiles, the highest values in Q4 (Table 1). There were no differen in bone histomorphometry or TBS based on glycemic status. Conclusion Chronic hyperglycemia associates with suppressed bone turnover and higher BMD in patients with ESKD, even in non-diabetics. Potential mechanisms linking glucose metabolism to bone quantity and quality requires further investigation.

ABSTRACTThe coexistence of osteoporosis and chronic kidney disease (CKD) is an evolving healthcare challenge in the face of increasingly aging populations. Globally, accelerating fracture incidence causes disability, impaired quality of life and increased mortality. Consequently, several novel diagnostic and therapeutic tools have been introduced for treatment and prevention of fragility fractures. Despite an especially high fracture risk in CKD, these patients are commonly excluded from interventional trials and clinical guidelines. While management of fracture risk in CKD has been discussed in recent opinion-based reviews and consensus papers in the nephrology literature, many patients with CKD stages 3–5D and osteoporosis are still underdiagnosed and untreated. The current review addresses this potential treatment nihilism by discussing established and novel approaches to diagnosis and prevention of fracture risk in patients with CKD stages 3–5D. Skeletal disorders are common in CKD. A wide variety of underlying pathophysiological processes have been identified, including premature aging, chronic wasting, and disturbances in vitamin D and mineral metabolism, which may impact bone fragility beyond established osteoporosis. We discuss current and emerging concepts of CKD–mineral and bone disorders (CKD-MBD) and integrate management of osteoporosis in CKD with current recommendations for management of CKD-MBD. While many diagnostic and therapeutic approaches to osteoporosis can be applied to patients with CKD, some limitations and caveats need to be considered. Consequently, clinical trials are needed that specifically study fracture prevention strategies in patients with CKD stages 3–5D.

Background Bone loss after kidney transplantation is highly variable. We investigated whether changes in bone turnover markers associate with bone loss during the first post-transplant year. Methods Bone mineral density (BMD) was measured at 0 and 12 months, with biointact parathyroid hormone, bone-specific alkaline phosphatase (BALP), intact procollagen type I N -terminal propeptide (PINP), and tartrate-resistant acid phosphatase isoform 5b (TRAP5b) measured at 0, 3, and 12 months post-transplant ( N =209). Paired transiliac bone biopsies were available in a subset ( n =49). Between-group differences were evaluated by Student's t test, Wilcoxon signed-rank test, or Pearson's chi-squared test. Results Changes in BMD varied from –22% to +17%/yr. Compared with patients with no change (±2.5%/yr), patients who gained BMD had higher levels of parathyroid hormone (236 versus 136 pg/ml), BALP (31.7 versus 18.8 μ g/L), and Intact PINP (121.9 versus 70.4 μ g/L) at time of transplantation; a greater decrease in BALP (−40% versus −21%) and Intact PINP (−43% versus −13%) by 3 months; and lower levels of Intact PINP (36.3 versus 60.0 μ g/L) at 12 months post-transplant. Patients who lost BMD had a less marked decrease, or even increase, in Intact PINP (+22% versus −13%) and TRAP5b (−27% versus −43%) at 3 months and higher Intact PINP (83.7 versus 60.0 μ g/L) and TRAP5b (3.89 versus 3.16 U/L) at 12 months compared with patients with no change. If none of the biomarkers decreased by the least significant change at 3 months, an almost two-fold (69% versus 36%) higher occurrence of bone loss was seen at 12 months post-transplant. Conclusions Bone loss after kidney transplantation was highly variable. Resolution of high bone turnover, as reflected by decreasing bone turnover markers, associated with BMD gain, while increasing bone turnover markers associated with bone loss.

Patients suffering from chronic kidney disease (CKD) often experience bone loss and arterial calcifications. It is unclear if hypogonadism contributes to the development of these complications and whether androgen therapy might prevent them. Male adult rats were randomized into four groups. The first group received standard chow (control), while three other groups were fed a 0.25% adenine/low vitamin K diet (CKD). Two CKD groups were treated with testosterone or dihydrotestosterone (DHT), whereas the control group and one CKD group received vehicle (VEH). CKD animals had 10-fold higher serum creatinine and more than 15-fold higher parathyroid hormone levels compared to controls. Serum testosterone levels were more than two-fold lower in the CKDVEH group compared to control + VEH and CKD + testosterone groups. Seminal vesicle weight was reduced by 50% in CKDVEH animals and restored by testosterone and DHT. CKD animals showed a low bone mass phenotype with decreased trabecular bone volume fraction and increased cortical porosity, which was not rescued by androgen treatment. Aortic calcification was much more prominent in CKD animals and not unequivocally prevented by androgens. Messenger RNA expression of the androgen receptor-responsive genes Acta1 and Col1a1 was reduced by CKD and stimulated by androgen treatment in levator ani muscle but not in the bone or aortic tissue. We conclude that adenine-induced CKD results in the development of hypogonadism in male rats. Androgen therapy is effective in restoring serum testosterone levels and androgen-sensitive organ weights but does not prevent bone loss or arterial calcifications, at least not in the presence of severe hyperparathyroidism.

Diabetic Kidney Disease (DKD) is a major microvascular complication for diabetic patients and is the most common cause of chronic kidney disease (CKD) and end-stage renal disease. Antidiabetic drugs, such as metformin and canagliflozin, have been shown to exert renoprotective effects. Additionally, quercetin recently showed promising results for the treatment of DKD. However, the molecular pathways through which these drugs exert their renoprotective effects remain partly unknown. The current study compares the renoprotective potential of metformin, canagliflozin, metformin + canagliflozin, and quercetin in a preclinical rat model of DKD. By combining streptozotocin (STZ) and nicotinamide (NAD) with daily oral N(ω)-Nitro-L-Arginine Methyl Ester (L-NAME) administration, DKD was induced in male Wistar Rats. After two weeks, rats were assigned to five treatment groups, receiving vehicle, metformin, canagliflozin, metformin + canagliflozin, or quercetin for a period of 12 weeks by daily oral gavage. Non-diabetic vehicle-treated control rats were also included in this study. All rats in which diabetes was induced developed hyperglycemia, hyperfiltration, proteinuria, hypertension, renal tubular injury and interstitial fibrosis, confirming DKD. Metformin and canagliflozin, alone or together, exerted similar renoprotective actions and similar reductions in tubular injury and collagen accumulation. Renoprotective actions of canagliflozin correlated with reduced hyperglycemia, while metformin was able to exert these effects even in the absence of proper glycemic control. Gene expression revealed that the renoprotective pathways may be traced back to the NF-κB pathway. No protective effect was seen with quercetin. In this experimental model of DKD, metformin and canagliflozin were able to protect the kidney against DKD progression, albeit in a non-synergistic way. These renoprotective effects may be attributable to the inhibition of the NF-κB pathway.

CINAC-patients present renal proximal tubular cell lysosomal lesions which are also observed in patients experiencing calcineurin inhibitor (CNI) nephrotoxicity, suggesting that CINAC is a toxin-induced nephropathy. An alternative hypothesis advocates chronic dehydration as a major etiological factor for CINAC. Here, we evaluated histological and molecular changes in dehydrated versus toxin exposed rats. Wistar rats were divided in 3 groups. Group 1 (n = 6) had free access to drinking water (control group). Group 2 (n = 8) was water deprived for 10 h per 24 h, 5 days/week and placed in an incubator (37 °C) for 30 min/h during water deprivation. Group 3 (n = 8) underwent daily oral gavage with cyclosporine (40 mg/kg body weight). After 28 days, renal function, histopathology and proteomic signatures were analysed. Cyclosporine-treated rats developed focal regions of atrophic proximal tubules with associated tubulo-interstitial fibrosis. PASM staining revealed enlarged argyrophilic granules in affected proximal tubules, identified as lysosomes by immunofluorescent staining. Electron microscopy confirmed the enlarged and dysmorphic phenotype of the lysosomes. Overall, these kidney lesions resemble those that have been previously documented in farmers with CINAC. Dehydration resulted in none of the above histopathological features. Proteomic analysis revealed that dehydration and cyclosporine both induce injury pathways, yet of a clear distinct nature with a signature of toxicity only for the cyclosporine group. In conclusion, both cyclosporine and dehydration are injurious to the kidney. However, dehydration alone does not result in kidney histopathology as observed in CINAC patients, whereas cyclosporine administration does. The histopathological analogy between CINAC and calcineurin inhibitor nephrotoxicity in rats and humans supports the involvement of an as-yet-unidentified environmental toxin in CINAC etiology.

Abstract Background and Aims Dysregulated mineral homeostasis is common in chronic kidney disease (CKD) and associated with bone demineralization and vascular calcification. The balance between bone formation and resorption, which reflect the bone calcium (Ca) balance (BCaB), cannot be determined without bone biopsy which is invasive and not easily repeatable. Recently, we have shown that stable (i.e. non-radioactive) Ca isotopes, 42Ca and 44Ca, can be measured in serum and their ratio (δ44/42Caserum) quantitatively determines net bone gain or loss of Ca. Thus, when bone formation exceeds bone resorption, the net BCaB is positive and δ44/42Caserum is high, and when bone resorption is the predominant process δ44/42Caserum is low compared to age-matched controls. In this study we compared δ44/42Caserum against δ44/42Cabone and arterial biopsy samples (δ44/42Caartery) and the sensitivity of δ44/42Ca in predicting changes in bone histology. Method Adults receiving chronic dialysis who underwent bone and arterial biopsies at the time of kidney transplantation were recruited. Patients who had parathyroidectomy or received cinacalcet or anti-resorptive agents were excluded. All participants had Dual Energy X-ray Absorptiometry (DXA) of the hip and lumbar spine. Ca44 and Ca42 measurements were performed in serum and bone and arterial biopsy samples using a multi-collector inductively-coupled plasma mass spectrometer (Thermo Fisher Scientific, Germany). Results Nineteen patients, median age 59.8 years, 84% male, median time on dialysis 3.3 years were included. δ44/42Ca was significantly higher in serum compared to bone or arterial biopsy samples (p < 0.0001), with the lowest isotope ratios in bone (Fig. 1A). δ44/42Cabone was significantly lighter than δ44/42Caartery (p = 0.0002; Fig. 1B). δ44/42Cabone correlated positively with the osteoblastic markers BAP and P1NP (p = 0.0006, R2 = 0.51 and p = 0.009, R2 = 0.31) and inversely with PTH and the osteoclastic marker RANKL (p = 0.0017, R2 = 52 and p = 0.02, R2 = 0.29 respectively; Fig. 2). Both the DXA hip and lumbar spine T-scores and z-scores correlated positively with δ44/42Cabone. δ44/42Caserum showed an inverse correlation with the osteoid area (p = 0.04, R2 = 0.22) and a positive correlation with the absolute mineralized area and the trabecular thickness (p = 0.0004, R2 = 0.58 and p = 0.013, R2 = 0.34 respectively. The were no significant correlations with δ44/42Caartery. On multivariable linear regression analysis significant predictors of δ44/42Caserum were δ44/42Cabone (p = 0.018, 95%CI −1.35 to −0.16), age (p = 0.02, 95%CI 0.002 to 0.02) and BAP (p = 0.019, 95%CI 0.04 to 0.38), together predicting 71% of the variability in δ44/42Caserum. The only significant predictor of δ44/42Cabone was the δ44/42Caserum: p = 0.004, 95%CI −1.6 to −0.37, model R2 = 69%. Conclusion δ44/42Caserum is a significant and independent maker of BCaB, correlating with bone histology measures, and may provide a more sensitive measure than DXA or bone biomarkers. Further studies are required to determine the clinical utility of using δ44/42Caserum to guide management of mineral bone disease in CKD.

Renal osteodystrophy (ROD) is a complex and serious complication of chronic kidney disease (CKD), a major global health problem caused by loss of renal function. Currently, the gold standard to accurately diagnose ROD is based on quantitative histomorphometric analysis of trabecular bone. Although this analysis encompasses the evaluation of osteoblast and osteoclast number/activity, tfigurehe interest in osteocytes remains almost nihil. Nevertheless, this cell type is evidenced to perform a key role in bone turnover, particularly through its production of various bone proteins, such as sclerostin. In this study, we aim to investigate, in the context of ROD, to which extent an association exists between bone turnover and the abundance of osteocytes and osteocytic sclerostin expression in both the trabecular and cortical bone compartments. Additionally, the effect of parathyroid hormone (PTH) on bone sclerostin expression was examined in parathyroidectomized rats. Our results indicate that PTH exerts a direct inhibitory function on sclerostin, which in turn negatively affects bone turnover and mineralization. Moreover, this study emphasizes the functional differences between cortical and trabecular bone, as the number of (sclerostin-positive) osteocytes is dependent on the respective bone compartment. Finally, we evaluated the potential of sclerostin as a marker for CKD and found that the diagnostic performance of circulating sclerostin is limited and that changes in skeletal sclerostin expression occur more rapidly and more pronounced. The inclusion of osteocytic sclerostin expression and cortical bone analysis could be relevant when performing bone histomorphometric analysis for diagnostic purposes and to unravel pathological mechanisms of bone disease.

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.

Calcification of the medial layer, inducing arterial stiffness, contributes significantly to cardiovascular mortality in patients with chronic kidney disease (CKD). Extracellular nucleotides block the mineralization of arteries by binding to purinergic receptors including the P2Y(2) receptor. This study investigates whether deletion of the P2Y(2) receptor influences the development of arterial media calcification in CKD mice. Animals were divided into: (i) wild type mice with normal renal function (control diet) (n =8), (ii) P2Y(2)R(-/-) mice with normal renal function (n =8), (iii) wild type mice with CKD (n =27), and (iv) P2Y(2)R(-/-) mice with CKD (n =22). To induce CKD, animals received an alternating (0.2-0.3%) adenine diet for 7weeks. All CKD groups developed a similar degree of chronic renal failure as reflected by high serum creatinine and phosphorus levels. Also, the presence of CKD induced calcification in the heart and medial layer of the aortic wall. However, deletion of the P2Y(2) receptor makes CKD mice more susceptible to the development of calcification in the heart and aorta (aortic calcium scores (median +/- IQR), CKD-wild type: 0.34 +/- 4.3 mg calcium/g wet tissue and CKD-P2Y(2)R(-/-): 4.0 +/- 13.2 mg calcium/g wet tissue). As indicated by serum and aortic mRNA markers, this P2Y(2)R(-/-) mediated increase in CKD-related arterial media calcification was associated with an elevation of calcification stimulators, including alkaline phosphatase and inflammatory molecules interleukin-6 and lipocalin 2. The P2Y(2) receptor should be considered as an interesting therapeutic target for tackling CKD-related arterial media calcification.

Sclerostin is a negative regulator of the Wnt/β-catenin signaling and is, therefore, an important inhibitor of bone formation and turnover. Because 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 bonelike cells. Therefore, we hypothesize that depletion/deactivation 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 (WT) 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. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).