Hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a complex disorder in need of precision medicine.

Hereditary hypophosphatemic rickets with hypercalciuria is an autosomal recessive phosphate-wasting disorder, associated with kidney and skeletal pathologies, which is caused by pathogenic variants of SLC34A3. In this issue, Zhu et al. describe a pooled analysis of 304 individuals carrying SLC34A3 variants. Their study underscores the complexity of hereditary hypophosphatemic rickets with hypercalciuria, as kidney and bone phenotypes generally do not coexist, heterozygous carriers of SLC34A3 variants also can be affected, and the response to oral phosphate supplementation is dependent on the genetic status.

Bone microarchitecture in patients with autoimmune hepatitis.

In patients with autoimmune hepatitis (AIH), osteoporosis represents a common extrahepatic complication, which we recently showed by an assessment of areal bone mineral density (aBMD) via dual-energy x-ray absorptiometry (DXA). However, it is well established that bone quality and fracture risk does not solely depend on aBMD, but also on bone microarchitecture. It is currently not known whether AIH patients exhibit a site-specific or compartment-specific deterioration in the skeletal microarchitecture. In order to assess potential geometric, volumetric, and microarchitectural changes, high-resolution peripheral quantitative computed tomography (HR-pQCT) measurements were performed at the distal radius and distal tibia in female patients with AIH (n = 51) and compared to age-matched female healthy controls (n = 32) as well as to female patients with AIH/primary biliary cholangitis (PBC) overlap syndrome (n = 25) and female patients with PBC alone (PBC, n = 36). DXA at the lumbar spine and hip, clinical characteristics, transient elastography (FibroScan) and laboratory analyses were also included in this analysis. AIH patients showed a predominant reduction of cortical thickness (Ct.Th) in the distal radius and tibia compared to healthy controls (p < .0001 and p = .003, respectively). In contrast, trabecular parameters such as bone volume fraction (BV/TV) did not differ significantly at the distal radius (p = .453) or tibia (p = .508). Linear regression models revealed significant negative associations between age and Ct.Th (95% confidence interval [CI], -14 to -5 µm/year, p < .0001), but not between liver stiffness, cumulative prednisolone dose (even after an adjustment for age), or disease duration with bone microarchitecture. The duration of high-dose prednisolone (≥7.5 mg) was negatively associated with trabecular thickness (Tb.Th) at the distal radius. No differences in bone microarchitecture parameters between AIH, AIH/PBC, and PBC could be detected. In conclusion, AIH patients showed a severe age-dependent deterioration of the cortical bone microarchitecture, which is most likely the major contribution to the observed increased fracture risk in these patients. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Response of the ENPP1-Deficient Skeletal Phenotype to Oral Phosphate Supplementation and/or Enzyme Replacement Therapy: Comparative Studies in Humans and Mice.

Inactivating mutations in human ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) may result in early-onset osteoporosis (EOOP) in haploinsufficiency and autosomal recessive hypophosphatemic rickets (ARHR2) in homozygous deficiency. ARHR2 patients are frequently treated with phosphate supplementation to ameliorate the rachitic phenotype, but elevating plasma phosphorus concentrations in ARHR2 patients may increase the risk of ectopic calcification without increasing bone mass. To assess the risks and efficacy of conventional ARHR2 therapy, we performed comprehensive evaluations of ARHR2 patients at two academic medical centers and compared their skeletal and renal phenotypes with ENPP1-deficient Enpp1asj/asj mice on an acceleration diet containing high phosphate treated with recombinant murine Enpp1-Fc. ARHR2 patients treated with conventional therapy demonstrated improvements in rickets, but all adults and one adolescent analyzed continued to exhibit low bone mineral density (BMD). In addition, conventional therapy was associated with the development of medullary nephrocalcinosis in half of the treated patients. Similar to Enpp1asj/asj mice on normal chow and to patients with mono- and biallelic ENPP1 mutations, 5-week-old Enpp1asj/asj mice on the high-phosphate diet exhibited lower trabecular bone mass, reduced cortical bone mass, and greater bone fragility. Treating the Enpp1asj/asj mice with recombinant Enpp1-Fc protein between weeks 2 and 5 normalized trabecular bone mass, normalized or improved bone biomechanical properties, and prevented the development of nephrocalcinosis and renal failure. The data suggest that conventional ARHR2 therapy does not address low BMD inherent in ENPP1 deficiency, and that ENPP1 enzyme replacement may be effective for correcting low bone mass in ARHR2 patients without increasing the risk of nephrocalcinosis. © 2021 American Society for Bone and Mineral Research (ASBMR).

Calcium and vitamin-D deficiency marginally impairs fracture healing but aggravates posttraumatic bone loss in osteoporotic mice.

Calcium and vitamin-D (Ca/VitD) deficiency is a major risk factor for osteoporosis. It may also contribute to the compromised bone healing frequently observed in osteoporotic patients, since calcium is essential for fracture-callus mineralization. Additionally, clinical data suggest systemic bone loss following fracture, which may aggravate osteoporosis and thus increase the risk for fragility fractures in osteoporotic patients further. However, the role of Ca/VitD in fracture healing and posttraumatic bone turnover has to date been poorly investigated. Here, we studied bone regeneration and posttraumatic bone turnover in C57BL/6 J mice with ovariectomy-induced osteoporosis. Mice were fed a standard or a Ca/VitD-deficient diet. Notably, fracture healing was only marginally disturbed in Ca/VitD-deficient mice. However, deficient mice displayed significantly increased serum parathyroid hormone levels and osteoclast activity, as well as reduced bone mass in the intact skeleton post-fracture, suggesting considerably enhanced calcium mobilization from the intact skeleton during bone regeneration. Ca/VitD supplementation initiated post-fracture prevented posttraumatic bone loss by reducing bone resorption and furthermore improved bone repair. These results imply that adequate Ca/VitD supply post-fracture is essential to provide sufficient calcium for callus-mineralization in order to prevent posttraumatic bone loss and to reduce the risk for secondary fractures in osteoporotic patients with Ca/VitD deficiency.

Hypochlorhydria-induced calcium malabsorption does not affect fracture healing but increases post-traumatic bone loss in the intact skeleton.

Efficient calcium absorption is essential for skeletal health. Patients with impaired gastric acidification display low bone mass and increased fracture risk because calcium absorption is dependent on gastric pH. We investigated fracture healing and post-traumatic bone turnover in mice deficient in Cckbr, encoding a gastrin receptor that affects acid secretion by parietal cells. Cckbr-/- mice display hypochlorhydria, calcium malabsorption, and osteopenia. Cckbr-/- and wildtype (WT) mice received a femur osteotomy and were fed either a standard or calcium-enriched diet. Healed and intact bones were assessed by biomechanical testing, histomorphometry, micro-computed tomography, and quantitative backscattering. Parathyroid hormone (PTH) serum levels were determined by enzyme-linked immunosorbent assay. Fracture healing was unaffected in Cckbr-/- mice. However, Cckbr-/- mice displayed increased calcium mobilization from the intact skeleton during bone healing, confirmed by significantly elevated PTH levels and osteoclast numbers compared to WT mice. Calcium supplementation significantly reduced secondary hyperparathyroidism and bone resorption in the intact skeleton in both genotypes, but more efficiently in WT mice. Furthermore, calcium administration improved bone healing in WT mice, indicated by significantly increased mechanical properties and bone mineral density of the fracture callus, whereas it had no significant effect in Cckbr-/- mice. Therefore, under conditions of hypochlorhydria-induced calcium malabsorption, calcium, which is essential for callus mineralization, appears to be increasingly mobilized from the intact skeleton in favor of fracture healing. Calcium supplementation during fracture healing prevented systemic calcium mobilization, thereby maintaining bone mass and improving fracture healing in healthy individuals whereas the effect was limited by gastric hypochlorhydria. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1914-1921, 2016.

The Protein Tyrosine Phosphatase Rptpζ Suppresses Osteosarcoma Development in Trp53-Heterozygous Mice.

Osteosarcoma (OS), a highly aggressive primary bone tumor, belongs to the most common solid tumors in growing children. Since specific molecular targets for OS treatment remain to be identified, surgical resection combined with multimodal (neo-)adjuvant chemotherapy is still the only way to help respective individuals. We have previously identified the protein tyrosine phosphatase Rptpζ as a marker of terminally differentiated osteoblasts, which negatively regulates their proliferation in vitro. Here we have addressed the question if Rptpζ can function as a tumor suppressor protein inhibiting OS development in vivo. We therefore analyzed the skeletal phenotype of mice lacking Ptprz1, the gene encoding Rptpζ on a tumor-prone genetic background, i.e. Trp53-heterozygosity. By screening a large number of 52 week old Trp53-heterozygous mice by contact radiography we found that Ptprz1-deficiency significantly enhanced OS development with 19% of the mice being affected. The tumors in Ptprz1-deficient Trp53-heterozygous mice were present in different locations (spine, long bones, ribs), and their OS nature was confirmed by undecalcified histology. Likewise, cell lines derived from the tumors were able to undergo osteogenic differentiation ex vivo. A comparison between Ptprz1-heterozygous and Ptprz1-deficient cultures further revealed that the latter ones displayed increased proliferation, a higher abundance of tyrosine-phosphorylated proteins and resistance towards the influence of the growth factor Midkine. Our findings underscore the relevance of Rptpζ as an attenuator of proliferation in differentiated osteoblasts and raise the possibility that activating Rptpζ-dependent signaling could specifically target osteoblastic tumor cells.

Low turnover osteoporosis in sheep induced by hypothalamic-pituitary disconnection.

The hypothalamus is of critical importance in regulating bone remodeling. This is underscored by the fact that intracerebroventricular-application of leptin in ewe leads to osteopenia. As a large animal model of osteoporosis, this approach has some limitations, such as high technical expenditure and running costs. Therefore we asked if a surgical ablation of the leptin signaling axis would have the same effects and would thereby be a more useful model. We analyzed the bone phenotype of ewe after surgical hypothalamo-pituitary disconnection (HPD + OVX) as compared to control ewe (OVX) after 3 and 12 months. Analyses included histomorphometric characterization, micro-CT and measurement of bone turnover parameters. Already 3 months after HPD we found osteopenic ewe with a significantly decreased bone formation (69%) and osteoclast activity (49%). After a period of 12 months the HPD group additionally developed an (preclinical) osteoporosis with significant reduction (33%) of femoral cortical thickness, as compared to controls (OVX). Taken together, HPD leads after 12 month to osteoporosis with a reduction in both trabecular and cortical bone caused by a low bone turnover situation, with reduced osteoblast and osteoclast activity, as compared to controls (OVX). The HPD-sheep is a suitable large animal model of osteoporosis. Furthermore our results indicate that an intact hypothalamo-pituitary axis is required for activation of bone turnover.

Impaired gastric acidification negatively affects calcium homeostasis and bone mass.

Activation of osteoclasts and their acidification-dependent resorption of bone is thought to maintain proper serum calcium levels. Here we show that osteoclast dysfunction alone does not generally affect calcium homeostasis. Indeed, mice deficient in Src, encoding a tyrosine kinase critical for osteoclast activity, show signs of osteopetrosis, but without hypocalcemia or defects in bone mineralization. Mice deficient in Cckbr, encoding a gastrin receptor that affects acid secretion by parietal cells, have the expected defects in gastric acidification but also secondary hyperparathyroidism and osteoporosis and modest hypocalcemia. These results suggest that alterations in calcium homeostasis can be driven by defects in gastric acidification, especially given that calcium gluconate supplementation fully rescues the phenotype of the Cckbr-mutant mice. Finally, mice deficient in Tcirg1, encoding a subunit of the vacuolar proton pump specifically expressed in both osteoclasts and parietal cells, show hypocalcemia and osteopetrorickets. Although neither Src- nor Cckbr-deficient mice have this latter phenotype, the combined deficiency of both genes results in osteopetrorickets. Thus, we find that osteopetrosis and osteopetrorickets are distinct phenotypes, depending on the site or sites of defective acidification.

Cloning and functional characterization of resistin-like molecule gamma.

Resistin, a recently discovered hormone that may play a crucial role in obesity-associated diabetes, is the founding member of a novel family of cysteine-rich proteins that are secreted by specific cell types. Three other members of this family have been described to date and were termed resistin-like molecules (RELMs). Here we describe the cloning and functional characterization of RELMgamma. The mouse RELMgamma-cDNA encodes a protein of 117 amino acids that contains a signal peptide leading to secretion of the protein. By Northern blotting the RELMgamma-mRNA is detectable in bone marrow, spleen, and lung as well as in peripheral blood granulocytes. Promyelocytic HL60 cells transfected with a RELMgamma expression plasmid have an increased proliferation rate compared to mock-transfected cells and display an altered response to retinoic acid-induced granulocytic differentiation. Taken together, these data provide the first experimental evidence that RELMgamma is a secreted molecule with a restricted expression pattern that may play a role in promyelocytic differentiation.