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.

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.

Cortical porosity development and progression is mitigated after etelcalcetide treatment in an animal model of chronic kidney disease.

PURPOSE: Chronic kidney disease (CKD) leads to increased bone fragility and risk of fracture. Cortical deteriorations, including cortical porosity, are key factors in fracture susceptibility in CKD. Since secondary hyperparathyroidism is common in CKD individuals and contributes to cortical deterioration, we hypothesized that reducing parathyroid hormone (PTH) may modulate CKD-induced cortical porosity. The goal of this pilot study was to assess the effects of lowering PTH, via the preclinical analogue of the FDA-approved calcimimetic etelcalcetide (KP-2326), on the development and progression of cortical pores in the setting of CKD. METHODS: Male Cy/+ Sprague Dawley rats with clinical biochemistries consistent with CKD (N = 8) were assigned to the study. At 30-32 weeks of age, cortical bone was assessed via In vivo µCT and blood collected for biochemistries to create baseline measures. Calcimimetic treatment with KP-2326 (KP) was then administered 3× weekly for 2-4 weeks. Cortical bone and biochemical parameters were repeated at study endpoint (33-37 wks of age). A group of age- and cohort-matched CKD rats (N = 4) were utilized as untreated controls. RESULTS: Untreated CKD rats had significantly increased cortical porosity over time, while porosity in KP-treated CKD rats was not significantly changed over time. Individual pore analysis revealed that pore area was significantly higher for expanding pores in untreated CKD rats compared to KP-treated CKD rats. Mechanical properties of KP-treated animal femora were similar to historical values of age-matched CKD animals and lower than those of age-matched non-diseased animals. CONCLUSION: Our pilot preclinical study demonstrates that etelcalcetide treatment can mitigate the progression of cortical bone changes in an animal model of CKD through suppression of pre-existing cortical pore expansion and limiting the size of new pore development. While stabilization of porosity is beneficial it remains likely that infilling of porosity will be needed to positively affect mechanical properties of bones in the setting of CKD.

Blueberry Polyphenols do not Improve Bone Mineral Density or Mechanical Properties in Ovariectomized Rats.

Osteoporosis-related bone fragility fractures are a major public health concern. Given the potential for adverse side effects of pharmacological treatment, many have sought alternative treatments, including dietary changes. Based on recent evidence that polyphenol-rich foods, like blueberries, increase calcium absorption and bone mineral density (BMD), we hypothesized that blueberry polyphenols would improve bone biomechanical properties. To test this, 5-month-old ovariectomized Sprague-Dawley rats (n = 10/gp) were orally gavaged for 90 days with either a purified extract of blueberry polyphenols (0-1000 mg total polyphenols/kg bw/day) or lyophilized blueberries (50 mg total polyphenols/kg bw/day). Upon completion of the dosing regimen, right femur, right tibia, and L1-L4 vertebrae were harvested and assessed for bone mineral density (BMD), with femurs being further analyzed for biomechanical properties via three-point bending. There were no differences in BMD at any of the sites analyzed. For bone mechanical properties, the only statistically significant difference was the high dose group having greater ultimate stress than the medium dose, although in the absence of differences in other measures of bone mechanical properties, we concluded that this result, while statistically significant, had little biological significance. Our results indicate that blueberry polyphenols had little impact on BMD or bone mechanical properties in an animal model of estrogen deficiency-induced bone loss.

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.

Age and sex effects on FGF23-mediated response to mild phosphate challenge.

BACKGROUND: During aging, there is a normal and mild loss in kidney function that leads to abnormalities of the kidney-bone metabolic axis. In the setting of increased phosphorus intake, hyperphosphatemia can occur despite increased concentrations of the phosphaturic hormone FGF23. This is likely from decreased expression of the FGF23 co-receptor Klotho (KL) with age; however, the roles of age and sex in the homeostatic responses to mild phosphate challenges remain unclear. METHODS: Male and female 16-week and 78-week mice were placed on either normal grain-based chow or casein (higher bioavailable phosphate) diets for 8 weeks. Gene expression, serum biochemistries, micro-computed tomography, and skeletal mechanics were used to assess the impact of mild phosphate challenge on multiple organ systems. Cell culture of differentiated osteoblast/osteocytes was used to test mechanisms driving key outcomes. RESULTS: Aging female mice responded to phosphate challenge by significantly elevating serum intact FGF23 (iFGF23) versus control diet; males did not show this response. Male mice, regardless of age, exhibited higher kidney KL mRNA with similar phosphate levels across both sexes. However, males and females had similar blood phosphate, calcium, and creatinine levels irrespective of age, suggesting that female mice upregulated FGF23 to maintain blood phosphorus, and compromised renal function could not explain the increased serum iFGF23. The 17ß-estradiol levels were not different between groups, and in vivo bone steroid receptor (estrogen receptor 1 [Esr1], estrogen receptor 2 [Esr2], androgen receptor [Ar]) expression was not different by age, sex, or diet. Trabecular bone volume was higher in males but decreased with both age and phosphate challenge in both sexes. Cortical porosity increased with age in males but not females. In vitro studies demonstrated that 17ß-estradiol treatment upregulated FGF23 and Esr2 mRNAs in a dose-dependent manner. CONCLUSIONS: Our study demonstrates that aging female mice upregulate FGF23 to a greater degree during a mild phosphate challenge to maintain blood phosphorus versus young female and young/old male mice, potentially due to direct estradiol effects on osteocytes. Thus, the control of phosphate intake during aging could have modifiable outcomes for FGF23-related phenotypes.

Voluntary Wheel Running Has Beneficial Effects in a Rat Model of CKD-Mineral Bone Disorder (CKD-MBD).

BACKGROUND: Reduced bone and muscle health in individuals with CKD contributes to their higher rates of morbidity and mortality. METHODS: We tested the hypothesis that voluntary wheel running would improve musculoskeletal health in a CKD rat model. Rats with spontaneous progressive cystic kidney disease (Cy/+ IU) and normal littermates (NL) were given access to a voluntary running wheel or standard cage conditions for 10 weeks starting at 25 weeks of age when the rats with kidney disease had reached stage 2-3 of CKD. We then measured the effects of wheel running on serum biochemistry, tissue weight, voluntary grip strength, maximal aerobic capacity (VO2max), body composition and bone micro-CT and mechanics. RESULTS: Wheel running improved serum biochemistry with decreased creatinine, phosphorous, and parathyroid hormone in the rats with CKD. It improved muscle strength, increased time-to-fatigue (for VO2max), reduced cortical porosity and improved bone microarchitecture. The CKD rats with voluntary wheel access also had reduced kidney cystic weight and reduced left ventricular mass index. CONCLUSIONS: Voluntary wheel running resulted in multiple beneficial systemic effects in rats with CKD and improved their physical function. Studies examining exercise interventions in patients with CKD are warranted.

Effect of ovariectomy on the progression of chronic kidney disease-mineral bone disorder (CKD-MBD) in female Cy/+ rats.

Male Cy/+ rats have shown a relatively consistent pattern of progressive kidney disease development that displays multiple key features of late stage chronic kidney disease-mineral bone disorder (CKD-MBD), specifically the development of cortical bone porosity. However, progression of disease in female Cy/+ rats, assessed in limited studies, is more heterogeneous and to date has failed to show development of the CKD-MBD phenotype, thus limiting their use as a practical model of progressive CKD-MBD. Animal and human studies suggest that estrogen may be protective against kidney disease in addition to its established protective effect on bone. Therefore, in this study, we aimed to determine the effect of ovariectomy (OVX) on the biochemical and skeletal manifestations of CKD-MBD in Cy/+ female rats. We hypothesized that OVX would accelerate development of the biochemical and skeletal features of CKD-MBD in female Cy/+ rats, similar to those seen in male Cy/+ rats. Female Cy/+ rats underwent OVX (n = 8) or Sham (n = 8) surgery at 15 weeks of age. Blood was collected every 5 weeks post-surgery until 35 weeks of age, when the rats underwent a 4-day metabolic balance, and the tibia and final blood were collected at the time of sacrifice. OVX produced the expected changes in trabecular and cortical parameters consistent with post-menopausal disease, and negative phosphorus balance compared with Sham. However, indicators of CKD-MBD were similar between OVX and Sham (similar kidney weight, plasma blood urea nitrogen, creatinine, creatinine clearance, phosphorus, calcium, parathyroid hormone, and no cortical porosity). Contrary to our hypothesis, OVX did not produce evidence of development of the CKD-MBD phenotype in female Cy/+ rats.