Roxadustat promotes osteoblast differentiation and prevents estrogen deficiency-induced bone loss by stabilizing HIF-1α and activating the Wnt/β-catenin signaling pathway

AUTHORS

Luyao Li, Afang Li, Li Zhu, Liangying Gan & Li Zuo

ABSTRACT

Background

Osteoporosis is a very common skeletal disorder that increases the risk of fractures. However, the treatment of osteoporosis is challenging. Hypoxia-inducible factor-1α (HIF-1α) plays an important role in bone metabolism. Roxadustat is a novel HIF stabilizer, and its effects on bone metabolism remain unknown. This study aimed to investigate the effects of roxadustat on osteoblast differentiation and bone remodeling in an ovariectomized (OVX) rat model.

Methods

In vitro, primary mouse calvarial osteoblasts were treated with roxadustat. Alkaline phosphatase (ALP) activity and extracellular matrix mineralization were assessed. The mRNA and protein expression levels of osteogenic markers were detected. The effects of roxadustat on the HIF-1α and Wnt/β-catenin pathways were evaluated. Furthermore, osteoblast differentiation was assessed again after HIF-1α expression knockdown or inhibition of the Wnt/β-catenin pathway. In vivo, roxadustat was administered orally to OVX rats for 12 weeks. Then, bone histomorphometric analysis was performed. The protein expression levels of the osteogenic markers HIF-1α and β-catenin in bone tissue were detected.

Results

In vitro, roxadustat significantly increased ALP staining intensity, enhanced matrix mineralization and upregulated the expression of osteogenic markers at the mRNA and protein levels in osteoblasts compared with the control group. Roxadustat activated the HIF-1α and Wnt/β-catenin pathways. HIF-1α knockdown or Wnt/β-catenin pathway inhibition significantly attenuated roxadustat-promoted osteoblast differentiation. In vivo, roxadustat administration improved bone microarchitecture deterioration and alleviated bone loss in OVX rats by promoting bone formation and inhibiting bone resorption. Roxadustat upregulated the protein expression levels of the osteogenic markers, HIF-1α and β-catenin in the bone tissue of OVX rats.

Conclusion

Roxadustat promoted osteoblast differentiation and prevented bone loss in OVX rats. The use of roxadustat may be a new promising strategy to treat osteoporosis.

Motor Impairments and Dopaminergic Defects Caused by Loss of Leucine-Rich Repeat Kinase 2 Function in Mice

AUTHORS

Guodong Huang, Daniel W. Bloodgood, Jongkyun Kang, Anu Shahapal, Phoenix Chen, Konstantin Kaganovsky, Jae-Ick Kim, Jun Ding and Jie Shen

ABSTRACT

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson’s disease (PD), but the pathogenic mechanism underlying LRRK2 mutations remains unresolved. In this study, we investigate the consequence of inactivation of LRRK2 and its functional homolog LRRK1 in male and female mice up to 25 months of age using behavioral, neurochemical, neuropathological, and ultrastructural analyses. We report that LRRK1 and LRRK2 double knock-out (LRRK DKO) mice exhibit impaired motor coordination at 12 months of age before the onset of DA neuron loss in the substantia nigra (SNpc). Moreover, LRRK DKO mice develop age-dependent, progressive loss of DA terminals in the striatum. Evoked dopamine release measured by fast-scan cyclic voltammetry in the dorsal striatum is also reduced in the absence of LRRK. Furthermore, LRRK DKO mice at 20–25 months of age show substantial loss of DA neurons in the SNpc. The surviving SNpc neurons in LRRK DKO mice at 25 months of age accumulate large numbers of autophagic and autolysosomal vacuoles and are accompanied with microgliosis. Surprisingly, the cerebral cortex is unaffected, as shown by normal cortical volume and neuron number as well as unchanged number of apoptotic cells and microglia in LRRK DKO mice at 25 months. These findings show that loss of LRRK function causes impairments in motor coordination, degeneration of DA terminals, reduction of evoked DA release, and selective loss of DA neurons in the SNpc, indicating that LRRK DKO mice are unique models for better understanding DA neurodegeneration in PD.

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

AUTHORS

Annabel Biruete, Corinne E Metzger, Neal X Chen, Elizabeth A Swallow, Curtis Vrabec, Erica L Clinkenbeard, Alexander J Stacy, Shruthi Srinivasan, Kalisha O'Neill, Keith G Avin, Matthew R Allen, Sharon M Moe

ABSTRACT

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) vs. IV iron on CKD-MBD and iron homeostasis in moderate CKD.

Methods

We tested the effects of 10 weeks of 2% FC vs. 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, PTH, and FGF23. Compared to untreated CKD rats, treatment with FC led to lower plasma phosphorus, intact FGF23, and a trend (p = 0.07) towards lower C-terminal FGF23. FC and IV iron equally reduced aorta and heart calcifications to levels similar to NL animals. Compared to 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 transferrin saturation rate compared to 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-to advanced CKD.

TiO2 Nanocoatings with Controllable Crystal Type and Nanoscale Topography on Zirconia Implants to Accelerate Bone Formation

AUTHORS

Nan Li, Zhichao Liu, Guanqi Liu, Zhi Wang, Xianwei Guo, Chuanbin Guo, Jianmin Han

ABSTRACT

In dentistry, zirconia implants have emerged as a promising alternative for replacing missing teeth due to their superior aesthetic performance and chemical stability. To improve the osseointegration of zirconia implants, modifying their surface with hierarchical micro/nanotopography and bioactive chemical composition are two effective ways. In this work, a microscale topography was prepared on a zirconia surface using hydrofluoric acid etching, and then a 50 nm TiO2 nanocoating was deposited via atomic layer deposition (ALD). Subsequently, an annealing treatment was used to transform the TiO2 from amorphous to anatase and simultaneously generate nanoscale topography. Various investigations into the coating surface morphology, topography, wettability, and chemical composition were carried out using scanning electron microscopy, white light interferometry, contact-angle measurement, X-ray diffraction, and X-ray photoelectron spectroscopy. In addition, in vitro cytocompatibility and osteogenic potential performance of the coatings were evaluated by human bone marrow mesenchymal stem cells (hBMSCs), and in vivo osseointegration performance was assessed in a rat femoral condyle model. Moreover, the possible mechanism was also investigated. The deposition of TiO2 film with/without annealing treatment did not alter the microscale roughness of the zirconia surface, whereas the nanotopography changed significantly after annealing. The in vitro studies revealed that the anatase TiO2 coating with regular wavelike nanostructure could promote the adhesion and proliferation of osteoblasts and further improve the osteogenic potential in vitro and osseointegration in vivo. These positive effects may be caused by nanoscale topography via the canonical Wnt/β-catenin pathway. The results suggest that using ALD in combination with annealing treatment to fabricate a nanotopographic TiO2 coating is a promising way to improve the osteogenic properties of zirconia implants.

Reduced bone mass in collagen prolyl 4-hydroxylase P4ha1+/-;P4ha2-/- compound mutant mice

AUTHORS

Jussi-Pekka Tolonen, Antti M. Salo, Mikko Finnilä, Ellinoora Aro, Emma Karjalainen, Veli-Pekka Ronkainen, Kati Drushinin, Christophe Merceron, Valerio Izzi, Ernestina Schipani, Johanna Myllyharju

ABSTRACT

Proper deposition of the extracellular matrix and its major components, the collagens, is essential for endochondral ossification and bone mass accrual. Collagen prolyl 4-hydroxylases (C-P4Hs) hydroxylate proline residues in the -X-Pro-Gly- repeats of all known collagen types. Their product, 4-hydroxyproline, is essential for correct folding and thermal stability of the triple-helical collagen molecules in physiological body temperatures. We have previously shown that inactivation of the mouse P4ha1 gene, which codes for the catalytic α subunit of the major C-P4H isoform, is embryonic lethal, while inactivation of the P4ha2 gene produced only a minor phenotype. Instead, mice with a haploinsufficiency of the P4ha1 gene combined with a homozygous deletion of the P4ha2 gene present with a moderate chondrodysplasia due to transient cell death of the growth plate chondrocytes. Here, to further characterize the bone phenotype of the P4ha1+/-;P4ha2-/- mice, we have carried out gene expression analyses at whole tissue and single cell levels, biochemical analyses, microcomputed tomography and histomorphometric analyses and second harmonic generation microscopy to show that C-P4H α subunit expression peaks early and that the C-P4H deficiency leads to reduced collagen amount, a reduced rate of bone formation and a loss of trabecular and cortical bone volume in the long bones. The total osteoblast number in the proximal P4ha1+/-;P4ha2-/- tibia and the C-P4H activity in primary P4ha1+/-;P4ha2-/- osteoblasts were reduced, while the population of osteoprogenitor colony forming-unit fibroblasts was increased in the P4ha1+/-;P4ha2-/- marrow. Thus, the P4ha1+/-;P4ha2-/- mouse model recapitulates key aspects of a recently recognized congenital connective tissue disorder with short stature and bone dysplasia caused by bi-allelic variants of the human P4HA1 gene. Altogether, the data demonstrate the allele-dose dependent importance of the C-P4Hs to the developing organism and a threshold effect of C-P4H activity in the proper production of bone matrix.

Bone volume, mineral density, and fracture risk after kidney transplantation

AUTHORS

Satu Keronen, Leena Martola, Patrik Finne, Inari S. Burton, Xiaoyu F. Tong, Heikki Kröger, Eero Honkanen

ABSTRACT

Background

Disordered mineral metabolism reverses incompletely after kidney transplantation in numerous patients. Post-transplantation bone disease is a combination of pre-existing chronic kidney disease and mineral disorder and often evolving osteoporosis. These two frequently overlapping conditions increase the risk of post-transplantation fractures.

Material and methods

We studied the prevalence of low bone volume in bone biopsies obtained from kidney transplant recipients who were biopsied primarily due to the clinical suspicion of persistent hyperparathyroidism between 2000 and 2015 at the Hospital District of Helsinki and Uusimaa. Parameters of mineral metabolism, results of dual-energy x-ray absorptiometry scans, and the history of fractures were obtained concurrently.

One hundred nine bone biopsies taken at a median of 31 (interquartile range, IQR, 18–70) months after transplantation were included in statistical analysis. Bone turnover was classified as high in 78 (72%) and normal/low in 31 (28%) patients. The prevalence of low bone volume (n = 47, 43%) was higher among patients with low/normal turnover compared to patients with high turnover [18 (58%) vs. 29 (37%), P = 0.05]. Thirty-seven fragility fractures in 23 (21%) transplant recipients corresponding to fracture incidence 15 per 1000 person-years occurred during a median follow-up 9.1 (IQR, 6.3–12.1) years. Trabecular bone volume did not correlate with incident fractures. Accordingly, low bone mineral density at the lumbar spine correlated with low trabecular bone volume, but not with incident fractures. The cumulative corticosteroid dose was an important determinant of low bone volume, but not of incident fractures.

Conclusions

Despite the high prevalence of trabecular bone loss among kidney transplant recipients, the number of fractures was limited. The lack of association between trabecular bone volume and fractures suggests that the bone cortical compartment and quality are important determinants of bone strength and post-transplantation fracture.