Two Weeks of Continuous Opioid Treatment in an Adenine-Induced Mouse Model of Chronic Kidney Disease Exacerbates the Bone Inflammatory State and Increases Osteoclasts.

Patients with chronic kidney disease (CKD) report high pain levels, but reduced renal clearance eliminates many analgesic options; therefore, 30-50% of CKD patients have chronic opioid prescriptions. Opioid use in CKD is associated with higher fracture rates. Opioids may directly alter bone turnover directly through effects on bone cells and indirectly via increasing inflammation. We hypothesized that continuous opioid exposure would exacerbate the high bone turnover state of CKD and be associated with elevated measures of inflammation. Male C57Bl/6J mice after 8 weeks of adenine-induced CKD (AD) and non-AD controls (CON) had 14-day osmotic pumps (0.25-µL/hr release) containing either saline or 50-mg/mL oxycodone (OXY) surgically implanted in the subscapular region. After 2 weeks, all AD mice had elevated blood urea nitrogen, parathyroid hormone, and serum markers of bone turnover compared to controls with no effect of OXY. Immunohistochemical staining of the distal femur showed increased numbers of osteocytes positive for the mu opioid and for toll-like receptor 4 (TLR4) due to OXY. Osteocyte protein expression of tumor necrosis factor-α (TNF-α) and RANKL were higher due to both AD and OXY so that AD + OXY mice had the highest values. Trabecular osteoclast-covered surfaces were also significantly higher due to both AD and OXY, resulting in AD + OXY mice having 4.5-fold higher osteoclast-covered surfaces than untreated CON. These data demonstrate that opioids are associated with a pro-inflammatory state in osteocytes which increases the pro-resorptive state of CKD.

Musculoskeletal Health Worsened from Carnitine Supplementation and Not Impacted by a Novel Individualized Treadmill Training Protocol.

INTRODUCTION: Chronic kidney disease (CKD) negatively affects musculoskeletal health, leading to reduced mobility, and quality of life. In healthy populations, carnitine supplementation and aerobic exercise have been reported to improve musculoskeletal health. However, there are inconclusive results regarding their effectiveness and safety in CKD. We hypothesized that carnitine supplementation and individualized treadmill exercise would improve musculoskeletal health in CKD. METHODS: We used a spontaneously progressive CKD rat model (Cy/+ rat) (n = 11-12/gr): (1) Cy/+ (CKD-Ctrl), (2) CKD-carnitine (CKD-Carn), and (3) CKD-treadmill (CKD-TM). Carnitine (250 mg/kg) was injected daily for 10 weeks. Rats in the treadmill group ran 4 days/week on a 5° incline for 10 weeks progressing from 30 min/day for week one to 40 min/day for week two to 50 min/day for the remaining 8 weeks. At 32 weeks of age, we assessed overall cardiopulmonary fitness, muscle function, bone histology and architecture, and kidney function. Data were analyzed by one-way ANOVA with Tukey's multiple comparisons tests. RESULTS: Moderate to severe CKD was confirmed by biochemistries for blood urea nitrogen (mean 43 ± 5 mg/dL CKD-Ctrl), phosphorus (mean 8 ± 1 mg/dL CKD-Ctrl), parathyroid hormone (PTH; mean 625 ± 185 pg/mL CKD-Ctrl), and serum creatinine (mean 1.1 ± 0.2 mg/mL CKD-Ctrl). Carnitine worsened phosphorous (mean 11 ± 3 mg/dL CKD-Carn; p < 0.0001), PTH (mean 1,738 ± 1,233 pg/mL CKD-Carn; p < 0.0001), creatinine (mean 1 ± 0.3 mg/dL CKD-Carn; p < 0.0001), cortical bone thickness (mean 0.5 ± 0.1 mm CKD-Ctrl, 0.4 ± 0.1 mm CKD-Carn; p < 0.05). Treadmill running significantly improves maximal aerobic capacity when compared to CKD-Ctrl (mean 14 ± 2 min CKD-TM, 10 ± 2 min CKD-Ctrl; p < 0.01). CONCLUSION: Carnitine supplementation worsened CKD progression, mineral metabolism biochemistries, and cortical porosity and did not have an impact on physical function. Individualized treadmill running improved maximal aerobic capacity but did not have an impact on CKD progression or bone properties. Future studies should seek to better understand carnitine doses in conditions of compromised renal function to prevent toxicity which may result from elevated carnitine levels and to optimize exercise prescriptions for musculoskeletal health.

Femoral Skeletal Perfusion is Reduced in Male Mice with Type 1 Diabetes.

The bone vasculature and blood flow are critical for bone modeling, remodeling, and regeneration. Vascular complications are one of the major health concerns of people with type 1 diabetes (T1D). Moreover, people with T1D have lower bone mineral density and increased bone fragility. The goal of this study was to understand whether bone perfusion was altered in a mouse model of T1D and how this related to changes in bone mass. T1D was induced via the administration of streptozotocin in 12-week-old C57BL/6NHsd male mice. The assessment of bone perfusion utilized the injection of fluorescent microspheres with assessment of levels in the bone. Femoral blood flow and VEGF-A expression in the cortical bone shafts were lower in the T1D mice, compared to healthy controls, in this pattern followed that of changes in bone mass. These data demonstrate a possible association between reduced skeletal perfusion and reduced bone mass in the setting of T1D.

Effects of ferric citrate and intravenous iron sucrose on markers of mineral, bone, and iron homeostasis in a rat model of CKD-MBD.

BACKGROUND: Anemia and chronic kidney disease-mineral and bone disorder (CKD-MBD) are common and begin early in CKD. Limited studies have concurrently compared the effects of ferric citrate (FC) versus intravenous (IV) iron on CKD-MBD and iron homeostasis in moderate CKD. METHODS: We tested the effects of 10 weeks of 2% FC versus IV iron sucrose in rats with moderate CKD (Cy/+ male rat) and untreated normal (NL) littermates. Outcomes included a comprehensive assessment of CKD-MBD, iron homeostasis and oxidative stress. RESULTS: CKD rats had azotemia, elevated phosphorus, parathyroid hormone and fibroblast growth factor-23 (FGF23). Compared with untreated CKD rats, treatment with FC led to lower plasma phosphorus, intact FGF23 and a trend (P = 0.07) toward lower C-terminal FGF23. FC and IV iron equally reduced aorta and heart calcifications to levels similar to NL animals. Compared with NL animals, CKD animals had higher bone turnover, lower trabecular volume and no difference in mineralization; these were unaffected by either iron treatment. Rats treated with IV iron had cortical and bone mechanical properties similar to NL animals. FC increased the transferrin saturation rate compared with untreated CKD and NL rats. Neither iron treatment increased oxidative stress above that of untreated CKD. CONCLUSIONS: Oral FC improved phosphorus homeostasis, some iron-related parameters and the production and cleavage of FGF23. The intermittent effect of low-dose IV iron sucrose on cardiovascular calcification and bone should be further explored in moderate-advanced CKD.

Iron deficiency and high-intensity running interval training do not impact femoral or tibial bone in young female rats.

In the USA, as many as 20 % of recruits sustain stress fractures during basic training. In addition, approximately one-third of female recruits develop Fe deficiency upon completion of training. Fe is a cofactor in bone collagen formation and vitamin D activation, thus we hypothesised Fe deficiency may be contributing to altered bone microarchitecture and mechanics during 12-weeks of increased mechanical loading. Three-week old female Sprague Dawley rats were assigned to one of four groups: Fe-adequate sedentary, Fe-deficient sedentary, Fe-adequate exercise and Fe-deficient exercise. Exercise consisted of high-intensity treadmill running (54 min 3×/week). After 12-weeks, serum bone turnover markers, femoral geometry and microarchitecture, mechanical properties and fracture toughness and tibiae mineral composition and morphometry were measured. Fe deficiency increased the bone resorption markers C-terminal telopeptide type I collagen and tartate-resistant acid phosphatase 5b (TRAcP 5b). In exercised rats, Fe deficiency further increased bone TRAcP 5b, while in Fe-adequate rats, exercise increased the bone formation marker procollagen type I N-terminal propeptide. In the femur, exercise increased cortical thickness and maximum load. In the tibia, Fe deficiency increased the rate of bone formation, mineral apposition and Zn content. These data show that the femur and tibia structure and mechanical properties are not negatively impacted by Fe deficiency despite a decrease in tibiae Fe content and increase in serum bone resorption markers during 12-weeks of high-intensity running in young growing female rats.

Elevations in Cortical Porosity Occur Prior to Significant Rise in Serum Parathyroid Hormone in Young Female Mice with Adenine-Induced CKD.

Chronic kidney disease (CKD) leads to significant bone loss primarily through the development of cortical porosity. In both patients and animal models of CKD, sustained elevations in serum parathyroid hormone (PTH) are associated with cortical porosity. In this study, we aimed to track the progression of cortical porosity and increased PTH utilizing the adenine-induced CKD model. Young female mice (8 weeks) were given 0.2% adenine to induce CKD. Tissues were collected from groups of adenine and age-matched control mice after 2, 6, and 10 weeks. Serum blood urea nitrogen was elevated at all time points in adenine mice, but serum PTH was only statistically elevated at the 10-week time point. Cortical porosity was sevenfold higher in 6-week adenine mice compared to age-matched controls and 14-fold higher in 10-week adenine mice vs. controls. Additionally, osteocyte receptor activator of nuclear factor κB ligand (RANKL) was elevated in adenine-fed mice, while annexin V, an early marker of cellular apoptosis, was mildly decreased in osteocytes in adenine-fed mice. Based on these results, we hypothesize high serum PTH signals to osteocytes prolonging their lifespan resulting in sustained RANKL which drives osteoclastic bone resorption in the cortex. In conclusion, our data show time-dependent elevations in serum PTH and cortical porosity in adenine-induced CKD mice and demonstrate changes in osteocyte RANKL and apoptosis which may contribute to the development of cortical pores.

Kidney Disease and Bone: Changing the Way We Look at Skeletal Health.

PURPOSE OF REVIEW: Kidney disease imparts profound skeletal changes, and unlike many other skeletal diseases, cortical bone is predominantly impacted. Significant advances in medical imaging have led to our ability to now obtain high-resolution three-dimensional views of cortical bone. This paper overviews recent work focused on cortical bone imaging, specifically cortical porosity, in kidney disease. RECENT FINDINGS: Although a number of clinical papers have used high-resolution imaging to assess cortical bone porosity, the most impactful work involves longitudinal study designs that have assessed cortical porosity changes over time. These latter studies demonstrate dramatic increases in cortical porosity in untreated individuals and a lack of clear efficacy in reversing porosity with treatment (although data are limited). Those papers providing longitudinal assessment, both clinical and pre-clinical, reveal powerful data about cortical porosity and provide a foundation upon which future studies can build.

Inflammation-induced lymphatic architecture and bone turnover changes are ameliorated by irisin treatment in chronic inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic disease with gastrointestinal dysfunction as well as comorbidities such as inflammation-induced bone loss and impaired immune response. Current treatments for IBD all have negative, potentially severe side effects. We aimed to test whether exogenous treatment with irisin, a novel immunomodulatory adipomyokine, could ameliorate IBD-induced lymphatic and bone alterations. Irisin treatment improved both gut and bone outcomes by mitigating inflammation and restoring structure. In the gut, IBD caused colonic lymphatic hyperproliferation into the mucosal and submucosal compartments. This proliferation in the rodent model is akin to what is observed in IBD patient case studies. In bone, IBD increased osteoclast surface and decreased bone formation. Both gut and osteocytes in bone exhibited elevated levels of TNF-α and receptor activator of NF-κB ligand (RANKL) protein expression. Exogenous irisin treatment restored normal colonic lymphatic architecture and increased bone formation rate concurrent with decreased osteoclast surfaces. After irisin treatment, gut and osteocyte TNF-α and RANKL protein expression levels were no different from vehicle controls. Our data indicate that the systemic immunologic changes that occur in IBD are initiated by damage in the gut and likely linked through the lymphatic system. Additionally, irisin is a potential novel intervention mitigating both local inflammatory changes in the gut and distant changes in bone.-Narayanan, S. A., Metzger, C. E., Bloomfield, S. A., Zawieja, D. C. Inflammation-induced lymphatic architecture and bone turnover changes are ameliorated by irisin treatment in chronic inflammatory bowel disease.

Inhibition of RANKL improves the skeletal phenotype of adenine-induced chronic kidney disease in mice.

Skeletal fragility and high fracture rates are common in CKD. A key component of bone loss in CKD with secondary hyperparathyroidism is high bone turnover and cortical bone deterioration through both cortical porosity and cortical thinning. We hypothesized that RANKL drives high bone resorption within cortical bone leading to the development of cortical porosity in CKD (study 1) and that systemic inhibition of RANKL would mitigate the skeletal phenotype of CKD (study 2). In study 1, we assessed the skeletal properties of male and female Dmp1-cre RANKLfl/fl (cKO) and control genotype (Ranklfl/fl; Con) mice after 10 wk of adenine-induced CKD (AD; 0.2% dietary adenine). All AD mice regardless of sex or genotype had elevated blood urea nitrogen and high PTH. Con AD mice in both sexes had cortical porosity and lower cortical thickness as well as high osteoclast-covered trabecular surfaces and higher bone formation rate. cKO mice had preserved cortical bone microarchitecture despite high circulating PTH as well as no CKD-induced increases in osteoclasts. In study 2, male mice with established AD CKD were either given a single injection of an anti-RANKL antibody (5 mg/kg) 8 wk post-induction of CKD or subjected to 3×/wk dosing with risedronate (1.2 µg/kg) for 4 wk. Anti-RANKL treatment significantly reduced bone formation rate as well as osteoclast surfaces at both trabecular and cortical pore surfaces; risedronate treatment had little effect on these bone parameters. In conclusion, these studies demonstrate that bone-specific RANKL is critical for the development of high bone formation/high osteoclasts and cortical bone loss in CKD with high PTH. Additionally, systemic anti-RANKL ligand therapy in established CKD may help prevent the propagation of cortical bone loss via suppression of bone turnover.

Romosozumab rescues impaired bone mass and strength in a murine model of diabetic kidney disease.

As international incidence of diabetes and diabetes-driven comorbidities such as chronic kidney disease (CKD) continue to climb, interventions are needed that address the high-risk skeletal fragility of what is a complex disease state. Romosozumab (Romo) is an FDA-approved sclerostin inhibitor that has been shown to increase bone mineral density and decrease fracture rates in osteoporotic patients with mild to severe CKD, but its effect on diabetes-weakened bone is unknown. We aimed to test Romo's performance in a model of combined diabetes and CKD. 6-week old male C57BL/6 mice were randomly divided into control (CON) and disease model (STZ-Ad) groups, using a previously established streptozotocin- and adenine-diet-induced model. After 16 weeks of disease induction, both CON and STZ-Ad groups were subdivided into two treatment groups and given weekly subcutaneous injections of 100 µL vehicle (phosphorus buffered saline, PBS) or 10 mg/kg Romo. Mice were euthanized after 4 weeks of treatment via cardiac exsanguination and cervical dislocation. Hindlimb bones and L4 vertebrae were cleaned of soft tissue, wrapped in PBS-soaked gauze and stored at -20C. Right tibiae, femora, and L4s were scanned via microcomputed tomography; tibiae were then tested to failure in 4-pt bending while L4s were compression tested. Romo treatment significantly increased cortical and trabecular bone mass in both STZ-Ad and CON animals. These morphological improvements created corresponding increases in cortical bending strength and trabecular compression strength, with STZ-Ad treated mice surpassing vehicle CON mice in all trabecular mechanics measures. These results suggest that Romo retains its efficacy at increasing bone mass and strength in diabetic kidney disease.

A moderate spinal contusion injury in rats alters bone turnover both below and above the level of injury with sex-based differences apparent in long-term recovery.

Spinal cord injury (SCI) leads to significant sublesional bone loss and high fracture rates. While loss of mechanical loading plays a significant role in SCI-induced bone loss, animal studies have demonstrated mechanical loading alone does not fully account for loss of bone following SCI. Indeed, we have shown that bone loss occurs below the level of an incomplete moderate contusion SCI, despite the resumption of weight-bearing and stepping. As systemic factors could also impact bone after SCI, bone alterations may also be present in bone sites above the level of injury. To examine this, we assessed bone microarchitecture and bone turnover in the supralesional humerus in male and female rats at two different ages following a moderate contusion injury in both sub-chronic (30 days) and chronic (180 days) time points after injury. At the 30-day timepoint, we found that both young and adult male SCI rats had decrements in trabecular bone volume at the supralesional proximal humerus (PH), while female SCI rats were not different from age-matched shams. At the 180-day timepoint, there were no statistical differences between SCI and sham groups, irrespective of age or sex, at the supralesional proximal humerus. At the 30-day timepoint, all SCI rats had lower BFR and higher osteoclast-covered trabecular surfaces in the proximal humerus compared to age-matched sham groups generally matching the pattern of SCI-induced changes in bone turnover seen in the sublesional proximal tibia. However, at the 180-day timepoint, only male SCI rats had lower BFR at the supralesional proximal humerus while female SCI rats had higher or no different BFR than their age-matched counterparts. Overall, this preclinical study demonstrates that a moderate contusion SCI leads to alterations in bone turnover above the level of injury within 30-days of injury; however male SCI rats maintained lower BFR in the supralesional humerus into long-term recovery. These data further highlight that bone loss after SCI is not driven solely by disuse. Additionally, these data allude to potential systemic factors exerting influence on bone following SCI and highlight the need to consider treatments for SCI-induced bone loss that impact both sublesional and systemic factors.

Clinically relevant doses of tiludronate do not affect bone remodelling in pasture-exercised horses.

BACKGROUND: Bisphosphonates are widely used in equine athletes to reduce lameness associated with skeletal disorders. Widespread off-label use has led to concern regarding potential negative effects on bone healing, but little evidence exists to support or refute this. OBJECTIVES: To investigate the influence of clinically relevant doses of tiludronate on bone remodelling and bone healing. STUDY DESIGN: Randomised, controlled in vivo experiments. METHODS: Each horse had a single tuber coxae biopsied (Day 0), then were divided into a treatment (IV tiludronate) or control (IV saline) group. Treatments were administered 30 and 90 days following initial biopsy. Biopsy of the tuber coxae was repeated on Day 60 to evaluate bone healing following a single treatment. Oxytetracycline was administered on Days 137 and 147 to label bone formation. The contralateral tuber coxae was biopsied on Day 150 to evaluate effects of repeated treatment. Bone biopsies were evaluated with micro-computed tomography and/or dynamic histomorphometry using standard techniques. RESULTS: Nineteen horses completed the study, with no complications following the biopsies and treatments. No significant differences in the trabecular bone parameters or bone formation rate were observed between treatment groups. MAIN LIMITATIONS: The use of a first-generation bisphosphonate may mean some effects of these drugs are underrepresented using this model. The results pertain to the tuber coxae and may not reflect injury or the healing response that occurs in long bones in training or racing. CONCLUSIONS: In this model, tiludronate did not affect normal bone remodelling in the horse, despite repeat dosages.

Estrogen deficiency induces changes in bone matrix bound water that do not closely correspond with bone turnover.

Postmenopausal osteoporosis, marked by estrogen deficiency, is a major contributor to osteoporotic fractures, yet early prediction of fractures in this population remains challenging. Our goal was to explore the temporal changes in bone-specific inflammation, oxidative stress, bone turnover, and bone-matrix water, and their relationship with estrogen deficiency-induced modifications in bone structure and mechanical properties. Additionally, we sought to determine if emerging clinically translatable imaging techniques could capture early bone modifications prior to standard clinical imaging. Two-month-old female Sprague Dawley rats (n = 48) underwent ovariectomy (OVX, n = 24) or sham operations (n = 24). A subgroup of n = 8 rats per group was sacrificed at 2-, 5-, and 10-weeks post-surgery to assess the temporal relationships of inflammation, oxidative stress, bone turnover, bone matrix water, mechanics, and imaging outcomes. OVX rats exhibited higher body weight compared to sham rats at all time points. By 5-weeks, OVX animals showed elevated markers of inflammation and oxidative stress in cortical bone, which persisted throughout the study, while cortical bone formation rate did not differ from sham until 10-weeks. DXA outcomes did not reveal differences between OVX and sham at any time point. Bound water, assessed using ultrashort echo time magnetic resonance imaging (UTE MRI), was lower in OVX at the earliest time point (2-weeks) and reduced again at 10-weeks with no difference at 5-weeks. These data demonstrate that bound water assessment using novel UTE MRI technology was lower at the earliest time point following OVX. However, no temporal relationship with bone turnover, inflammation, or oxidative stress was observed at the time points assessed in this study. These findings underscore both the increased need to understand bone hydration changes and highlight the usefulness of UTE MRI for non-invasive bone hydration measurements.

Inflammaging and Bone Loss in a Rat Model of Spinal Cord Injury.

Spinal cord injury (SCI) results in significant loss of sublesional bone, adding to the comorbidity of SCI with an increased risk of fracture and post-fracture complications. Unfortunately, the effect of SCI on skeletal health is also likely to rise, as the average age of SCI has increased and there are well-known negative effects of age on bone. To date, however, the impact of age and age-associated inflammation (inflammaging) on skeletal health after SCI remains largely unknown. To address this, we compared bone parameters in young (3 month) and middle-aged (9 month) male and female rats with a moderate thoracic contusion injury, to age- and sex-matched sham-operated controls. Skeletal parameters, locomotor function, and serum cytokine levels were assessed at both subchronic (30 days) and chronic (180 days) time points post-injury. We hypothesized that SCI would lead to a dramatic loss of bone immediately after injury in all SCI groups, with inflammaging leading to greater loss in middle-aged SCI rats. We also predicted that whereas younger rats might re-establish bone properties in more chronic phases of SCI, middle-aged rats would not. Supporting these hypothesis, trabecular bone volume was significantly lower in male and young female SCI rats early after injury. Contrary to our hypothesis, however, there was greater loss of trabecular bone volume, relative to age-matched shams, in young compared with middle-aged SCI rats, with no effects of SCI on trabecular bone volume in middle-aged female rats. Moreover, despite recovery of weight-supported locomotor activity, bone loss persisted into the chronic phase of injury for the young rats. Bone formation rates were lower in young male SCI rats, regardless of the time since injury, whereas both young and middle-aged female SCI rats had lower bone formation in the subchronic but not the chronic phase of SCI. In middle-aged rats, SCI-induced higher osteoclast surfaces, which also persisted into the chronic phase of SCI in middle-aged females. Neither age nor SCI-induced increases in inflammation seemed to be associated with bone loss. In fact, SCI had more dramatic and persistent effects on bone in male rats, whereas aging and SCI elevated serum cytokines only in female rats. Overall, this study demonstrates SCI-induced loss of bone and altered bone turnover in male and female rats that persists into the chronic phase post-injury. The sex- and age-dependent variations in bone turnover and serum cytokines, however, underscore the need to further explore both mechanisms and potential therapeutics in multiple demographics.

Exercise training modifies the bone and endocrine response to graded reductions in energy availability in skeletally mature female rodents.

Introduction: Reductions in energy availability leading to weight loss can induce loss of bone and impact important endocrine regulators of bone integrity. We sought to elucidate whether endurance exercise (EX) can mitigate bone loss observed in sedentary (SED) skeletally mature rodents subjected to graded energy deficits. Methods: Female virgin rats (n=84, 5-mo-old; 12/group) were randomized to baseline controls and either sedentary (SED) or exercise (EX) conditions, and within each exercise status to adlib-fed (ADLIB), or moderate (MOD) or severe (SEV) energy restriction diets for 12 weeks. Rats assigned to EX groups performed treadmill running to increase weekly energy expenditure by 10%. MOD-ER-SED, SEV-ER-SED, MOD-ER-EX and SEV-ER-EX were fed modified AIN93M diets with 20%, 40% 10%, and 30% less energy content, respectively, with 100% of all other nutrients provided. Results: Energy availability (EA) was effectively reduced by ~14% and ~30% in the MOD-ER and SEV-ER groups, respectively. MOD-ER for 12 weeks resulted in few negative impacts on bone and, except for serum leptin in MOD-ER-SED rats, no significant changes in endocrine factors. By contrast, SEV-ER in SED rats resulted in significantly lower total body and femoral neck bone mass, and reduced serum estradiol, IGF-1 and leptin. EX rats experiencing the same reduction in energy availability as SEV-ER-SED exhibited higher total body mass, lean mass, total BMC, and higher serum IGF-1 at the end of 12 weeks. Bone mechanical properties at 3 bone sites (mid-femur, distal femur, femoral neck) were minimally impacted by ER but positively affected by EX. Discussion: These findings indicate that combining increased EX energy expenditure with smaller reductions in energy intake to achieve a targeted reduction in EA provides some protection against loss of bone mass and lean mass in skeletally mature female rats, likely due to better preservation of circulating IGF-1, and that bone mechanical integrity is not significantly degraded with either moderate or severe reduced EA.

Ex vivo exposure to calcitonin or raloxifene improves mechanical properties of diseased bone through non-cell mediated mechanisms.

Raloxifene (RAL) reduces clinical fracture risk despite modest effects on bone mass and density. This reduction in fracture risk may be due to improved material level-mechanical properties through a non-cell mediated increase in bone hydration. Synthetic salmon calcitonin (CAL) has also demonstrated efficacy in reducing fracture risk with only modest bone mass and density improvements. This study aimed to determine if CAL could modify healthy and diseased bone through cell-independent mechanisms that alter hydration similar to RAL. 26-week-old male C57BL/6 mice induced with chronic kidney disease (CKD) beginning at 16 weeks of age via 0.2 % adenine-laced casein-based (0.9 % P, 0.6 % C) chow, and their non-CKD control littermates (Con), were utilized. Upon sacrifice, right femora were randomly assigned to the following ex vivo experimental groups: RAL (2 µM, n = 10 CKD, n = 10 Con), CAL (100 nM, n = 10 CKD, n = 10 Con), or Vehicle (VEH; n = 9 CKD, n = 9 Con). Bones were incubated in PBS + drug solution at 37 °C for 14 days using an established ex vivo soaking methodology. Cortical geometry (µCT) was used to confirm a CKD bone phenotype, including porosity and cortical thinning, at sacrifice. Femora were assessed for mechanical properties (3-point bending) and bone hydration (via solid state nuclear magnetic resonance spectroscopy with magic angle spinning (ssNMR)). Data were analyzed by two-tailed t-tests (µCT) or 2-way ANOVA for main effects of disease, treatment, and their interaction. Tukey's post hoc analyses followed a significant main effect of treatment to determine the source of the effect. Imaging confirmed a cortical phenotype reflective of CKD, including lower cortical thickness (p < 0.0001) and increased cortical porosity (p = 0.02) compared to Con. In addition, CKD resulted in weaker, less deformable bones. In CKD bones, ex vivo exposure to RAL or CAL improved total work (+120 % and +107 %, respectively; p < 0.05), post-yield work (+143 % and +133 %), total displacement (+197 % and +229 %), total strain (+225 % and +243 %), and toughness (+158 % and +119 %) vs. CKD VEH soaked bones. Ex vivo exposure to RAL or CAL did not impact any mechanical properties in Con bone. Matrix-bound water by ssNMR showed CAL treated bones had significantly higher bound water compared to VEH treated bones in both CKD and Con cohorts (p = 0.001 and p = 0.01, respectively). RAL positively modulated bound water in CKD bone compared to VEH (p = 0.002) but not in Con bone. There were no significant differences between bones soaked with CAL vs. RAL for any outcomes measured. RAL and CAL improve important post-yield properties and toughness in a non-cell mediated manner in CKD bone but not in Con bones. While RAL treated CKD bones had higher matrix-bound water content in line with previous reports, both Con and CKD bones exposed to CAL had higher matrix-bound water. Therapeutic modulation of water, specifically the bound water fraction, represents a novel approach to improving mechanical properties and potentially reducing fracture risk.

The Dietary Fermentable Fiber Inulin Alters the Intestinal Microbiome and Improves Chronic Kidney Disease Mineral-Bone Disorder in a Rat Model of CKD.

Background: Dietary fiber is important for a healthy diet, but intake is low in CKD patients and the impact this has on the manifestations of CKD-Mineral Bone Disorder (MBD) is unknown. Methods: The Cy/+ rat with progressive CKD was fed a casein-based diet of 0.7% phosphate with 10% inulin (fermentable fiber) or cellulose (non-fermentable fiber) from 22 weeks to either 30 or 32 weeks of age (~30 and ~15 % of normal kidney function). We assessed CKD-MBD, cecal microbiota, and serum gut-derived uremic toxins. Two-way ANOVA was used to evaluate the effect of age and inulin diet, and their interaction. Results: In CKD animals, dietary inulin led to changes in microbiota alpha and beta diversity at 30 and 32 weeks, with higher relative abundance of several taxa, including Bifidobacterium and Bacteroides , and lower Lactobacillus . Inulin reduced serum levels of gut-derived uremic toxins, phosphate, and parathyroid hormone, but not fibroblast growth factor-23. Dietary inulin decreased aorta and cardiac calcification and reduced left ventricular mass index and cardiac fibrosis. Bone turnover and cortical bone parameters were improved with inulin; however, bone mechanical properties were not altered. Conclusions: The addition of the fermentable fiber inulin to the diet of CKD rats led to changes in the gut microbiota composition, lowered gut-derived uremic toxins, and improved most parameters of CKD-MBD. Future studies should assess this fiber as an additive therapy to other pharmacologic and diet interventions in CKD. Significance Statement: Dietary fiber has well established beneficial health effects. However, the impact of fermentable dietary fiber on the intestinal microbiome and CKD-MBD is poorly understood. We used an animal model of progressive CKD and demonstrated that the addition of 10% of the fermentable fiber inulin to the diet altered the intestinal microbiota and lowered circulating gut-derived uremic toxins, phosphorus, and parathyroid hormone. These changes were associated with improved cortical bone parameters, lower vascular calcification, and reduced cardiac hypertrophy, fibrosis and calcification. Taken together, dietary fermentable fiber may be a novel additive intervention to traditional therapies of CKD-MBD.

Assessing cortical bone porosity with MRI in an animal model of chronic kidney disease.

Chronic kidney disease (CKD) is characterized by secondary hyperparathyroidism and an increased risk of hip fractures predominantly related to cortical porosity. Unfortunately, bone mineral density measurements and high-resolution peripheral computed tomography (HR-pQCT) imaging have shortcomings that limit their utility in these patients. Ultrashort echo time magnetic resonance imaging (UTE-MRI) has the potential to overcome these limitations by providing an alternative assessment of cortical porosity. The goal of the current study was to determine if UTE-MRI could detect changes in porosity in an established rat model of CKD. Cy/+ rats (n = 11), an established animal model of CKD-MBD, and their normal littermates (n = 12) were imaged using microcomputed tomography (microCT) and UTE-MRI at 30 and 35 weeks of age (which approximates late-stage kidney disease in humans). Images were obtained at the distal tibia and the proximal femur. Cortical porosity was assessed using the percent porosity (Pore%) calculated from microCT imaging and the porosity index (PI) calculated from UTE-MRI. Correlations between Pore% and PI were also calculated. Cy/+ rats had higher Pore% than normal rats at both skeletal sites at 35 weeks (tibia = 7.13 % +/- 5.59 % vs. 0.51 % +/- 0.09 %, femur = 19.99 % +/- 7.72 % vs. 2.72 % +/- 0.32 %). They also had greater PI at the distal tibia at 30 weeks of age (0.47 +/- 0.06 vs. 0.40 +/- 0.08). However, Pore% and PI were only correlated in the proximal femur at 35 weeks of age (ρ = 0.929, Spearman). These microCT results are consistent with prior studies in this animal model utilizing microCT imaging. The UTE-MRI results were inconsistent, resulting in variable correlations with microCT imaging, which may be related to suboptimal bound and pore water discrimination at higher magnetic field strengths. Nevertheless, UTE-MRI may still provide an additional clinical tool to assess fracture risk without using ionizing radiation in CKD patients.

The Dietary Fiber Inulin Slows Progression of Chronic Kidney Disease-Mineral Bone Disorder (CKD-MBD) in a Rat Model of CKD.

Chronic kidney disease (CKD)-mineral bone disorder (CKD-MBD) leads to fractures and cardiovascular disease. Observational studies suggest beneficial effects of dietary fiber on both bone and cardiovascular outcomes, but the effect of fiber on CKD-MBD is unknown. To determine the effect of fiber on CKD-MBD, we fed the Cy/+ rat with progressive CKD a casein-based diet of 0.7% phosphate with 10% inulin (fermentable fiber) or cellulose (non-fermentable fiber) from 22 weeks to either 30 or 32 weeks of age (~30% and ~15% of normal kidney function; CKD 4 and 5). We assessed CKD-MBD end points of biochemistry, bone quantity and quality, cardiovascular health, and cecal microbiota and serum gut-derived uremic toxins. Results were analyzed by two-way analysis of variance (ANOVA) to evaluate the main effects of CKD stage and inulin, and their interaction. The results showed that in CKD animals, inulin did not alter kidney function but reduced the increase from stage 4 to 5 in serum levels of phosphate and parathyroid hormone, but not fibroblast growth factor-23 (FGF23). Bone turnover and cortical bone parameters were similarly improved but mechanical properties were not altered. Inulin slowed progression of aorta and cardiac calcification, left ventricular mass index, and fibrosis. To understand the mechanism, we assessed intestinal microbiota and found changes in alpha and beta diversity and significant changes in several taxa with inulin, together with a reduction in circulating gut derived uremic toxins such as indoxyl sulfate and short-chain fatty acids. In conclusion, the addition of the fermentable fiber inulin to the diet of CKD rats led to a slowed progression of CKD-MBD without affecting kidney function, likely mediated by changes in the gut microbiota composition and lowered gut-derived uremic toxins. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Single cell cortical bone transcriptomics define novel osteolineage gene sets altered in chronic kidney disease.

Introduction: Due to a lack of spatial-temporal resolution at the single cell level, the etiologies of the bone dysfunction caused by diseases such as normal aging, osteoporosis, and the metabolic bone disease associated with chronic kidney disease (CKD) remain largely unknown. Methods: To this end, flow cytometry and scRNAseq were performed on long bone cells from Sost-cre/Ai9+ mice, and pure osteolineage transcriptomes were identified, including novel osteocyte-specific gene sets. Results: Clustering analysis isolated osteoblast precursors that expressed Tnc, Mmp13, and Spp1, and a mature osteoblast population defined by Smpd3, Col1a1, and Col11a1. Osteocytes were demarcated by Cd109, Ptprz1, Ramp1, Bambi, Adamts14, Spns2, Bmp2, WasI, and Phex. We validated our in vivo scRNAseq using integrative in vitro promoter occupancy via ATACseq coupled with transcriptomic analyses of a conditional, temporally differentiated MSC cell line. Further, trajectory analyses predicted osteoblast-to-osteocyte transitions via defined pathways associated with a distinct metabolic shift as determined by single-cell flux estimation analysis (scFEA). Using the adenine mouse model of CKD, at a time point prior to major skeletal alterations, we found that gene expression within all stages of the osteolineage was disturbed. Conclusion: In sum, distinct populations of osteoblasts/osteocytes were defined at the single cell level. Using this roadmap of gene assembly, we demonstrated unrealized molecular defects across multiple bone cell populations in a mouse model of CKD, and our collective results suggest a potentially earlier and more broad bone pathology in this disease than previously recognized.