Combining raloxifene and mechanical loading improves bone composition and mechanical properties in a murine model of chronic kidney disease (CKD).

INTRODUCTION: Patients with chronic kidney disease (CKD) are at an alarming risk of fracture compared to age and sex-matched non-CKD individuals. Clinical and preclinical data highlight two key factors in CKD-induced skeletal fragility: cortical porosity and reduced matrix-level properties including bone hydration. Thus, strategies are needed to address these concerns to improve mechanical properties and ultimately lower fracture risk in CKD. We sought to evaluate the singular and combined effects of mechanical and pharmacological interventions on modulating porosity, bone hydration, and mechanical properties in CKD. METHODS: Sixteen-week-old male C57BL/6J mice underwent a 10-week CKD induction period via a 0.2 % adenine-laced casein-based diet (n = 48) or remained as non-CKD littermate controls (Con, n = 48). Following disease induction (26 weeks of age), n = 7 CKD and n = 7 Con were sacrificed (baseline cohort) to confirm a steady-state CKD state was achieved prior to the initiation of treatment. At 27 weeks of age, all remaining mice underwent right tibial loading to a maximum tensile strain of 2050 µÆ 3× a week for five weeks with the contralateral limb as a non-loaded control. Half of the mice (equal number CKD and Con) received subcutaneous injections of 0.5 mg/kg raloxifene (RAL) 5× a week, and the other half remained untreated (UN). Mice were sacrificed at 31 weeks of age. Serum biochemistries were performed, and bi-lateral tibiae were assessed for microarchitecture, whole bone and tissue level mechanical properties, and composition including bone hydration. RESULTS: Regardless of intervention, BUN and PTH were higher in CKD animals throughout the study. In CKD, the combined effects of loading and RAL were quantified as lower cortical porosity and improved mechanical, material, and compositional properties, including higher matrix-bound water. Loading was generally responsible for positive impacts in cortical geometry and structural mechanical properties, while RAL treatment improved some trabecular outcomes and material-level mechanical properties and was responsible for improvements in several compositional parameters. While control animals responded positively to loading, their bones were less impacted by the RAL treatment, showing no deformation, toughness, or bound water improvements which were all evident in CKD. Serum PTH levels were negatively correlated with matrix-bound water. DISCUSSION: An effective treatment program to improve fracture risk in CKD ideally focuses on the cortical bone and considers both cortical porosity and matrix properties. Loading-induced bone formation and mechanical improvements were observed across groups, and in the CKD cohort, this included lower cortical porosity. This study highlights that RAL treatment superimposed on active bone formation may be ideal for reducing skeletal complications in CKD by forming new bone with enhanced matrix properties.

Six months of voluntary alcohol consumption in male cynomolgus macaques reduces intracortical bone porosity without altering mineralization or mechanical properties.

Chronic heavy alcohol consumption is a risk factor for low trauma bone fracture. Using a non-human primate model of voluntary alcohol consumption, we investigated the effects of 6 months of ethanol intake on cortical bone in cynomolgus macaques (Macaca fascicularis). Young adult (6.4 ± 0.1 years old, mean ± SE) male cynomolgus macaques (n = 17) were subjected to a 4-month graded ethanol induction period, followed by voluntary self-administration of water or ethanol (4 % w/v) for 22 h/d, 7 d/wk. for 6 months. Control animals (n = 6) consumed an isocaloric maltose-dextrin solution. Tibial response was evaluated using densitometry, microcomputed tomography, histomorphometry, biomechanical testing, and Raman spectroscopy. Global bone response was evaluated using biochemical markers of bone turnover. Monkeys in the ethanol group consumed an average of 2.3 ± 0.2 g/kg/d ethanol resulting in a blood ethanol concentration of 90 ± 12 mg/dl in longitudinal samples taken 7 h after the daily session began. Ethanol consumption had no effect on tibia length, mass, density, mechanical properties, or mineralization (p > 0.642). However, compared to controls, ethanol intake resulted in a dose-dependent reduction in intracortical bone porosity (Spearman rank correlation = -0.770; p < 0.0001) and compared to baseline, a strong tendency (p = 0.058) for lower plasma CTX, a biochemical marker of global bone resorption. These findings are important because suppressed cortical bone remodeling can result in a decrease in bone quality. In conclusion, intracortical bone porosity was reduced to subnormal values 6 months following initiation of voluntary ethanol consumption but other measures of tibia architecture, mineralization, or mechanics were not altered.

A novel murine model of combined insulin-dependent diabetes and chronic kidney disease has greater skeletal detriments than either disease individually.

Diabetes and chronic kidney disease (CKD) consistently rank among the top ten conditions in prevalence and mortality in the United States. Insulin-dependent diabetes (IDD) and CKD each increase the risk of skeletal fractures and fracture-related mortality. However, it remains unknown whether these conditions have interactive end-organ effects on the skeleton. We hypothesized that combining IDD and CKD in mice would cause structural and mechanical bone alterations that are more deleterious compared to the single disease states. Female C57BL6/J mice were divided into four groups: 1) N = 12 Control (CTRL), 2) N = 10 Streptozotocin-induced IDD (STZ), 3) N = 10 Adenine diet-induced CKD (AD), and 4) N = 9 Combination (STZ+AD). STZ administration resulted in significantly higher blood glucose, HbA1c (p < 0.0001), and glucose intolerance (p < 0.0001). AD resulted in higher blood urea nitrogen (p = 0.0002) while AD, but not STZ+AD mice, had high serum parathyroid hormone (p < 0.0001) and phosphorus (p = 0.0005). STZ lowered bone turnover (p = 0.001). Trabecular bone volume was lowered by STZ (p < 0.0001) and increased by AD (p = 0.003). Tissue mineral density was lowered by STZ (p < 0.0001) and AD (p = 0.02) in trabecular bone but only lowered by STZ in cortical bone (p = 0.002). Cortical porosity of the proximal tibia was increased by AD, moment of inertia was lower in both disease groups, and most cortical properties were lower in all groups vs CTRL. Ultimate force, stiffness, toughness, and total displacement/strain were lowered by STZ and AD. Fracture toughness was lower by AD (p = 0.003). Importantly, Cohen's D indicated that STZ+AD most strongly lowered bone turnover and mechanical properties. Taken together, structural and material-level bone properties are altered by STZ and AD while their combination resulted in greater detriments, indicating that improving bone health in the combined disease state may require novel interventions.

Segregating the effects of ferric citrate-mediated iron utilization and FGF23 in a mouse model of CKD.

Ferric citrate (FC) is an approved therapy for chronic kidney disease (CKD) patients as a phosphate (Pi) binder for dialysis-dependent CKD, and for iron deficiency anemia (IDA) in non-dialysis CKD. Elevated Pi and IDA both lead to increased FGF23, however, the roles of iron and FGF23 during CKD remain unclear. To this end, iron and Pi metabolism were tested in a mouse model of CKD (0.2% adenine) ± 0.5% FC for 6 weeks, with and without osteocyte deletion of Fgf23 (flox-Fgf23/Dmp1-Cre). Intact FGF23 (iFGF23) increased in all CKD mice but was lower in Cre+ mice with or without FC, thus the Dmp1-Cre effectively reduced FGF23. Cre+ mice fed AD-only had higher serum Pi than Cre- pre- and post-diet, and the Cre+ mice had higher BUN regardless of FC treatment. Total serum iron was higher in all mice receiving FC, and liver Tfrc, Bmp6, and hepcidin mRNAs were increased regardless of genotype; liver IL-6 showed decreased mRNA in FC-fed mice. The renal 1,25-dihydroxyvitamin D (1,25D) anabolic enzyme Cyp27b1 had higher mRNA and the catabolic Cyp24a1 showed lower mRNA in FC-fed mice. Finally, mice with loss of FGF23 had higher bone cortical porosity, whereas Raman spectroscopy showed no changes in matrix mineral parameters. Thus, FC- and FGF23-dependent and -independent actions were identified in CKD; loss of FGF23 was associated with higher serum Pi and BUN, demonstrating that FGF23 was protective of mineral metabolism. In contrast, FC maintained serum iron and corrected inflammation mediators, potentially providing ancillary benefit.

Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) R47H Variant Causes Distinct Age- and Sex-Dependent Musculoskeletal Alterations in Mice.

Previous studies proposed the Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), a receptor expressed in myeloid cells including microglia in brain and osteoclasts in bone, as a link between brain and bone disease. The TREM2 R47H variant is a known risk factor for Alzheimer's disease (AD), the most common form of dementia. To investigate whether altered TREM2 signaling could contribute to bone and skeletal muscle loss, independently of central nervous system defects, we used mice globally hemizygous for the TREM2 R47H variant (TREM2R47H/+ ), which do not exhibit AD pathology, and wild-type (WT) littermate control mice. Dxa/Piximus showed bone loss in female TREM2R47H/+ animals between 4 and 13 months of age and reduced cancellous and cortical bone (measured by micro-computed tomography [µCT]) at 13 months, which stalled out by 20 months of age. In addition, they exhibited decreased femoral biomechanical properties measured by three-point bending at 13 months of age, but not at 4 or 20 months. Male TREM2R47H/+ animals had decreased trabecular bone geometry but increased ultimate strain and failure force at 20 months of age versus WT. Only male TREM2R47H/+ osteoclasts differentiated more ex vivo after 7 days with receptor activator of nuclear factor κB ligand (RANKL)/macrophage colony-stimulating factor (M-CSF) compared to WT littermates. Yet, estrogen receptor alpha expression was higher in female and male TREM2R47H/+ osteoclasts compared to WT mice. However, female TREM2R47H/+ osteoclasts expressed less complement 3 (C3), an estrogen responsive element, and increased protein kinase B (Akt) activity, suggesting altered estrogen signaling in TREM2R47H/+ cells. Despite lower bone volume/strength in TREM2R47H/+ mice, skeletal muscle function measured by plantar flexion and muscle contractility was increased in 13-month-old female mutant mice. Overall, these data demonstrate that an AD-associated TREM2 variant can alter bone and skeletal muscle strength in a sex-dimorphic manner independent of central neuropathology, potentially mediated through changes in osteoclastic intracellular signaling. © 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).

Calcimimetics Alter Periosteal and Perilacunar Bone Matrix Composition and Material Properties in Early Chronic Kidney Disease.

Chronic kidney disease (CKD) affects 15% of Americans and greatly increases fracture risk due to elevated parathyroid hormone, cortical porosity, and reduced bone material quality. Calcimimetic drugs are used to lower parathyroid hormone (PTH) in CKD patients, but their impact on bone matrix properties remains unknown. We hypothesized that tissue-level bone quality is altered in early CKD and that calcimimetic treatment will prevent these alterations. To test this hypothesis, we treated Cy/+ rats, a model of spontaneous and progressive CKD-mineral and bone disorder (CKD-MBD), with KP-2326, a preclinical analogue of etelcalcetide, early in the CKD disease course. To measure tissue-level bone matrix composition and material properties, we performed colocalized Raman spectroscopy and nanoindentation on new periosteal bone and perilacunar bone using hydrated femur sections. We found that CKD and KP treatment lowered mineral type B carbonate substitution whereas KP treatment increased mineral crystallinity in new periosteal bone. Reduced elastic modulus was lower in CKD but was not different in KP-treated rats versus CTRL. In perilacunar bone, KP treatment lowered type B carbonate substitution, increased crystallinity, and increased mineral-to-matrix ratio in a spatially dependent manner. KP treatment also increased reduced elastic modulus and hardness in a spatially dependent manner. Taken together, these data suggest that KP treatment improves material properties on the tissue level through a combination of lowering carbonate substitution, increasing mineral crystallinity, and increasing relative mineralization of the bone early in CKD. As a result, the mechanical properties were improved, and in some regions, were the same as control animals. Therefore, calcimimetics may help prevent CKD-induced bone deterioration by improving bone quality in new periosteal bone and in bone tissue near osteocyte lacunae. © 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).

Non-Additive Effects of Combined NOX1/4 Inhibition and Calcimimetic Treatment on a Rat Model of Chronic Kidney Disease-Mineral and Bone Disorder (CKD-MBD).

Chronic kidney disease-mineral and bone disorder (CKD-MBD) increases cardiovascular calcification and skeletal fragility in part by increasing systemic oxidative stress and disrupting mineral homeostasis through secondary hyperparathyroidism. We hypothesized that treatments to reduce reactive oxygen species formation and reduce parathyroid hormone (PTH) levels would have additive beneficial effects to prevent cardiovascular calcification and deleterious bone architecture and mechanics before end-stage kidney disease. To test this hypothesis, we treated a naturally progressive model of CKD-MBD, the Cy/+ rat, beginning early in CKD with the NADPH oxidase (NOX1/4) inhibitor GKT-137831 (GKT), the preclinical analogue of the calcimimetic etelcalcetide, KP-2326 (KP), and their combination. The results demonstrated that CKD animals had elevated blood urea nitrogen, PTH, fibroblast growth factor 23 (FGF23), and phosphorus. Treatment with KP reduced PTH levels compared with CKD animals, whereas GKT treatment increased C-terminal FGF23 levels without altering intact FGF23. GKT treatment alone reduced aortic calcification and NOX4 expression but did not alter the oxidative stress marker 8-OHdG in the serum or aorta. KP treatment reduced aortic 8-OHdG and inhibited the ability for GKT to reduce aortic calcification. Treatments did not alter heart calcification or left ventricular mass. In the skeleton, CKD animals had reduced trabecular bone volume fraction and trabecular number with increased trabecular spacing that were not improved with either treatment. The cortical bone was not altered by CKD or by treatments at this early stage of CKD. These results suggest that GKT reduces aortic calcification while KP reduces aortic oxidative stress and reduces PTH, but the combination was not additive. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Effects of Raloxifene and tibial loading on bone mass and mechanics in male and female mice.

Purpose: Raloxifene (RAL) is a selective estrogen receptor modulator (SERM) that has previously been shown to cause acellular benefits to bone tissue. Due to these improvements, RAL was combined with targeted tibial loading to assess if RAL treatment during periods of active bone formation would allow for further mechanical enhancements.Methods: Structural, mechanical, and microstructural effects were assessed in bone from C57BL/6 mice that were treated with RAL (0.5 mg/kg), tibial loading, or both for 6 weeks, beginning at 10 weeks of age.Results:Ex vivo microcomputed tomography (CT) images indicated RAL and loading work together to improve bone mass and architecture, especially within the cancellous region of males. Increases in cancellous bone volume fraction were heavily driven by increases in trabecular thickness, though there were some effects on trabecular spacing and number. In the cortical regions, RAL and loading both increased cross-sectional area, cortical area, and cortical thickness. Whole-bone mechanical testing primarily indicated the effects of loading. Further characterization through Raman spectroscopy and nanoindentation showed load-based changes in mineralization and micromechanics, while both loading and RAL caused changes in the secondary collagen structure. In contrast to males, in females, there were large load-based effects in the cancellous and cortical regions, resulting in increased whole-bone mechanical properties. RAL had less of an effect on cancellous and cortical architecture, though some effects were still present.Conclusion: RAL and loading work together to impact bone architecture and mechanical integrity, leading to greater improvements than either treatment individually.

The impact of advanced glycation end products on bone properties in chronic kidney disease.

PURPOSE OF REVIEW: Chronic kidney disease (CKD) affects over 15% of Americans and results in an increased risk of skeletal fractures and fracture-related mortality. However, there remain great challenges in estimating fracture risk in CKD patients, as conventional metrics such as bone density assess bone quantity without accounting for the material quality of the bone tissue. The purpose of this review is to highlight the detrimental effects of advanced glycation end products (AGEs) on the structural and mechanical properties of bone, and to demonstrate the importance of including bone quality when assessing fracture risk in CKD patients. RECENT FINDINGS: Increased oxidative stress and inflammation drive the production of AGEs in CKD patients that form nonenzymatic crosslinks between type I collagen fibrils in the bone matrix. Nonenzymatic crosslinks stiffen and embrittle the bone, reducing its ability to absorb energy and resist fracture. Clinical measurement of AGEs is typically indirect and fails to distinguish the identity and properties of the various AGEs. SUMMARY: Accounting for the impact of AGEs on the skeleton in CKD patients may improve our estimation of overall bone quality, fracture risk, and treatments to improve both bone quantity and quality by reducing AGEs in patients with CKD merit investigation in order to improve our understanding of the etiology of increased fracture risk.

Control of Bone Matrix Properties by Osteocytes.

Osteocytes make up 90-95% of the cellular content of bone and form a rich dendritic network with a vastly greater surface area than either osteoblasts or osteoclasts. Osteocytes are well positioned to play a role in bone homeostasis by interacting directly with the matrix; however, the ability for these cells to modify bone matrix remains incompletely understood. With techniques for examining the nano- and microstructure of bone matrix components including hydroxyapatite and type I collagen becoming more widespread, there is great potential to uncover novel roles for the osteocyte in maintaining bone quality. In this review, we begin with an overview of osteocyte biology and the lacunar-canalicular system. Next, we describe recent findings from in vitro models of osteocytes, focusing on the transitions in cellular phenotype as they mature. Finally, we describe historical and current research on matrix alteration by osteocytes in vivo, focusing on the exciting potential for osteocytes to directly form, degrade, and modify the mineral and collagen in their surrounding matrix.

The Importance of Biologically Active Vitamin D for Mineralization by Osteocytes After Parathyroidectomy for Renal Hyperparathyroidism.

Hypomineralized matrix is a factor determining bone mineral density. Increased perilacunar hypomineralized bone area is caused by reduced mineralization by osteocytes. The importance of vitamin D in the mineralization by osteocytes was investigated in hemodialysis patients who underwent total parathyroidectomy (PTX) with immediate autotransplantation of diffuse hyperplastic parathyroid tissue. No previous reports on this subject exist. The study was conducted in 19 patients with renal hyperparathyroidism treated with PTX. In 15 patients, the serum calcium levels were maintained by subsequent administration of alfacalcidol (2.0 µg/day), i.v. calcium gluconate, and oral calcium carbonate for 4 weeks after PTX (group I). This was followed in a subset of 4 patients in group I by a reduced dose of 0.5 µg/day until 1 year following PTX; this was defined as group II. In the remaining 4 patients, who were not in group I, the serum calcium (Ca) levels were maintained without subsequent administration of alfacalcidol (group III). Transiliac bone biopsy specimens were obtained in all groups before and 3 or 4 weeks after PTX to evaluate the change of the hypomineralized bone area. In addition, patients from group II underwent a third bone biopsy 1 year following PTX. A significant decrease of perilacunar hypomineralized bone area was observed 3 or 4 weeks after PTX in all group I and II patients. The area was increased again in the group II patients 1 year following PTX. In group III patients, an increase of the hypomineralized bone area was observed 4 weeks after PTX. The maintenance of a proper dose of vitamin D is necessary for mineralization by osteocytes, which is important to increase bone mineral density after PTX for renal hyperparathyroidism. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

A novel fizzy/Cdc20-dependent mechanism suppresses necrosis in neural stem cells.

Cancer stem cells likely survive chemotherapy or radiotherapy by acquiring mutations that inactivate the endogenous apoptotic machinery or by cycling slowly. Thus, knowledge about the mechanisms linking the activation of an alternative cell death modality and the cell cycle machinery could have a transformative impact on the development of new cancer therapies, but the mechanisms remain completely unknown. We investigated the regulation of alternative cell death in Drosophila larval brain neural stem cells (neuroblasts) in which apoptosis is normally repressed. From a screen, we identified two novel loss-of-function alleles of the Cdc20/fizzy (fzy) gene that lead to premature brain neuroblast loss without perturbing cell proliferation in other diploid cell types. Fzy is an evolutionarily conserved regulator of anaphase promoting complex/cyclosome (APC/C). Neuroblasts carrying the novel fzy allele or exhibiting reduced APC/C function display hallmarks of necrosis. By contrast, neuroblasts overexpressing the non-degradable form of canonical APC/C substrates required for cell cycle progression undergo mitotic catastrophe. These data strongly suggest that Fzy can elicit a novel pro-survival function of APC/C by suppressing necrosis. Neuroblasts experiencing catastrophic cellular stress, or overexpressing p53, lose Fzy expression and undergo necrosis. Co-expression of fzy suppresses the death of these neuroblasts. Consequently, attenuation of the Fzy-dependent survival mechanism functions downstream of catastrophic cellular stress and p53 to eliminate neuroblasts by necrosis. Strategies that target the Fzy-dependent survival mechanism might lead to the discovery of new treatments or complement the pre-existing therapies to eliminate apoptosis-resistant cancer stem cells by necrosis.