Introduction: Patients receiving hemodialysis (HD) experience higher mortality on dialysis days, implicating potential dialysis-specific factors. Phosphate is a critical component of ATP, and is rapidly removed with HD. We aimed to examine the relationship between the magnitude of phosphate reduction during HD and change in left ventricular strain measurements as a reflection of cardiac function. Methods: Prevalent ESKD patients on maintenance HD were prospectively recruited from a single center. Echocardiograms were performed pre- and mid-dialysis, with concurrent laboratory measurements including phosphate, potassium, troponin, brain natriuretic peptide, hemoglobin. Left ventricular global longitudinal strain (LVGLS), ejection fraction (LVEF), and global circumferential strain (LVGCS) were assessed. In those with worsening LVGLS (N=39), univariate and multivariate linear regression models assessed associations between changes in laboratory values and LVGLS adjusting for demographics, comorbidities, and HD-specific factors. Sensitivity analyses included adjustment for baseline LVEF and volume status (inferior vena cava measurements). Results: The 54 HD participants had mean age 71 years, 96% male, reflective of a veterans-specific cohort. During HD, 75% (39/52) experienced worsening LVGLS (pre-HD: -12.64 ± 3.69, mid-HD: -9.56 ± 3.12, p<0.001), while 23% (12/52) improved (pre-HD: -11.35 ± 5.12, mid-HD: -12.79 ± 5.61, p<0.001). LVEF decreased overall (49.39% to 44.01%, p<0.001). No significant overall change was seen in LVGCS (mean 15.66 ± 4.49 to 15.54 ± 5.65). In multivariate models (Table 1), greater phosphate reduction was independently associated with worsening LVGLS (p<0.05), even after adjusting for changes in other labs, demographics, HD vintage, session duration, ultrafiltration volume, residual renal function, and albumin. Results were similar when adjusted for baseline LVEF, and volume status. Phosphate reduction was not significantly associated with changes in LVEF, LVGCS. Hs-troponin and BNP were not significantly altered. Conclusion: Greater phosphate reduction during HD is independently associated with acute worsening of LVGLS, a sensitive marker of myocardial dysfunction, but not with changes in LVEF, LVGCS. These findings suggest that rapid phosphate shifts during HD may acutely contribute to adverse reduction in global myocardial deformation and highlight the need for further investigation into dialysis protocols that mitigate this risk.

Pseudohypoparathyroidism type 1B (PHP1B) is a multihormone resistance disorder caused by aberrant GNAS methylation. Characteristic epigenetic changes at GNAS differentially methylated regions (DMRs), i.e., NESP, AS1, AS2, XL, and A/B, are associated with specific structural defects in different autosomal dominant PHP1B (AD-PHP1B) subtypes. However, mechanisms underlying abnormal GNAS methylation remain incompletely defined, largely because viable PHP1B mouse models are lacking. Using lymphoblastoid cells and induced pluripotent stem cells, we show that various GNAS methylation patterns in PHP1B reflect differential disruption of sense and antisense GNAS transcripts. In cases with broad GNAS methylation changes, loss of the maternal, sense-transcribed exon H/AS region impairs methylation of the AS1 DMR, which results in biallelic expression of an antisense transcript, GNAS-AS1, and NESP hypermethylation. In contrast, cases with normal AS1 methylation, including STX16 deletions, show monoallelic GNAS-AS1 expression and normal NESP methylation. The roles of these GNAS transcripts were confirmed by a retrotransposon in GNAS-AS1 intron 1, identified in an AD-PHP1B family. This insertion impaired exon H/AS transcription when located on the maternal allele, thus preventing the complete establishment of methylation at all maternal GNAS DMRs, leading to biallelic GNAS-AS1 transcription. However, maternal GNAS-AS1 transcription was profoundly attenuated, thus allowing only a small gain-of-methylation at NESP. Likewise, on the paternal allele, the retrotransposon attenuated GNAS-AS1 transcription, thus preventing complete NESP methylation. Our findings support a model of bidirectional transcription-mediated regulation of methylation at GNAS DMRs and will help to refine systematic approaches for establishing molecular defects underlying different PHP1B subtypes.

Bone turnover assessment and monitoring are essential for chronic kidney disease (CKD)-associated bone care. Patients with CKD suffer from significantly elevated fracture risk due to abnormally high or low bone turnover, which benefits from diametrically opposite treatments informed by patient-specific bone turnover data. However, a reliable, accessible, noninvasive bone turnover assessment and monitoring tool remains an unmet clinical need. Combining time-lapse (TL) analysis with high-resolution peripheral quantitative computed tomography (HR-pQCT) scans obtained over time allows for in vivo temporospatial bone remodeling assessment. This study aimed to evaluate the feasibility of applying TL HR-pQCT to assess and monitor local bone formation and resorption in patients with CKD. A customized TL HR-pQCT pipeline was developed on a second-generation HR-pQCT platform and optimized using both ex vivo cadaveric phantom and in vivo scan-rescan HR-pQCT images. The annualized least significant changes in bone formation and resorption were evaluated using in vivo longitudinal reproducibility images. Finally, the feasibility of the TL HR-pQCT pipeline in assessing and monitoring bone turnover was evaluated in patients with end-stage kidney disease (ESKD; n = 9). We found that a 2-month TL period was sufficient for the TL HR-pQCT pipeline to reliably assess and monitor local bone turnover in a cohort of patients with ESKD. We also demonstrated the importance of characterizing TL HR-pQCT precision metrics using longitudinal baseline/follow-up rather than short-term scan-rescan datasets. The TL HR-pQCT pipeline assessed a range of bone formation metrics agreeing with the gold-standard histomorphometry bone formation reported in the literature for patients with CKD and ESKD. Our findings highlight that TL HR-pQCT holds promise as a "virtual bone biopsy" that reliably assesses and monitors local bone turnover for CKD bone care. Subsequent work will focus on validating this TL HR-pQCT pipeline against the gold-standard bone biopsy with quantitative histomorphometry.

Context Jansen metaphyseal chondrodysplasia (JMC) is an ultra-rare autosomal dominant disease that is caused by heterozygous, activating PTH1R mutations resulting in PTH- and PTHrP-independent hypercalcemia and hypercalciuria, leading to nephrocalcinosis and impaired renal function later in life. The activated PTH1R plays critical roles in mineral ion homeostasis and bone lengthening, as well as bone formation and resorption. Currently, little is known about bone turnover markers and bone histomorphometric changes in JMC patients. Objective This study aimed to assess changes in bone microarchitecture, bone formation, and bone protein expression in 2 pediatric patients with JMC harboring the H223R-PTHR1 mutation. Methods Bone histomorphometry, immunohistochemistry, and histologic analyses were conducted on iliac crest biopsy samples from 2 male siblings affected by JMC (ages 6 and 8 years) and 9 healthy control males of similar age, with normal kidney function. Results Both patients with JMC displayed irregular bone architecture, increased osteoid, and a prolonged osteoid maturation process. While trabecular volume remained normal, immunohistochemical analysis demonstrated increased in PTH1R expression in both osteoblasts and fibroblastic cells on the bone surface. Cortical bone displayed areas of intense osteoclast activity and scattered marrow fibrosis. Remarkably, osteocytes in samples from patients with JMC had osteoid buildup within their lacunae and canaliculi that were both shorter and less abundant. DMP1 immunohistochemistry highlighted the abnormal canalicular network in patients. FGF23 staining in osteocytes was enhanced while sclerostin was diminished. Conclusion The H223R-PTH1R mutation in patients with JMC leads to bone structural irregularities, hypomineralization, abnormal osteocyte morphology, and altered expression of osteocyte-derived proteins. These findings underscore the multifaceted impact of the mutant PTH1R on bone physiology and focus attention on the osteocyte as a cellular target for therapeutic intervention. Whether normalizing gene expression in osteocytes is possible and can improve bone health in patients with JMC remains to be seen. Assessment of osteocyte morphology and function may provide novel diagnostic endpoints for future clinical trials with JMC therapeutics.

Hemoglobin affinity for oxygen is modulated by ambient oxygen tension, acid/base status, 2,3 diphosphoglycerate (2,3DPG) concentrations and other factors, facilitating tissue oxygenation under changing conditions. 2,3DPG is a key regulator of oxygen affinity within red blood cells and its levels are affected by blood phosphate. P50, the partial pressure of oxygen at which 50% of its hemoglobin binding sites are occupied, is a marker of oxygen delivery to tissues. We measured P50 during hemodialysis and explored its relationship with mineral metabolites and left ventricular strain as a marker of cardiac function. Venous blood gas and other laboratory parameters were measured in 20 prevalent patients pre- and post-hemodialysis. To avoid arterio-venous mixing, we selected patients dialyzing through tunneled dialysis catheters. Associations of P50 with demographics, laboratory parameters and echocardiographic measurements were examined using linear regression models. P50 levels decreased from 27.1 ± 0.9 mmHg to 26.2 ± 0.7 mmHg during hemodialysis (P < .001). Among 18 predictors evaluated, older age, and greater reductions in phosphate during hemodialysis were the strongest predictors of P50 changes in multivariate models. There was acute worsening in left ventricular global longitudinal strain (LVGLS) during hemodialysis (reduction of 1.4 ± 3.9%; P = .03). Greater reductions in P50 during hemodialysis and older age were significantly associated with greater reductions in LVGLS. Hemodialysis consistently reduces P50. The magnitude of P50 change was strongly associated with concurrent phosphate changes. P50 reductions correlated with acute lowering of LVGLS. These observations illuminate a potential cause of systemic tissue hypoxia and potential cardiac dysfunction during hemodialysis.

Introduction Tetracycline labeling for bone biopsy facilitates quantification of the pace of new bone production. As tetracycline labeling needs to be done prior to biopsy, it cannot be used to assess bone turnover in patients presenting with fractures, yet knowing turnover rate in patients experiencing fractures - especially in those with chronic kidney disease (CKD) - may guide appropriate medical therapy after surgical repair. Therefore, we sought to determine the diagnostic accuracy of static markers of bone turnover relative to tetracycline labeling in a pediatric and adult cohort of patients with chronic kidney disease (CKD) undergoing iliac crest biopsy with histomorphometry. Methods We evaluated two cohorts, one of 147 children and young adults ages 18±10 and another of 151 adults ages 49±13 who had undergone iliac crest biopsy with tetracycline labeling for clinical indications of CKD-mineral and bone disorders. We used bone formation rate relative to bone surface (BFR/BS) based on double tetracycline labeling as our gold standard marker of bone turnover. A blinded investigator used light microscopy without fluorescence to measure static bone turnover parameters. We compared the area under the ROC curve (AUC), sensitivity, and specificity of each static parameter with low and high bone turnover based on BFR/BS. Results In the pediatric and adult cohorts, 35 (24 %) and 70 (46 %) had low bone turnover, respectively, and 18 (12 %) and 30 (20 %) had high bone turnover, respectively. The static parameters with the greatest AUCs for low and high turnover were osteoblast surface/bone surface (Ob.S/BS), osteoclast surface/bone surface (Oc.S/BS), eroded surface/bone surface (ES/BS), osteoid surface/bone surface (OS/BS), osteoid volume/bone volume (OV/BV), and osteoid thickness (O.Th.) in both cohorts. Ob.S/BS had the highest AUC for low and high turnover in the pediatric cohort (0.8204 and 0.8678, respectively) whereas Oc.S/BS had the highest AUC for low turnover (0.8325) and ES/BS had the highest AUC for high turnover (0.7360) in the adult cohort. Discussion Static measures of histomorphometry that do not rely on tetracycline bone labeling can identify low and high bone turnover in children and adults with CKD with moderate to high accuracy. This approach may allow assessment of bone turnover in the setting of clinical fractures where clinicians may have access to bone tissue but where tetracycline labeling is not available.

Background: Idiopathic juvenile osteoporosis (IJO) is a rare condition characterized by low bone mass that can increase the risk of fractures in children. Treatment options for these patients are limited as the molecular mechanisms of disease initiation and progression are incompletely understood. Sclerostin inhibits canonical Wnt signaling, which is important for the bone formation activity of osteoblasts, and elevated sclerostin has been implicated in adult osteoporosis. Objective: To evaluate the role of sclerostin in IJO, high-resolution confocal microscopy analyses were performed on bone biopsies collected from 13 pediatric patients. Methods: Bone biopsies were stained with sclerostin, and β-catenin antibodies showed elevated expression across osteocytes and increased sclerostin-positive osteocytes in 8 of the 13 total IJO patients (62%). Results: Skeletal sclerostin was associated with static and dynamic histomorphometric parameters. Further, colocalization analyses showed that bone sclerostin colocalized with phosphorylated β-catenin, a hallmark of Wnt signaling that indicates Wnt inhibition. In contrast, sclerostin-positive osteocytes were not colocalized with an “active” unphosphorylated form of β-catenin. Conclusions: These results support a model that altered levels of sclerostin and Wnt signaling activity occur in IJO patients.

Maturation defects are intrinsic features of osteoblast lineage cells in CKD patients. These defects persist ex vivo, suggesting that CKD induces epigenetic changes in bone cells. To gain insights into which signaling pathways contribute to CKD-mediated, epigenetically driven, impairments in osteoblast maturation, we characterized RNA expression and DNA methylation patterns by RNA-Seq and MethylationEpic in primary osteoblasts from nine adolescent and young adult dialysis patients with end-stage kidney disease and three healthy references. ATAC-Seq was also performed on a subset of osteoblasts. Bone matrix protein expression was extracted from the iliac crest and evaluated by proteomics. Gene set enrichment analysis was used to establish signaling pathways consistently altered in chromatin accessibility, DNA methylation, and RNA expression patterns. Single genes were suppressed in primary osteoblasts using shRNA and mineralization characterized in vitro. The effect of nuclear factor of activated T cells (NFAT) signaling suppression was also assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) incorporation. We found that signaling pathways critical for osteoblast differentiation were strongly downregulated in CKD osteoblasts. Gene set enrichment analysis identified highly significant methylation changes, differential chromatin accessibility, and altered RNA expression in NFAT signaling targets. NFAT inhibition reduced osteoblast proliferation. Combined analysis of osteoblast RNA expression and whole bone matrix composition identified 13 potential ligand-receptor pairs. In summary, epigenetic changes in CKD osteoblasts associate with altered expression of multiple osteoblast genes and signaling pathways. An increase in NFAT signaling may play a role in impaired CKD osteoblast maturation. Epigenetic changes also associate with an altered bone matrix, which may contribute to bone fragility. Further studies are necessary to elucidate the pathways affected by these genetic alterations since elucidating these pathways will be vital to correcting the underlying biology of bone disease in the CKD population.

Background: Iron deficiency is common in children with kidney failure, but current guidelines are based on biomarkers of iron stores that may be influenced by inflammation. This is the first study that examined which serum iron indices were associated with stainable marrow iron stores (the gold standard) in this population with kidney failure who underwent bone biopsies. Methods: This cross-sectional study enrolled 71 clinically stable children and young adults receiving dialysis who underwent bone biopsy for chronic kidney disease-mineral bone disorder between 2007 through 2011. Bone biopsies were stained with Perls' Prussian blue and independently interpreted by a pathologist blinded to participants' iron parameters and clinical status. Marrow staining was scored absent vs. present to facilitate receiver operator curve (ROC) analysis. In ROC analysis, the ability of serum ferritin to detect stainable marrow iron stores was compared with that of transferrin saturation (TSAT), serum hepcidin, and clinical guideline-based iron deficiency cut-offs for serum iron, TSAT, and their combinations. Results: Mean age was 17.2 +/- 4.4 years (range 2-28), and 30% of patients were female. Median dialysis vintage was 1.2 (IQR 0.7, 2.0) years, and 56% were supported by peritoneal dialysis. Mean hemoglobin was 12.4 +/- 1.7 g/dl, and 35% were receiving iron supplementation at the time of biopsy. Based on the gold standard of depleted marrow iron stores, 46.5% of patients were iron-deficient. As an indicator of marrow iron staining, serum ferritin provided a higher area under the ROC curve than serum hepcidin, TSAT, or clinical guidelines-based evaluation of TSAT + ferritin. Conclusions: In this cohort of children and young adults with kidney failure, serum ferritin provided the best indication of stainable marrow iron stores, followed by transferrin saturation.

Osteocytes are the most abundant type of bone cell and play crucial roles in bone health. Osteocytes sense mechanical stress and orchestrate osteoblasts and osteoclasts to maintain bone density and strength. Beyond this, osteocytes have also emerged as key regulators of organ crosstalk, and they function as endocrine organs via their roles in secreting factors that mediate signaling within their neighboring bone cells and in distant tissues. As such, osteocyte dysfunction has been associated with the bone abnormalities seen across a spectrum of chronic kidney disease. Specifically, dysregulated osteocyte morphology and signaling have been observed in the earliest stages of chronic kidney disease and have been suggested to contribute to kidney disease progression. More important, US Food and Drug Administration-approved inhibitors of osteocytic secreted proteins, such as fibroblast growth factor 23 and sclerostin, have been used to treat bone diseases. The present mini review highlights new research that links dysfunctional osteocytes to the pathogenesis of chronic kidney disease mineral and bone disorder.

Purpose of review Renal osteodystrophy (ROD) is a complex disorder of bone metabolism that affects virtually all adults and children with chronic kidney disease (CKD). ROD is associated with adverse clinical outcomes including bone loss, mineralization and turnover abnormalities, skeletal deformities, fractures, cardiovascular events, and death. Despite current therapies, fracture incidence is 2-fold to 100-fold higher in adults and 2-fold to 3-fold higher in children when compared to without CKD. Limited knowledge of ROD pathogenesis, due to the lack of patient-derived large-scale multimodal datasets, impedes development of therapeutics aimed at reducing morbidity and mortality of CKD patients. The purpose of the review is to define the much needed infrastructure for the advancement of RDO treatment. Recent findings Recently, we created a large-scale data and tissue biorepository integrating clinical, bone quality, transcriptomic, and epigenomic data along with stored urine, blood, and bone samples. This database will provide the underpinnings for future research endeavors leading to the elucidation and characterization of the pathogenesis of ROD in CKD patients with and without dialysis. Summary The availability of an open-access NIH-funded resource that shares bone-tissue-based information obtained from patients with ROD with the broad scientific community represents a critical step in the process of discovering new information regarding unrecognized bone changes that have severe clinical complications. This will facilitate future high-impact hypothesis-driven research to redefine our understanding of ROD pathogenesis and pathophysiology and inform the development of disease-modifying and prevention strategies

The pathophysiology of chronic kidney disease-mineral and bone disorder (CKD-MBD) is not well understood. Specific factors secreted by osteocytes are elevated in the serum of adults and pediatric patients with CKD-MBD, including FGF-23 and sclerostin, a known inhibitor of the Wnt signaling pathway. The molecular mechanisms that promote bone disease during the progression of CKD are incompletely understood. In this study, we performed a cross-sectional analysis of 87 pediatric patients with pre-dialysis CKD and post-dialysis (CKD 5D). We assessed the associations between serum and bone sclerostin levels and biomarkers of bone turnover and bone histomorphometry. We report that serum sclerostin levels were elevated in both early and late CKD. Higher circulating and bone sclerostin levels were associated with histomorphometric parameters of bone turnover and mineralization. Immunofluorescence analyses of bone biopsies evaluated osteocyte staining of antibodies towards the canonical Wnt target, β-catenin, in the phosphorylated (inhibited) or unphosphorylated (active) forms. Bone sclerostin was found to be colocalized with phosphorylated β-catenin, which suggests that Wnt signaling was inhibited. In patients with low serum sclerostin levels, increased unphosphorylated “active” β-catenin staining was observed in osteocytes. These data provide new mechanistic insight into the pathogenesis of CKD-MBD and suggest that sclerostin may offer a potential biomarker or therapeutic target in pediatric renal osteodystrophy.

Fibroblast growth factor 23 (FGF23) plays a significant role in phosphate homeostasis but data on children are limited. We aimed to detect FGF23 levels in 107 healthy children aged 6-16 years and evaluate its correlation with markers of phosphate and calcium metabolism, and the dietary intake of calcium, phosphate, and proteins. Height, weight, and Tanner stages were measured, and dietary intake was calculated. Biochemical analyses of hemoglobin, serum calcium, phosphate, creatinine, Vitamin D, and plasma parathyroid hormone (PTH) and FGF23 levels were performed, alongside their associations with FGF23. Of the children, 65.4% were males. Their mean body mass index was 15.79 ± 2.96 for males and 16.5 ± SD 2.72 for females. The mean Vitamin D and PTH levels were 29.7 ± 1.1 ng/mL and 29.2 ± 1.2 pg/mL, respectively. The mean FGF23 levels were 159 ± 15.2 reference units (RU)/mL. The mean FGF23 levels were significantly higher in females (209.3 ± 31 RU/mL) than in males (132.3 ± 15.1 RU/mL). All biochemical parameters were within the normal range. FGF23 correlated with age, weight, and height, but not Vitamin D, PTH, or dietary calcium and phosphate. FGF23 showed a negative correlation with hemoglobin levels (r = -0.23). Since most children had a nonvegetarian diet, the FGF23 levels were not assessed in vegetarians. These observations were attributed to the rural lifestyle favoring adequate exposure to sunlight and physical activity. The increased FGF23 levels in females, the trends in urban settings, and the levels in strictly vegetarian diets need further study.

Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that plays a central role in chronic kidney disease-mineral bone disorder and is associated with CKD progression and cardiovascular morbidity. Factors related to CKD-associated anemia, including iron deficiency, can increase FGF23 production. This study aimed to assess whether anemia and/or iron deficiency are associated with increased circulating concentrations of FGF23 in the large, well-characterized Chronic Kidney Disease in Children (CKiD) study cohort. Hemoglobin concentrations, iron parameters, C-terminal (total) FGF23, intact FGF23, and relevant covariables were measured in cross-sectional analysis of CKiD study subjects. In 493 pediatric patients with CKD (median [interquartile range] age 13 [9, 16] years), the median estimated glomerular filtration rate was 48 [35, 61] ml/min/1.73 m2, and 103 patients (21

Background: Iron deficiency is common in children with end-stage kidney disease (ESKD) but diagnosing iron deficiency in ESKD is challenging as serum indicators of iron stores may be influenced by inflammation and other factors. We examined which of the clinically used serum iron indices were predictive of bone marrow iron stores (the gold standard) in dialysis-dependent children and young adults with ESKD, in the setting of increased nutritional requirements and co-existing inflammation. Methods: This cross-sectional study enrolled 71 clinically-stable children and young adults (age range 2 to 28 years) receiving dialysis, who underwent bone biopsy for CKD-mineral bone disorders between 2007 through 2011. Congenital anomalies of the kidney and urinary tract (CAKUT) and hereditary diseases (63.3%) were the main causes of renal failure, followed by primary glomerulonephritis (36.7%). Bone samples were stained with Perls' Prussian blue and independently interpreted by a pathologist unaware of the patients' iron parameters and clinical status. Staining was scored absent vs present for receiver operator curve (ROC) analysis. In ROC analysis, the ability of serum ferritin to predict bone sample iron stores was compared with serum hepcidin, transferrin saturation (TSAT), and clinical guideline-based iron deficiency cut-offs for serum iron, TSAT, and their combination. We generated optimal cut off points using the Youden's J statistic. Results: Mean age was 17.2 ± 4.4 years, and 30% of subjects were female. Median dialysis vintage was 1.2 (IQR 0.7, 2.0) years, and 56% were supported by peritoneal dialysis. Mean hemoglobin was 12.4 ± 1.7 g/dl, and 35% were receiving iron supplementation at the time of biopsy. Based on gold standard of depleted bone iron stores, 46.5% patients were iron-deficient. As a predictor of bone marrow iron staining, serum ferritin provided the highest area under the ROC curve compared to other tests (Table), but at the usual clinical ferritin cutoff (100 ng/ml) only 22% of true iron-deficient subjects were diagnosed compared to 52% of true iron-deficient subjects using a higher optimal cutoff point for ferritin (188 ng/ml). Conclusions: In children with ESKD, iron deficiency as determined by the gold standard bone iron staining was highly prevalent (close to 50%), and serum ferritin provided the best prediction of bone sample iron stores. Optimal ferritin cutoff for decision-making about iron administration should reflect the current relative risks and benefits of iron therapy. Future trials testing higher ferritin cut-offs and patient centered outcomes in children with ESKD are timely.