Bone Perfusion Alterations in Chronic Kidney Disease

Indiana University-Purdue University Indianapolis (IUPUI)Patients with chronic kidney disease (CKD) are at an alarming risk of fracture and cardiovascular disease-associated mortality. There is a critical need to better understand the underlying mechanism driving altered cardiovascular and skeletal homeostasis, as well as any connection between the two. CKD has been shown to have negative effects on many vascular properties including endorgan perfusion. Surprisingly, exploration of skeletal perfusion and vasculature has not been undertaken in CKD. Alterations in bone perfusion are linked to dysregulation of bone remodeling and mass in multiple conditions. An understanding of the detrimental impact of CKD on bone perfusion is a crucial step in understanding bone disease in these patients. The goal of this series of studies was to test the global hypothesis that skeletal perfusion is altered in CKD and that alterations can be modulated through treatments that affect metabolic dysfunction. These studies utilized a rat model of CKD to conduct metabolic assessments, bone perfusion measurements, bone imaging studies, and isolated vessel reactivity experiments. Our results showed that animals with CKD had higher levels of parathyroid hormone (PTH), leading to substantial bone resorption. Bone perfusion measurements showed CKD-induced elevations in cortical bone perfusion with levels progressing alongside CKD severity. Conversely we show that marrow perfusion was lower in advanced CKD. PTH suppression therapy in animals with CKD resulted in the normalization of cortical bone perfusion and cortical bone mass, but did not normalize marrow bone perfusion. These results show a clear association between bone deterioration and altered bone perfusion in CKD. While the relationship of altered bone perfusion and bone deterioration in CKD necessitates further work, these results indicate that determining the mechanisms of bone perfusion alterations and whether they are drivers, propagators, or consequences of skeletal deterioration in CKD could help untangle a key player in CKD-induced bone alterations

What Animal Models Have Taught Us About the Safety and Efficacy of Bisphosphonates in Chronic Kidney Disease

Purpose of Review Bisphosphonates (BPs) have long been the gold-standard anti-remodeling treatment for numerous metabolic bone diseases. Since these drugs are excreted unmetabolized through the kidney, they are not recommended for individuals with compromised kidney function due to concerns of kidney and bone toxicity. The goal of this paper is to summarize the preclinical BP work in models of kidney disease with particular focus on the bone, kidney, and vasculature. Recent Findings Summative data exists showing positive effects on bone and vascular calcifications with minimal evidence for bone or kidney toxicity in animal models. Summary Preclinical data suggest it may be worthwhile to take a step back and reconsider the use of bisphosphonates to lessen skeletal/vascular complications associated with compromised kidney function

Assessment of regional bone tissue perfusion in rats using fluorescent microspheres

Disturbances in bone blood flow have been shown to have deleterious effects on bone properties yet there remain many unanswered questions about skeletal perfusion in health and disease, partially due to the complexity of measurement methodologies. The goal of this study was use fluorescent microspheres in rats to assess regional bone perfusion by adapting mouse-specific fluorescent microsphere protocol. Ten fifteen-week old Sprague Dawley rats were injected with fluorescent microspheres either via cardiac injection (n = 5) or via tail vein injection (n = 5). Femora and tibiae were harvested and processed to determine tissue fluorescence density (TFD) which is proportional to the number of spheres trapped in the tissue capillaries. Right and left total femoral TFD (2.77 ± 0.38 and 2.70 ± 0.24, respectively) and right and left tibial TFD (1.11 ± 0.26 and 1.08 ± 0.34, respectively) displayed bilateral symmetry in flow when assessed in cardiac injected animals. Partitioning of the bone perfusion into three segments along the length of the bone showed the distal femur and proximal tibia received the greatest amount of perfusion within their respective bones. Tail vein injection resulted in unacceptably low TFD levels in the tibia from 4 of the 5 animals. In conclusion this report demonstrates the viability of cardiac injection of fluorescent microspheres to assess bone tissue perfusion in rats

Zoledronate treatment has different effects in mouse strains with contrasting baseline bone mechanical phenotypes

Aref, M. W., McNerny, E. M. B., Brown, D., Jepsen, K. J., & Allen, M. R. (2016). Zoledronate treatment has different effects in mouse strains with contrasting baseline bone mechanical phenotypes. Osteoporosis International : A Journal Established as Result of Cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA, 27(12), 3637–3643. https://doi.org/10.1007/s00198-016-3701-

Raloxifene neutralizes bone brittleness induced by anti-remodeling treatment and increases fatigue life through non-cell mediated mechanisms

Pre-clinical data have shown that tissue level effects stemming from bisphosphonateinduced suppression of bone remodeling can result in bone that is stronger yet more brittle. Raloxifene has been shown to reduce bone brittleness through non-cellular mechanisms. The goal of this work was to test the hypothesis that raloxifene can reverse the bone brittleness resulting from bisphosphonate treatment. Dog and mouse bone from multiple bisphosphonate dosing experiments were soaked in raloxifene and then assessed for mechanical properties. Mice treated with zoledronate in vivo had lower post-yield mechanical properties compared to controls. Raloxifene soaking had significant positive effects on select mechanical properties of bones from both vehicle and zoledronate treated mice. Although the effects were blunted in zoledronate bones relative to vehicle, the soaking was sufficient to normalize properties to control levels. Additional studies showed that raloxifene-soaked bones had a significant positive effect on cycles to failure (+114%) compared to control-soaked mouse bone. Finally, raloxifene soaking significantly improved select properties of ribs from dogs treated for 3 years with alendronate. These data show that ex vivo soaking in raloxifene can act through non-cellular mechanisms to enhance mechanical properties of bone previously treated with bisphosphonate. We also document that the positive effects of raloxifene soaking extend to enhancing fatigue properties of bone

Reference point indentation is insufficient for detecting alterations in traditional mechanical properties of bone under common experimental conditions

Reference point indentation (RPI) was developed as a novel method to assess mechanical properties of bone in vivo, yet it remains unclear what aspects of bone dictate changes/differences in RPI-based parameters. The main RPI parameter, indentation distance increase (IDI), has been proposed to be inversely related to the ability of bone to form/tolerate damage. The goal of this work was to explore the relationshipre-intervention RPI measurebetween RPI parameters and traditional mechanical properties under varying experimental conditions (drying and ashing bones to increase brittleness, demineralizing bones and soaking in raloxifene to decrease brittleness). Beams were machined from cadaveric bone, pre-tested with RPI, subjected to experimental manipulation, post-tested with RPI, and then subjected to four-point bending to failure. Drying and ashing significantly reduced RPI's IDI, as well as ultimate load (UL), and energy absorption measured from bending tests. Demineralization increased IDI with minimal change to bending properties. Ex vivo soaking in raloxifene had no effect on IDI but tended to enhance post-yield behavior at the structural level. These data challenge the paradigm of an inverse relationship between IDI and bone toughness, both through correlation analyses and in the individual experiments where divergent patterns of altered IDI and mechanical properties were noted. Based on these results, we conclude that RPI measurements alone, as compared to bending tests, are insufficient to reach conclusions regarding mechanical properties of bone. This proves problematic for the potential clinical use of RPI measurements in determining fracture risk for a single patient, as it is not currently clear that there is an IDI, or even a trend of IDI, that can determine clinically relevant changes in tissue properties that may contribute to whole bone fracture resistance

Parathyroid suppression therapy normalizes chronic kidney disease-induced elevations in cortical bone vascular perfusion: a pilot study

Patients with chronic kidney disease (CKD) have accelerated bone loss, vascular calcification and abnormal biochemistries, together contributing to an increased risk of cardiovascular disease and fracture-associated mortality. Despite evidence of vascular pathologies and dysfunction in CKD, our group has shown that cortical bone tissue perfusion is higher in a rat model of high-turnover CKD. The goal of the present study was to test the hypothesis that parathyroid hormone (PTH) suppressive interventions would normalize cortical bone vascular perfusion in the setting of CKD. In two separate experiments, 35-week old CKD animals and their normal littermates, underwent intra-cardiac fluorescent microsphere injection to assess the effect of 10 weeks of PTH suppression (Experiment 1: calcium supplementation, Experiment 2: calcimimetic treatment) on alterations in bone tissue perfusion. In Experiment 1, CKD animals had serum blood urea nitrogen (BUN) and PTH levels significantly higher than NL (+182% and +958%; p<0.05). CKD+Ca animals had BUN levels that were similar to CKD, while PTH levels were significantly lower and comparable to NL. Both femoral cortex (+220%, p=0.003) and tibial cortex (+336, p=0.005) tissue perfusion were significantly higher in CKD animals when compared to NL; perfusion was normalized to those of NL in CKD+Ca animals. MicroCT analysis of the proximal tibia cortical porosity showed a trend toward higher values in CKD (+401%; p=0.017) but not CKD+Ca (+111%; p = 0.38) compared to NL. Experiment 2, using an alternative method of PTH suppression, showed similar results as those of Experiment 1. These data demonstrate that PTH-suppression based interventions normalize cortical bone perfusion in the setting of CKD.This work was supported by a United States (U.S.) Department of Veterans Affairs grant (BX003025) to MRA. MWA was supported by F30 DK115162 and T32 AR065971 during separate portions of this work. KP-2326 was provided through a material transfer agreement with Amgen

Raloxifene improves bone mechanical properties in mice previously treated with zoledronate

Bisphosphonates represent the gold-standard pharmaceutical agent for reducing fracture risk. Long-term treatment with bisphosphonates can result in tissue brittleness which in rare clinical cases manifests as atypical femoral fracture. Although this has led to an increasing call for bisphosphonate cessation, few studies have investigated therapeutic options for follow-up treatment. The goal of this study was to test the hypothesis that treatment with raloxifene, a drug that has cell-independent effects on bone mechanical material properties, could reverse the compromised mechanical properties that occur following zoledronate treatment. Skeletally mature male C57Bl/6J mice were treated with vehicle (VEH), zoledronate (ZOL), or ZOL followed by raloxifene (RAL; 2 different doses). At the conclusion of 8 weeks of treatment, femora were collected and assessed with microCT and mechanical testing. Trabecular BV/TV was significantly higher in all treated animals compared to VEH with both RAL groups having significantly higher BV/TV compared to ZOL (+21%). All three drug-treated groups had significantly more cortical bone area, higher cortical thickness, and greater moment of inertia at the femoral mid-diaphysis compared to VEH with no difference among the three treated groups. All three drug-treated groups had significantly higher ultimate load compared to VEH-treated animals (+14 to 18%). Both doses of RAL resulted in significantly higher displacement values compared to ZOL-treated animals (+25 to +50%). In conclusion, the current work shows beneficial effects of raloxifene in animals previously treated with zoledronate. The higher mechanical properties of raloxifene-treated animals, combined with similar cortical bone geometry compared to animals treated with zoledronate, suggest that the raloxifene treatment is enhancing mechanical material properties of the tissue

Time course of rapid bone loss and cortical porosity formation observed by longitudinal μCT in a rat model of CKD

Background Rodent studies of bone in chronic kidney disease have primarily relied on end-point examinations of bone microarchitecture. This study used longitudinal in vivo microcomputed tomography (in vivo μCT) to characterize the onset and progression of bone loss, specifically cortical porosity, in the Cy/+ rat of model of CKD. Methods Male CKD rats and normal littermates were studied. In vivo μCT scans of the right distal tibia repeated at 25, 30, and 35 weeks were analyzed for longitudinal changes in cortical and trabecular bone morphometry. In vitro μCT scans of the tibia and femur identified spatial patterns of bone loss across distal, midshaft and proximal sites. Results CKD animals had reduced BV/TV and cortical BV at all time points but developed cortical porosity and thinning between 30 and 35 weeks. Cortical pore formation was localized near the endosteal surface. The severity of bone loss was variable across bone sites, but the distal tibia was representative of both cortical and trabecular changes. Conclusions The distal tibia was found to be a sensitive suitable site for longitudinal imaging of both cortical and trabecular bone changes in the CKD rat. CKD trabecular bone loss progressed through ~30 weeks followed by a sudden acceleration in cortical bone catabolism. These changes varied in timing and severity across individuals, and cortical bone loss and porosity progressed rapidly once initiated. The inclusion of longitudinal μCT in future studies will be important for both reducing the number of required animals and to track individual responses to treatment