Bispecific antibody against sclerostin and DKK1 improves bone health and reduces bone marrow adipose tissue accumulation in experimental chronic kidney disease

Bispecific antibody against sclerostin and DKK1 improves bone health and reduces bone marrow adipose tissue accumulation in experimental chronic kidney disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Bispecific antibody against sclerostin and DKK1 improves bone health and reduces bone marrow adipose tissue accumulation in experimental chronic kidney disease Worachet Promruk, Soher Jayash, Chartinun Chutoe, Hua Zhu Ke, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7788058/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Chronic kidney disease (CKD) leads to bone loss and bone marrow adipose tissue (BMAT) accumulation. Sclerostin and dickkopf-1 (DKK1) are two inhibitors of Wnt signalling, which suppress bone formation, promote bone marrow adipogenesis, and are elevated in CKD. However, therapies targeting sclerostin have shown limited efficacy in improving bone health in CKD animal models. Herein, we explored whether dual inhibition of sclerostin and DKK1 via a rodent bispecific antibody (rbsAb) could prevent bone loss and suppress BMAT accumulation in a CKD mouse model. CKD was induced using an adenine-supplemented diet in male mice, with CKD and control mice treated weekly for 6-weeks with vehicle or 30 mg/kg body weight of rbsAb. Circulating sclerostin and DKK1 were ~ 2- and ~ 3-fold higher, respectively, in CKD mice compared to controls. Proteomic profiling by LC-MS/MS and functional enrichment analysis suggested that in CKD mice, adipogenesis, osteoclast differentiation and bone resorption were increased whereas osteoblast differentiation was inhibited. These changes were prevented by antibody treatment. MicroCT revealed that long bones of CKD mice were characterised by lower bone mineral density, trabecular and cortical bone, and impaired biomechanical properties, but their vertebrae were unaffected. RbsAb treatment prevented cortical and trabecular bone loss and restored biomechanical properties. BMAT, as visualised by microCT imaging of osmium-stained bones, was elevated in CKD but reduced to control levels by rbsAb treatment. In conclusion, dual inhibition of sclerostin and DKK1 improved bone integrity and suppressed BMAT in experimental CKD, suggesting a promising therapeutic avenue for renal osteodystrophy. Biological sciences/Physiology/Bone quality and biomechanics Health sciences/Diseases/Endocrine system and metabolic diseases/Metabolic syndrome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The progressive loss of kidney function that occurs in chronic kidney disease (CKD) leads to disturbed mineral metabolism, with CKD patients commonly presenting with hyperphosphatemia, hypercalcemia, hyperparathyroidism and increased fibroblastic growth factor-23 (FGF23) levels [ 1 ]. These systemic changes are the primary indicators for the diagnosis of CKD–mineral bone disorder (CKD–MBD), which develops in the early stages of CKD. Disease progression results in a myriad of complications including vascular calcification and a wide spectrum of bone disorders characterised by abnormalities in bone quantity and quality, and generally referred to as renal osteodystrophy (ROD) [ 2 , 3 ]. The prevalence of osteoporosis in CKD patients ranges from 18–32% and fracture incidence of patients with end-stage kidney disease is 8-fold higher than that of the general population [ 4 ]. A histopathological classification system based on abnormalities in bone turnover, volume and mineralisation is used to diagnose ROD and categorise it into various diseases states. These include high turnover with either fibrosis (osteitis fibrosa) or abnormal mineralisation (mixed disease), and low turnover with either normal (adynamic bone disease) or abnormal mineralisation (osteomalacia) [ 5 ]. The stimuli for increased bone remodelling are unclear, but enhanced osteoclastic bone resorption in response to elevated parathyroid hormone (PTH) is associated with a net loss of bone mass despite an accelerating bone turnover [ 6 – 8 ]. Rapid remodelling also results in bone that is both immature and poorly mineralised [ 3 ]. Nevertheless, while high-turnover bone lesions induced by secondary hyperparathyroidism are common in late-stage CKD, alternative causative mechanisms are likely as very low serum PTH levels correlate with adynamic bone disease and an increased fracture rate [ 9 , 10 ]. This is clinically important, as accumulating evidence suggests that low bone turnover disease is the dominant form of ROD in the early stages of CKD, making it an ideal period to develop targeted therapeutic interventions [ 10 ]. The factors contributing to the development of low bone turnover disease are unclear but may include elevated levels of phosphate, FGF23 and/or protein-bound uremic toxins [ 11 – 14 ]. Uremic toxins can induce skeletal resistance to PTH and oxidative stress and mitochondrial dysfunction in osteoblasts/osteocytes [ 15 – 19 ]. Circulating concentrations of sclerostin, a Wnt/β-catenin antagonist, are also increased in CKD patients and animal models, and these changes, which are inversely correlated to estimated glomerular filtration rate in CKD patients, are increased during adynamic bone disease and prior to rises in circulating PTH and FGF-23 [ 20 – 29 ]. The relationship between CKD and another Wnt/β-catenin inhibitor, dickkopf-related protein 1 (DKK1), is less clear, with studies reporting unchanged [ 28 , 30 ] decreased [ 29 , 31 ] or increased [ 26 , 27 ] serum levels. Disturbed Wnt/β-catenin signalling may contribute to the low bone mass characteristic of ROD. Although Sost-deficient ( Sost −/− ) mice have increased bone mass, they have limited protection against nephrectomy-induced cortical and trabecular bone loss [ 32 , 33 ]; however, the high bone mass of Sost −/− mice at baseline may confound the interpretation of these data. Therefore, approaches using a sclerostin-neutralising antibody (Scl-Ab) such as romosozumab, which is widely recognised to increase bone mineral density (BMD) and reduce fractures in healthy men and postmenopausal women, may be more informative and offer potential therapeutic options to prevent bone loss in CKD [ 34 – 37 ]. Studies on CKD patients are limited. Two studies reported that romosozumab improved BMD at the lumbar spine, total hip, and femoral neck and reduced the relative risk of new vertebral fractures in postmenopausal women with osteoporosis and mild-moderate CKD [ 38 , 39 ]. However, in a pre-clinical rat model of progressive ROD, administration of a Scl-Ab provided limited protection to the architecture of the long bones and no improvement in their biomechanical properties [ 26 ]. Interestingly, DKK1 serum levels are raised with prolonged romosozumab treatment and also in patients with sclerosteosis and van Buchem disease [ 40 , 41 ]. Similarly, Sost −/− mice and mice administered sclerostin antibody also present with a compensatory increase in the expression of DKK1 [ 26 , 42 , 43 ] raising the possibility that elevated DKK1 may attenuate the anabolic effects of sclerostin inhibition and negate major improvement in skeletal health in Scl-Ab-treated mice [ 26 ]. In addition to bone loss, CKD can also lead to bone marrow adipose tissue (BMAT) accumulation in animal models [ 44 – 46 ] and humans [ 47 , 48 ]. The mechanism(s) responsible are unclear but may also involve Wnt/β-catenin signalling, which can inhibit adipogenesis and adipocyte-specific gene expression in white adipose tissue [ 49 – 51 ]. Congruently, Scl-Ab treatment reduced BMAT accumulation in ovariectomised rabbits [ 52 ] and irradiated [ 53 ], diabetic [ 54 ] and rosiglitazone-treated [ 55 ] mice; however, the changes were modest, suggesting that additional mechanisms may be involved and/or a compensatory increase in DKK1 may blunt the neutralising effects of the Scl-Ab. The increased DKK1 expression in response to Scl-Ab treatment may limit the ability of the antibody to protect bone health in CKD mice and decrease BMAT accumulation. Therefore, in this study we tested whether a rodent bispecific antibody (rbsAb) against sclerostin and DKK1 can improve bone health and decrease BMAT accumulation in an experimental model of CKD. Results Disease profile is similar in vehicle- and rbsAb-treated CKD mice We first confirmed the development of the CKD-MBD phenotype in the experimental mice. Dietary adenine-supplementation led to a loss of bodyweight within the first week and by end of the study the CKD mice were ~ 40% lighter than control (CTRL) mice. This was reflected in a decreased mass of the inguinal and gonadal white adipose tissue depots (iWAT and gWAT, respectively) of the vehicle-treated CKD mice (Figs. 1 A - C). The CKD mice also presented with hyperphosphatemia and increased BUN, creatinine and calcium (Figs. 1 D - F). Similar effects were observed between CTRL and CKD mice administered the rbsAb. The rbsAb treatment also modestly decreased iWAT and gWAT masses and increased serum phosphate in CTRL mice (Figs. 1 B, C, G). Plasma PTH levels were similar in vehicle-treated CTRL and CKD mice but were raised in both CTRL and CKD mice with the rbsAb administration (Fig. 1 H). Plasma DKK1 and sclerostin levels are elevated in CKD mice The ability of the rbsAb to reduce circulating levels of sclerostin and DKK1 was next investigated. In vehicle-treated mice, plasma levels of sclerostin were higher in CKD compared to CTRL mice (Figs. 1 I). Sclerostin concentrations in rbsAb-treated mice were extremely high, reaching supraphysiological concentrations and with no evidence of antibody-mediated neutralisation (Fig. 1 I). This is likely due to the antibody-sclerostin complex having cross-reactivity with the sclerostin ELISA, as reported previously [ 26 , 41 ]. In vehicle-treated mice, CKD increased median DKK1 concentrations by almost 3-fold. However, the unadjusted P value for this relatively large effect ( P = 0.0297) was no longer below the significance threshold after adjusting for multiple comparisons (Fig. 1 J). Moreover, treatment with the rbsAb strongly and significantly decreased DKK1 concentrations in both CTRL and CKD mice to levels that were indistinguishable from each other (Fig. 1 J). This demonstrates effective DKK1 neutralisation by the rbsAb. Plasma sclerostin and DKK1 levels of vehicle treated CTRL and CKD mice were not correlated to plasma PTH levels (Suppl Figs. 1A, B). RbsAb treatment prevents tibial bone loss in CKD mice Given that CKD causes bone loss [ 17 , 56 ], we next tested if rbsAb treatment was able to prevent bone loss in the tibia and vertebrae of CKD mice. Trabecular structure and BMD of the tibia were compromised in vehicle- but not rbsAb-treated CKD mice (Figs. 2 A – F). Moreover, while rbsAb administration increased trabecular BMD, bone volume fraction, thickness and number in the tibia of CTRL mice, these rbsAb effects were even greater in the CKD mice. This resulted in higher values for these skeletal properties in rbsAb-treated CKD mice compared to similarly treated CTRL mice (Figs. 2 B - E). The decrease in trabecular separation in rbsAb-treated CTRL and CKD mice showed a similar effect (Fig. 2 F). In cortical bone of vehicle-treated CKD mice, bone area, thickness, periosteal perimeter and polar moment of inertia were reduced whereas medullary area, endosteal perimeter and porosity were increased when compared to the tibia of vehicle-treated CTRL mice (Figs. 2 G - N). RbsAb treatment increased bone area, thickness, periosteal perimeter, porosity and polar moment of inertia but reduced medullary area and endosteal perimeter along the tibial length in both CTRL and CKD mice. The cortical bone changes in response to rbsAb treatment were similar in CTRL and CKD mice and accordingly the magnitude of differences noted between vehicle-treated CTRL and CKD mice were maintained in the rbsAb-treated mice (Figs. 2 G - N). This suggests that, within long bones, CKD alters the skeletal effects of sclerostin and DKK1 in a bone-type-specific manner. Furthermore, the influence of CKD and/or rbsAb treatment on cortical architecture appeared to be site dependent. For example, differences in cortical porosity were greater in the proximal tibia (Fig. 2 N) whereas more marked differences in cortical thickness were noted in the distal tibia (Fig. 2 I). Neither plasma sclerostin or plasma DKK1 had any correlation with trabecular BV/TV (Suppl Figs. 1C, D). In L4 vertebrae, CKD also had significant effects on trabecular and cortical BMD and bone architecture (Figs. 3 A-H). In vehicle-treated mice, only cortical thickness was decreased by CKD, whereas the effects of CKD were more pronounced in rbsAb-treated mice (Figs. 3 A-H). RbsAb administration increased trabecular BMD, bone volume fraction, thickness and number whereas trabecular separation was unaffected (Figs. 3 A – E). Similarly, cortical BMD, volume and thickness were also increased in response to the rbsAb (Figs. 3 F – H). However, the anabolic effects of the rbsAb on vertebral trabecular and cortical bone differed between CTRL and CKD mice: for trabecular bone, the rbsAb had a stronger anabolic effect in CKD than in CTRL mice (Figs. 3 A, B, D, E), while the rbsAb effects on cortical bone were stronger in CTRL than in CKD mice (Figs. 3 F - H). This bone-type specific response, within the vertebrae, to rbsAb treatment is similar to that observed in the tibia (Fig. 2 ). RbsAb treatment improves tibiae and vertebrae biomechanical properties of CKD mice As bone structure and geometry influence biomechanical properties, we next examined the response of the femur and L4 vertebrae to mechanical loading. Femur stiffness, maximum load and work to fracture were all lower in vehicle-treated CKD mice when compared to their respective CTRLs (Figs. 3 I, J, L). All biomechanical properties apart from post-yield displacement were increased in rbsAb-treated CTRL and CKD mice and, in all cases, the values from the rbsAb-treated CKD mice remained lower than similarly treated CTRL mice (Figs. 3 I - M) and reflect the structural changes observed in the cortical bone of the tibia (Figs. 2 G – M). In contrast, compression loading of L4 vertebrae revealed that stiffness, maximum load, yield load and work to fracture were similar in vertebrae from vehicle-treated CTRL and CKD mice (Figs. 3 N - Q) and reflect their similar trabecular and cortical architecture (Figs. 3 A – H). Similarity in biomechanical properties was also observed in vertebrae from rbsAb-administered CTRL and CKD mice, albeit all were increased when compared to their respective vehicle-treated mice (Figs. 3 N - Q). Gene and proteomic profiling of cortical bone reveals rbsAb-mediated prevention of CKD-induced dysregulation of osteoblast differentiation To further understand the cellular events responsible for bone loss in CKD mice and its prevention by rbsAb administration, we performed gene expression and proteomic analyses of cortical bone. The expression of osteoblast/osteocyte genes was similar in tibial cortical bone of vehicle-treated CTRL and CKD mice (Figs. 4 A - J), while the effects of rbsAb treatment were variable: it increased the expression of Sost in both CTRL and CKD mice (Fig. 4 I); increased Sp7, Bglap, Alpl, Col1a1 and Dkk1 in tibiae of CTRL mice but not CKD mice (Figs. 4 B – E, J); and increased Tnfrsf11b expression in CKD mice only (Fig. 4 H). The proteomic analysis by LC-MS/MS revealed that 199 proteins were down regulated and 189 proteins were upregulated in bone from vehicle-treated CKD mice compared to vehicle-treated CTRL mice (Fig. 5 A, C). Moreover, in CKD mice, the expression of 123 proteins was downregulated and 336 proteins were upregulated in bone by the administration of rbsAb compared with vehicle (Fig. 5 B, D). To identify the osteoblast/osteocyte-related pathways implicated in the aetiology of ROD, we performed functional enrichment analysis. From the top 8 ontology terms, the osteoblast differentiation pathway was depleted, whereas both bone resorption and bone remodelling pathways were enriched in vehicle-treated CKD mice (Fig. 5 E). The enrichment analysis of CKD mice further indicated that the rbsAb was able to prevent the defective osteoblast differentiation and excessive bone resorption and bone remodelling which is consistent with the increased cortical bone mass observed in the rbsAb-treated mice (Figs. 2 G – L). Given that the osteoblast differentiation pathway was notably affected by CKD and rbsAb treatment, we further assessed the expression patterns of some of the significantly altered proteins associated with this pathway (Figs. 5 F – M). The expression of COL1A1 and TNAP, were both down regulated in vehicle-treated CKD mice but the osteoblast transcription factor, SP7 was unchanged (Figs. 5 F – H). With regard to Wnt signalling, the expression of LRP5, LRP6 and sclerostin, but not CTNNB1 and DKK1, was decreased in vehicle-treated CKD mice (Figs. 5 I – M). The rbsAb increased TNAP, SOST and DKK1 expression which is in accord with the gene expression results (Figs. 4 D, I, J). RbsAb treatment prevents BMAT accumulation in CKD mice We next quantified BMAT along the tibial length as visualised by microCT imaging of osmium-stained bones to determine if the accumulation of BMAT that occurs in CKD animal models and patients was prevented by rbsAb treatment [ 44 , 46 , 48 ]. Total BMAT area, or BMAT area normalised to bone marrow area, of vehicle-treated CKD mice was increased compared to vehicle-treated CTRL mice (Figs. 6 A - C). RbsAb treatment reduced BMAT accumulation along the entire length of both CTRL and CKD tibiae (Figs. 6 A - C). Moreover, rbsAb treatment of CKD mice decreased the relative BMAT volume to levels similar to those observed in vehicle-treated CTRL mice (Fig. 6 C). Correlation analysis revealed an inverse relationship between rBMAT and trabecular BV/TV, which is in agreement with previous studies [ 46 ] (Fig. 6 D). Bone marrow adiposity can be decreased by PTH [ 57 , 58 ], but no correlation was found between plasma PTH and rBMAT (Suppl Fig. 1E); this is in broad agreement with prior observations [ 47 , 59 ]. Similarly, neither plasma sclerostin nor plasma DKK1 had any correlation with rBMAT (Suppl Figs. 1F, G). Bone marrow adipocyte and osteoclast gene and protein expression are dysregulated in CKD but normalised by rbsAb treatment We next investigated the cellular and molecular mechanisms responsible for the accumulation of BMAT in CKD and its prevention by the rbsAb. To do so, we assessed the expression of key adipocyte transcription factors and phenotype-specific genes and proteins in bone marrow from CTRL and CKD mice, both with and without rbsAb treatment. In comparison to vehicle-treated CTRL mice, the expression of Pparg2 and Adipoq , but not Cebpa , Fabp4 or the lipase-encoding genes, Pnpla2 and Lipe , was increased in vehicle-treated CKD mice (Figs. 6 E - J). RbsAb treatment down regulated Pparg2 and Adipoq expression in CKD mice, resulting in similar levels to those in rbsAb -treated CTRL mice; this is consistent with the effects on relative marrow adiposity (Fig. 6 C). Fabp4 expression was also down regulated by antibody treatment of both CTRL and CKD mice (Fig. 6 H). Sclerostin can promote the differentiation of bone marrow stem cells into osteoclasts; thus, we also quantified the expression of osteoclast transcription factors and mature osteoclast genes. The expression of the master transcription regulator of osteoclast differentiation, Nfatc1 , was similar in CTRL and CKD samples and was not affected by rbsAb treatment (Fig. 6 L). In contrast, the expression of Ctsk, Mmp9, Acp5 and Tnfsf11 was increased by CKD in vehicle-treated mice and rbsAb treatment reduced Tnfsf11 and Ctsk expression in CKD samples resulting in similar expression levels between CTRL and CKD mice (Figs. 6 K - O). We further interrogated these bone marrow effects using proteomics. The volcano plots (Figs. 7 A, B) and Venn diagrams (Figs. 7 C, D) illustrate the global proteomic alterations observed under CKD conditions and the modulatory effects of rbsAb treatment in CKD mice. Functional enrichment analysis of the top 6 ontology terms disclosed that, under vehicle treatment, adipogenesis, osteoclast differentiation and signalling, and bone resorption pathways were enriched in CKD vs CTRL mice (Fig. 7 E). These enriched pathways were depleted by rbsAb treatment of CKD mice (Fig. 7 E) and together these data are in accord with the ability of the rbsAb to protect CKD mice from bone loss and BMAT accumulation (Figs. 2 and 6 ), respectively. Adipocyte proteins were then analysed, which illustrated upregulation of FABP4, PLIN1, LEPR, ADIPOQ, LPL, LIPE and PNPLA2 in CKD mice. These increases were significantly prevented by rbsAb treatment, thereby cancelling or minimising the difference in their expression levels between CTRL and CKD mice (Figs. 7 F-L). In addition, proteins associated with osteoclast differentiation and bone resorption were upregulated in vehicle-treated CKD vs CTRL mice (Figs. 7 O – R). This CKD effect was generally prevented by rbsAb administration, resulting in expression levels of NFKB, NFATC and CTSK that were similar between rbsAb -treated CTRL and CKD mice (Figs. 7 O – R). SOST and DKK1 expression were similar in vehicle-treated CTRL and CKD mice but, in agreement with the bone gene expression data (Figs. 5 J, K), the expression of both β-catenin inhibitors was increased by rbsAb treatment (Fig. 7 M, N). Discussion Therapies for the treatment of ROD are currently focussed on the use of calcitriol and calcimimetics to lower circulating levels of PTH and reduce the adverse skeletal effects of secondary hyperparathyroidism [ 60 , 61 ]. This approach is recommended prior to consideration of other more targeted interventions but as elevated PTH levels are a poor indicator of low bone turnover disease there is a requirement for alternative treatments that can improve bone health regardless of PTH status [ 9 ]. Furthermore, CKD often coexists with osteoporosis in the ageing population, resulting in an increased fracture risk in patients with these combined comorbidities [ 62 ]. While there are some recognised limitations and caveats, many established therapeutic approaches to osteoporosis can improve bone health in CKD patients [ 63 ]. In patients with mild to-moderate CKD, bisphosphonates raise BMD and reduce fractures but their administration to patients with pre-existing very low bone turnover is a concern, as is their accumulation in CKD patients with impaired renal clearance [ 64 , 65 ]. Other anti-osteoporosis drugs such as denosumab, teriparatide and abaloparatide increase BMD and reduce fracture risk in patients with mild to moderate CKD. However, PTH analogues may aggravate existing hyperparathyroidism and denosumab, like bisphosphonates, can induce hypocalcemia [ 63 , 66 – 68 ]. An attractive alternative approach may involve the neutralisation of sclerostin; an inhibitor of bone formation and recognised to be elevated in bone [ 3 , 20 ] and serum [ 20 , 22 , 25 ] of CKD patients. Romosozumab is a humanised antibody that targets sclerostin resulting in the transient activation of bone formation and inhibition of bone resorption [ 34 , 69 ]. Romosozumab can reduce fracture risk in CKD but evidence is limited to a recent post hoc analysis of patients from the phase 3 clinical trials, FRAME and ARCH [ 38 ]. Preclinical studies have also reported that romosozumab can improve bone health in murine models of diabetes and CKD [ 70 , 71 ]. A limitation of targeting sclerostin alone to promote bone formation is the up-regulation of DKK1 expression, which may dampen the bone formation response to sclerostin neutralisation [ 26 , 41 – 43 ]. Therefore, one of the aims of this present study was to examine the ability of a bispecific antibody to sclerostin and DKK1 to improve bone health in a mouse model of CKD. It has been widely reported that the structure and biomechanical properties of long bones are compromised in CKD rodents [ 17 , 72 – 74 ]. A role for secondary hyperparathyroidism is often implicated in the progression of ROD but structural and mechanical integrity defects are also found in the tibia of this study despite normal levels of circulating PTH. However, in contrast to previous animal studies in which PTH levels were elevated we do not observe structural or biomechanical abnormalities in the vertebrae of the CKD mice [ 75 – 77 ]. The gene expression and functional pathway analysis suggest that rbsAb treatment promotes osteoblast differentiation and inhibits bone resorption and this consistent with the rbsAb profound effects on both cortical geometry and trabecular architecture of the vertebrae and long bones of CTRL and CKD mice. The efficacy, of the rbsAb to improve biomechanical and structural properties of the tibia of CKD mice is in stark contrast to that observed in a rat model of CKD where the effects of a Scl-Ab on the structure and biomechanical properties of vertebrae and long bones were limited and only observed in low PTH conditions [ 26 , 75 ]. This superior skeletal response in CKD mice to the neutralisation of sclerostin and DKK1 rather than sclerostin alone is consistent with previous studies where a bispecific antibody targeting sclerostin and DKK1 was more effective than monotherapy treatment in increasing BMD, biomechanical properties and bone repair activity in rats and nonhuman primates [ 42 , 78 – 80 ]. Also, a 3:1 mixture of Scl-Ab and DKK1-Ab promotes the formation of 2–3 times more trabecular bone than an equivalent dose of Scl-Ab alone despite DKK1-Ab having no consistent osteoanabolic effects [ 42 , 81 – 83 ]. The increased cortical porosity in the rbsAb treated mice was however unexpected as previous studies have indicated that when corrected for bone volume, cortical porosity in Sost −/− mice was similar to wild-type mice [ 84 ]. In this study we also aimed to verify if the rbsAb was able to reduce the amount of BMAT that is known to accumulate in CKD patients and animal models [ 44 , 45 , 47 , 48 ]. Though Wnt/β-catenin signalling is known to block expression of C/EBPα and PPARγ in white adipocytes and the loss of Wnt/β-catenin signalling causes a cell fate shift of pre-osteoblasts from osteoblasts to adipocytes, less is known about the regulatory effects of this pathway on bone marrow adipocytes (BMad) [ 85 , 86 ]. Sclerostin levels positively associate with higher vertebral marrow fat in men and in cell culture studies, adipogenesis is promoted in sclerostin challenged bone marrow mesenchymal stromal cells [ 87 , 88 ]. Decreased BMAT accumulation in Sost −/− mice provides further evidence that BMad accumulation in CKD could be a consequence of increased sclerostin levels [ 87 ]. Nevertheless, romosozumab has no effect on bone marrow adiposity of the iliac crest in postmenopausal osteoporotic women and the ability of Scl-Ab to decrease BMAT in irradiated or rosiglitazone treated mice was modest and limited to specific regions of the long bones [ 53 , 55 , 89 ]. These subtle, depot specific effects of Scl-Ab on BMad contrast with the results of this present study. Gene and protein expression data suggest an inhibition of bone marrow adipogenesis with rbsAb treatment and this is consistent with the reduced BMAT accumulation along the entire tibia of rbsAb -treated CTRL and CKD mice. The decrease in total BMAT in rbsAb -treated mice is similar to that reported in Scl-Ab-treated normal adult rats [ 90 ] but this reduction may in part be a consequence of an increase in trabecular bone volume and a corresponding reduction in marrow space in which adipocytes can inhabit. However, when normalised to marrow area, BMAT is still decreased in rbsAb -treated mice and suggests that an increase in Wnt/β-catenin signalling has a direct negative effect on BMad, independent of bone changes. When normalised to marrow area a reduction in BMAT was not observed in Scl-Ab-treated rats [ 90 ] and suggests that the ability of the Scl-Ab alone to restore normal amounts of BMAT is impeded by a compensatory increase in the expression of DKK1. Wnt pathway inhibition increases gradually as kidney function declines and is considered an early event in the pathogenesis of CKD [ 24 ]. Nevertheless, the relationship between blood sclerostin and PTH levels in CKD patients and animal models is unclear. A positive, negative or, as found in this present study, no correlation between circulating levels of sclerostin and PTH have been reported [ 26 , 30 ]. Although PTH is known to supress Sost and Dkk1 expression by bone cells [ 91 , 92 ], a regulatory role for PTH in this study is unclear as circulating PTH levels in the CKD mice is normal. Possibly, the chronic elevation of circulating sclerostin and DKK1 in CKD overwhelms the ability of PTH to regulate their circulating levels leading to PTH resistance and the promotion/aggravation of adynamic bone disease as well as an inability to decrease marrow adipogenesis [ 57 , 58 , 93 ]. Nevertheless, an explanation for the higher circulating levels of sclerostin and DKK1 in CKD remains unclear [ 20 – 29 ]. Reduced renal excretion is unlikely [ 25 ], and others have reported immunohistochemistry data indicating increased osteocyte expression of sclerostin in CKD [ 3 , 20 , 25 ]. Alternatively, the increased expression of sclerostin and DKK1 may involve TGF-β, which is elevated in serum and bone in CKD and promotes osteocyte sclerostin expression [ 94 , 95 ]. Furthermore, sclerostin levels are positively correlated with inflammation markers, phosphate and uremic toxins whereas FGF23 can induce DKK1 and inhibit osteoblast Wnt/β-catenin signalling via a soluble Klotho/MAPK–mediated process [ 96 – 98 ]. The upregulation of Sost and Dkk1 expression and their encoded proteins in bone of rbsAb -treated mice may be a consequence of lowered circulating levels of the proteins although for sclerostin we were unable to confirm this due to possible ELISA cross-reactivity with the bispecific antibody [ 26 , 41 ]. A similar increase in osteocyte Sost expression has been observed previously in Scl-Ab-treated CKD rats [ 26 ]. While the rbsAb improves bone health and decreases BMad accumulation in an experimental model of CKD, it is important to recognise that there may be limitations to this possible therapeutic approach. Cardiovascular adverse events have been reported with romosozumab in some (ARCH and BRIDGE) but not all (FRAME) clinical trials [ 35 – 37 , 99 ]. The presence of sclerostin [ 100 , 101 ] and DKK1 [ 102 ] in cardiovascular tissue may protect against vascular calcification and while theoretically romosozumab inhibition may aggravate the progression of vascular calcification in CKD there is no evidence of aortic mineralisation in rats or cynomolgus monkeys treated long-term with romosozumab [ 103 – 105 ]. Furthermore, neutralisation of DKK1 prevents vascular calcification in mice with renal insufficiency [ 27 ] and to the best of our knowledge, cardiovascular events have not been reported in Sost deficient mice or individuals with sclerosteosis or van Buchem disease [ 106 – 108 ]. Nevertheless, further animal and clinical studies focused on potential vascular effects in the setting of sclerostin and/or DKK1 blockade are required to evaluate the effect of prolonged treatment on cardiovascular health. In conclusion, mice fed an adenine enriched diet present with BMAT accumulation, trabecular and cortical bone loss and impaired biomechanical properties. A bispecific antibody to sclerostin and DKK1 was able to improve bone structure and biomechanical properties of bone and suppress BMAT accumulation. These results highlight a novel therapeutic strategy to enhance bone health in patients with CKD and pave the way for future translational applications in ROD management. Materials and Methods Mice To induce CKD, eight-week-old male C57BL/6JCrl mice (Charles River Laboratories, Margate, UK) were fed a diet supplemented with 0.2% adenine for 6-weeks (Envigo, Bicester, UK). Each week the adenine diet was offered for 5-days and replaced by a normal diet for 2 days. This modification of our previous protocol was based on the studies of Lair and colleagues and introduced to induce CKD but avoid pathological weight loss [ 109 , 110 ]. The CTRL mice received the same diet without adenine (Envigo). The bispecific antibody against sclerostin and DKK1 (Angitia Biopharmaceuticals, Guangzhou, China) or vehicle (PBS) was administered (30 mg/kg body weight) by subcutaneous injection to CTRL and CKD mice once a week for 6 weeks (n = 10/group). Body weights were obtained from mice twice weekly until sacrifice at 14-weeks of age. All mouse studies were approved by the University of Edinburgh Animal Welfare and Ethical Review Board and were conducted under a project license granted by the UK Home Office. Animal studies were conducted and are reported in line with the ARRIVE guidelines. Plasma biochemistry All mice were sacrificed after 6-weeks treatment and blood was collected by cardiac puncture under terminal anaesthesia. Plasma creatinine, blood urea nitrogen (BUN), phosphate and calcium were quantified using a biochemistry analyser (Beckman Coulter AU480). Intact PTH (QuidelOrtho, San Diego, USA), sclerostin and DKK1 (R&D Systems, Abbington, UK) were measured by ELISA according to the manufacturers’ instructions. Micro computed tomography (microCT) The changes in trabecular and cortical bone structure and BMD of L4 vertebrae (unfixed) and left tibia (fixed in 10% formaldehyde for 24 hours) were assessed by microCT (NeoScan N80, Mechelen, Belgium). Briefly, the bones were scanned with an isotropic voxel size of 5 µm (60 kV, 167 µA and 0.5 mm aluminium filter, 0.6° rotation angle) and the scans were reconstructed using the NRecon 1.7.3.0 program (Bruker, Kontich, Belgium) to remove artefacts, including beam-hardening and ring artefacts. CTAn software 1.15.4.0 (Skyscan, Kontich, Belgium) was used to evaluate bone histomorphometric parameters. To generate three-dimensional (3D) images, the scans were reconstructed and 3D images were created using the Neoscan80 software package (NeoScan). For L4 vertebrae, a 300-slice subset through the middle of the vertebrae’s body was analysed and the following trabecular [bone mineral density (BMD; g/cm 2 ), bone volume/tissue volume (BV/TV; %), thickness (Tb. Th; mm), number (Tb. N; 1/mm) and separation (Tb. Sp; mm)] and cortical [BMD; g/cm 2 , bone volume (BV; mm 3 ) and Th (mm)] parameters were calculated [ 56 ]. Each tibia was aligned along its longitudinal axis and the trabecular volume of interest (VOI) in the proximal metaphysis was a 1000-µm section of the metaphysis, 250 µm subjacent to the growth plate. The same trabecular parameters measured in the L4 vertebrae were quantified in the tibial reconstructions. For cortical analysis of the tibia, the proximal and distal portions were digitally cropped to exclude the epiphysis, growth plates and trabecular bone from the analysis [ 57 ]. Bone area (B.Ar; mm 2 ), thickness (Th; mm), polar moment of inertia ( J ; mm 4 ), medullary area (Med.Ar; mm 2 ), periosteal perimeter (P.Pm; mm), endosteal perimeter (E.Pm; mm) and porosity (%) were determined. Hydroxyapatite phantoms of known densities (0.25 and 0.75 g/cm 3 ) were scanned and reconstructed under identical conditions as the experimental samples to allow the calculation of BMD. R studio was used to create the line graph of the cortical bone parameters along the tibia length [ 55 , 58 ]. Quantification of BMAT After initial microCT scanning, the bone marrow adipocytes within the left tibiae were stained with osmium tetroxide as previously described [ 46 ]. In brief, the decalcified bones were incubated with 1% osmium tetroxide for 48h, washed and stored in Sorensens’ buffer at 4°C. The osmium-stained bones were re-scanned by microCT and total BMAT area was calculated as well as being normalised to the size of the bone marrow cavity with trabecular bone was excluded. BMAT volume was quantified in two distinct anatomical regions: the growth plate to tibia/fibula junction (GP-T/F J), which contains regulated BMAT (rBMAT); and the tibia/fibula junction to the end of distal bone (T/F J-End), which contains constitutive BMAT (cBMAT) [ 111 ]. R studio was used to create the line graph of BMAT accumulation along the tibia length. Biomechanical testing of tibia and vertebrae The L4 vertebrae and right femora were stored, unfixed, at − 20°C in water and their biomechanical properties were evaluated by a LS5 Lloyds materials testing machine with NEXYGEN Plus software (Ametek, Leicester, UK). For the 3-point bending, the femora were positioned horizontally on custom supports and a 100N load cell was applied perpendicular to the mid-diaphysis at a speed of 10 mm/min. For compression loading, the vertebral body was isolated from the spinal processes and prepared with flat and parallel ends using a polishing wheel and finally bonded to a fixed bottom plate with cyanoacrylate glue. A 500N load cell at a speed of 10 mm/min, compressed the vertebra. Each femur and vertebra were tested to fracture and data recorded after every 0.2 N change in load. The load–displacement curve for each bone was analysed, and stiffness, maximum load, yield load, work to fracture and post-yield displacement were calculated [ 112 ]. RT-qPCR The proximal and distal ends of the right tibiae were removed and the bone marrow was flushed out by centrifugation. Both the tibial shaft and the bone marrow were snap frozen in liquid nitrogen and stored at -80 0 C. The tissue was homogenised by a Rotor-Stator Homogenizer and RNA was extracted using a Qiagen RNeasy Mini kit (Qiagen, Manchester, UK) and quantified by nanodrop spectrophotometry (Thermo Fisher Scientific, Loughborough, UK). RNA quality was evaluated by the 260/280 nm ratio. After reverse transcription, gene expression was quantified using the SYBER green method and an Agilent Aria 2.1 real-time qPCR system (Agilent Technologies, Cheadle, UK). Target gene expression was normalised to a housekeeping gene ( Ppia ) and analysed using the ΔΔCt method. Oligonucleotide primers (Supp. table 1) were obtained from Sigma-Aldrich (Gillingham, UK) and Thermo Fisher Scientific. Proteomics Both proximal and distal ends of the right tibia were removed and bone morrow isolated by centrifugation. Cortical bone and bone marrow tissue were homogenised by a Rotor-Stator Homogenizer in a 100 µl of lysis buffer containing 5% sodium deoxycholate, 100 mM Tris-HCl (pH 8.5), 1 mg/mL chloroacetamide, and 1.5 mg/mL tris(2-carboxyethyl)phosphine. The lysates were subsequently heated at 95°C for 15 minutes. Protein capture and digestion from bone and bone marrow lysates were performed using an automated KingFisher Flex system (Thermo Fisher Scientific) [ 113 ]. Briefly, proteins were captured using MagReSyn HILIC magnetic microspheres (ReSyn Biosciences, Pretoria, South Africa). Protein-bound beads were then digested with 0.5 µg of MS-grade trypsin (Thermo Fisher Scientific) in 50 mM triethylammonium bicarbonate buffer (Sigma-Aldrich) at 37°C. The tryptic peptides were sequentially washed with 95% (v/v) acetonitrile (ACN) and 70% ethanol (EtOH). Digestion was terminated by acidification with 2% formic acid. Peptides were subsequently desalted using a C18-based desalting procedure, eluted with 0.1% trifluoroacetic acid (TFA) in 50% ACN, and dried by speed vacuum. Thereafter, dried peptides were reconstituted in 0.1% TFA for subsequent mass spectrometry analysis. One microgram of desalted peptides was loaded onto 25 cm Aurora columns (IonOptiks, Australia) using an RSLC nano µ HPLC system coupled to a Fusion Lumos mass spectrometer. Peptide separation was achieved with a 70-min linear gradient ranging from 5% to 30% acetonitrile in 0.5% acetic acid. The mass spectrometer was operated in data-independent acquisition (DIA) mode, collecting MS scans from 350–1650 Da at 120k resolution, followed by MS/MS acquisition across 45 windows with 0.5 Da overlap (200–2000 Da range) at 30k resolution and a normalized collision energy (NCE) of 28. The raw data were processed using DIA-NN 2.0 software with spectral matching performed against the Mus musculus UniProt protein database [ 114 ]. Normalization was conducted based on the total peptide abundance across LC-MS runs. GSEA was done using GSEA software (version 4.4.3, Broad institute, USA). Statistical analysis Data are presented as mean ± SEM or as violin plots, as indicated in the figure legends, with individual data points representing each biological replicate. Statistical analysis was performed using a two-way analysis of variance (ANOVA) to determine the effect of the rbsAb treatment and CKD status on bone and BMAT alterations. Correlations between individual parameters were performed using Spearman correlation. As the plasma DKK1 levels were not normally distributed the data were analysed using the Mann–Whitney test with Bonferroni correction for multiple comparisons. Statistical analysis was implemented using GraphPad Prism software (GraphPad Software, Inc., USA) and R studio (for cortical bone and BMAT analysis) and statistical significance was shown as; * p < 0.05; ** p < 0.01 and *** p < 0.001. For differential expression of proteomic profile, the data were processed using Perseus software [ 115 ]. Log₂ transformation was applied to the data, and missing values were imputed based on a normal distribution. Pairwise comparisons between groups were conducted using a two-sample t-test. Proteins exhibiting a p-value of less than 0.05 and a fold-change greater than 1.5 were considered significantly altered between the compared groups. Data Availability The proteomics data including raw files and search results have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD069117. Declarations Conflict of Interest HZK and XL are employees of Angitia Biopharmaceuticals (Angitia Incorporated Limited). All other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Author contributions Conceptualisation: WP, CF, WPC, KAS, LAS, HZK, XL Methodology: WP, SNJ, CC, AVK, HZK, XL. Formal analysis: WP, CF, CC, AVK, WPC, KS, LAS. Writing—original draft: WP and CF. Writing—review and editing: all authors. Visualisation: WP, CC. Supervision: KAS, LAS, WPC and CF. Funding acquisition: WP and CF. All authors read and approved the final manuscript. Acknowledgements We thank Heather Warnock and the staff of the Biological Research Facility (BRF) at the University of Edinburgh for providing invaluable animal support, Colin Wood and the staff at Easter Bush Pathology, Royal (Dick) School of Veterinary Studies, University of Edinburgh for conducting mouse serum biochemistries. We acknowledge financial support from Chulabhorn Royal Academy to WP and the Biotechnology and Biological Sciences Research Council (BBSRC) for supporting LAS via a Discovery Fellowship (BB/X009904/1) and for Institute Strategic Programme Grant Funding (BBS/E/RL/230001C) to CF and SNJ. 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Jayash, S.N., et al., Anti-RANKL Therapy Prevents Glucocorticoid-Induced Bone Loss and Promotes Muscle Function in a Mouse Model of Duchenne Muscular Dystrophy . Calcif Tissue Int, 2023. 113(4): p. 449–468. Batth, T.S., et al., Protein Aggregation Capture on Microparticles Enables Multipurpose Proteomics Sample Preparation . Mol Cell Proteomics, 2019. 18(5): p. 1027–1035. Demichev, V., et al., DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput . Nat Methods, 2020. 17(1): p. 41–44. Tyanova, S., et al., The Perseus computational platform for comprehensive analysis of (prote)omics data . Nat Methods, 2016. 13(9): p. 731–40. Additional Declarations There is no conflict of interest Supplementary Files RevieweraccessdetailsforProteomicsdata.docx eviewer access details for Proteomics data SupplFigure1forWPromruk.pdf Suppl Figure 1. Correlation analysis. SupplTable1PrimersWPromruk.pdf Suppl Table 1 Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: revise 04 Dec, 2025 Review # 3 received at journal 02 Dec, 2025 Review # 1 received at journal 23 Nov, 2025 Reviewer # 3 agreed at journal 21 Nov, 2025 Review # 2 received at journal 12 Nov, 2025 Reviewer # 2 agreed at journal 29 Oct, 2025 Reviewer # 1 agreed at journal 27 Oct, 2025 Reviewers invited by journal 27 Oct, 2025 Submission checks completed at journal 06 Oct, 2025 Editor assigned by journal 06 Oct, 2025 First submitted to journal 06 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7788058","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":525296056,"identity":"59774e14-94da-484c-882e-55d545f10ef3","order_by":0,"name":"Worachet 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10:01:40","extension":"xml","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":201949,"visible":true,"origin":"","legend":"","description":"","filename":"BONERES052190structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7788058/v1/1c0f0388f21b319829cb7b66.xml"},{"id":95523498,"identity":"43aca940-fda5-4161-9a3f-29150a12fb6a","added_by":"auto","created_at":"2025-11-10 09:57:08","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":223823,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7788058/v1/abed1951847817109a80f274.html"},{"id":95523586,"identity":"1d4b1d93-b392-466f-8188-da188e2d65d0","added_by":"auto","created_at":"2025-11-10 09:58:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":460006,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of CKD development in adenine-treated mice and effect of rbsAb on plasma concentrations of sclerostin and DKK1. \u0026nbsp;\u0026nbsp;\u003c/strong\u003e(A) Body weight of CKD mice was decreased over the 6-weeks of the study. The administration of antibody to either CTRL or CKD mice had no additional effect on body weight. (B, C) Inguinal (iWAT) and gonadal (gWAT) white adipose depots were decreased in vehicle and antibody-treated CKD mice. (D – G) Plasma concentrations of blood urea nitrogen (BUN), creatinine, calcium (Ca) and phosphate (Pi) were increased in vehicle and antibody-treated CKD mice. Pi levels were increased in antibody-treated control, but not CKD, mice. (H) Plasma concentrations of PTH were similar in vehicle-treated control and CKD mice but increased following antibody administration. (I, J) Plasma levels of sclerostin and DKK1 were both higher in vehicle-treated CKD compared to vehicle-treated control mice. The antibody lowered DKK1 levels in CTRL and CKD mice to levels that were indistinguishable from each other however sclerostin levels in antibody-treated mice were erronously high possibly due to cross-reactivity with the antigen‐antibody complex and the sclerostin ELISA. \u0026nbsp;For the data shown in the violin plots (C – I), significant effects of CKD, antibody treatment, and CKD - antibody treatment interaction were assessed using two-way ANOVA and overall p values for each variable, and their interactions, are shown beneath each graph. For plasma DKK1 data (J), the Mann–Whitney test with Bonferroni correction was used to test non-normally distributed data. \u0026nbsp;Significant differences between comparable groups were assessed using Tukey’s multiple comparison test and are indicated by * P \u0026lt; 0.05, **P \u0026lt; 0.01 or ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7788058/v1/5f9fd5106e74195841c61433.jpg"},{"id":95523583,"identity":"171e5289-1790-44ac-a735-047ab71a894e","added_by":"auto","created_at":"2025-11-10 09:58:42","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":778785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroCT analysis of tibial trabecular and cortical bone. \u003c/strong\u003e(A) Representative microCT images of trabecular bone from mice of the four experimental groups. Trabecular (Tb) (B) bone mineral density (BMD), (C) bone volume fraction (BV/TV), (D) thickness (Th) were decreased in vehicle-treated CKD mice whereas (F) Tb separation (Sp) was increased in vehicle-treated CKD mice. No effect on (E) Tb number (N) was observed. Although antibody treated increased Tb. BMD, BV/TV, Th, and N and decreased Tb.Sp in control and CKD mice the effect was greater in the CKD mice. (G – N) Quantification of whole cortical bone analysis excluding proximal and distal metaphyseal bone (G). Cortical (H) bone area (B.Ar), (I) thickness, (J) polar moment of inertia (J) and (L) periosteal perimeter (P.Pm) of vehicle-treated CKD mice were all reduced in various regions whereas (K) medullary area (Med.Ar), (M) endosteal perimeter (E.Pm) and (M) porosity were increased when compared to bones of vehicle-treated CTRL mice. Antibody treatment increased B.Ar, thickness, P.Pm, porosity and J but reduced Med.Ar and E.Pm along the tibial length in both CTRL and CKD mice. For the data shown in the violin plots (B - F), significant effects of CKD, antibody treatment, and CKD – antibody treatment interaction were assessed using two-way ANOVA and overall p values for each variable, and their interactions, are shown beneath each graph. Significant differences between comparable groups were assess using Tukey’s multiple comparison test and are indicated by * P \u0026lt; 0.05, **P \u0026lt; 0.01 or ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7788058/v1/5c16c7254d0ef437645d9a88.jpg"},{"id":95524230,"identity":"a72be419-561a-4c37-b3ad-eb1111462a3e","added_by":"auto","created_at":"2025-11-10 10:02:32","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":607684,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroCT analysis of vertebrae trabecular and cortical bone and biomechanical properties of tibia and vertebrae\u003c/strong\u003e. Analysis of (A – E) trabecular and (F – H) cortical bone indicated that only (H) cortical thickness of L4 vertebrae was affected in vehicle-treated CKD mice compared to similarly treated CTRL mice. Antibody administration increased trabecular (Tb) (A) bone mineral density (BMD), (B) bone volume fraction (BV/TV), (C) thickness (Th), (E) number (N) and cortical (cort) (F) BMD, (G) BV and (H) Th in CTRL and CKD mice whereas (D) Tb separation (sp) was unaffected. Biomechanical analysis indicated that femur (I) stiffness, (J) maximum load, (L) work to fracture but not (K) yield load were lower in vehicle-treated CKD mice when compared to their respective CTRLs and all biomechanical properties apart from (M) post-yield displacement were increased in CTRL and CKD mice treated with antibody. \u0026nbsp;L4 vertebrae (N) stiffness, (O) maximum load, (P) yield load and (Q) work to fracture of vehicle-treated CTRL and CKD mice were similar and all biomechanical properties were increased in all mice treated with antibody. All data are shown as violin plots and significant effects of CKD, antibody treatment, and CKD – antibody treatment interaction were assessed using two-way ANOVA and overall p values for each variable, and their interactions, are shown beneath each graph. Significant differences between comparable groups were assess using Tukey’s multiple comparison test and are indicated by * P \u0026lt; 0.05, **P \u0026lt; 0.01 or ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7788058/v1/5d044d61439b6337a222b99f.jpg"},{"id":95320687,"identity":"ad626b54-eec8-4392-9daf-4be12d34d9ff","added_by":"auto","created_at":"2025-11-06 16:39:58","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":405002,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOsteogenic gene expression in cortical bone of the tibia.\u003c/strong\u003e The expression of the osteoblast/osteocyte genes (A) \u003cem\u003eRunx2\u003c/em\u003e, (B) \u003cem\u003eSp7\u003c/em\u003e, (C) \u003cem\u003eBglap\u003c/em\u003e (D) \u003cem\u003eAlpl \u003c/em\u003e\u0026nbsp;(E) \u003cem\u003eCol1a1\u003c/em\u003e (F) \u003cem\u003eMepe\u003c/em\u003e (G) \u003cem\u003eTnfsf11 \u003c/em\u003e(H) \u003cem\u003eTnfrsf11b \u003c/em\u003e(I) \u003cem\u003eSost \u003c/em\u003eand (J) \u003cem\u003eDkk1 \u003c/em\u003ewere similar in vehicle-treated CTRL and CKD mice. Antibody treatment increased the expression of \u003cem\u003eSp7, Bglap, Alpl, Col1a1 \u003c/em\u003eand\u003cem\u003eDkk1\u003c/em\u003e in CTRL mice, \u003cem\u003eTnfrsf11b\u003c/em\u003e expression in CKD mice and \u003cem\u003eSost\u003c/em\u003e expression CTRL and CKD mice. \u0026nbsp;\u003cem\u003eRunx2, Mepe \u003c/em\u003eand \u003cem\u003eTnfsf11 \u003c/em\u003eexpression by CTRL and CKD mice were altered by antibody treatment. All data are shown as violin plots and significant effects of CKD, antibody treatment, and CKD – antibody treatment interaction were assessed using two-way ANOVA and overall p values for each variable, and their interactions, are shown beneath each graph. Significant differences between comparable groups were assess using Tukey’s multiple comparison test and are indicated by * P \u0026lt; 0.05, **P \u0026lt; 0.01 or ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7788058/v1/acdb78cff40db53a83a15b56.jpg"},{"id":95523942,"identity":"fc0ab760-adb7-4d4e-91c9-ebdaff15b454","added_by":"auto","created_at":"2025-11-10 10:01:34","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":561471,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProteomic profiling of cortical bone. \u003c/strong\u003e(A, B)\u003cstrong\u003e \u003c/strong\u003eVolcano plots and (C, D) Venn diagrams illustrate global differential protein expression in the tibia of CKD mice and mice treated with antibody. \u0026nbsp;(E) Gene set enrichment analysis identified the osteoblast/osteocyte-related pathways implicated in the aetiology of ROD and those affected by antibody-treatment. From the proteomics analysis the expression of some of the significantly altered proteins associated with osteoblast differentiation such as (G) TNAP and (H) COL1A1, but not (F) SP7 were reduced in vehicle-treated CKD mice. The expression of proteins involved in Wnt-signalling, (I) LRP5, (J) LRP6, and (L) SOST were reduced in vehicle-treated CKD mice whereas (K) CTNNB1 and (M) DKK1 were unchanged. \u0026nbsp;Antibody treatment increased (G) TNAP, (L) SOST and (M) DKK1 expression in both control and CKD bones. Comparison of the expression of individual protein are shown as violin plots and significant effects of CKD, antibody treatment, and CKD – antibody treatment interaction were assessed using two-way ANOVA and overall p values for each variable, and their interactions, are shown beneath each graph. Significant differences between comparable groups were assessed using Tukey’s multiple comparison test and are indicated by * P \u0026lt; 0.05, **P \u0026lt; 0.01 or ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"15.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7788058/v1/3157d8b212f05b03f9eb55dc.jpg"},{"id":95320692,"identity":"fb871974-760d-4535-91ef-a012aad27445","added_by":"auto","created_at":"2025-11-06 16:39:58","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":716206,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBMAT distribution along the tibial bone shaft and adipogenic and osteoclastogenic gene expression in bone marrow tissue. \u003c/strong\u003e(A)\u003cstrong\u003e \u003c/strong\u003eVisualisation of osmium-stained bone marrow adipose tissue (BMAT) present between the growth plate to tibia/fibula junction (GP-T/F J), which contains regulated BMAT (rBMAT) and the tibia/fibula junction to the end of distal bone (T/F J-End), which contains constitutive BMAT (cBMAT) of vehicle and antibody treated CTRL and CKD mice. (B) Total BMAT and (C) BMAT normalised to bone marrow area (with trabecular bone excluded) was increased in vehicle-treated CKD mice compared to similarly treated CTRL mice. Antibody treatment reduced BMAT accumulation along the entire length of the tibia of CTRL and CKD mice. (D) Spearman correlations between trabecular bone volume fraction (Tb.BV/TV) and rBMAT indicated a negative correlation. The expression of the adipogenic genes (E) \u003cem\u003ePparg2\u003c/em\u003e and (G) \u003cem\u003eAdipoq\u003c/em\u003e but not (F) \u003cem\u003eCebpa\u003c/em\u003e, (H) \u003cem\u003eFabp4\u003c/em\u003e, (I) \u003cem\u003eLipe\u003c/em\u003eor (J) \u003cem\u003ePnpla2\u003c/em\u003e was increased in vehicle-treated CKD mice. Antibody treatment had no effect on the expression of \u003cem\u003eCebpa, Lipe\u003c/em\u003eor \u003cem\u003ePnpla2\u003c/em\u003e but the difference in expression of \u003cem\u003ePparg2\u003c/em\u003e and \u003cem\u003eAdipoq\u003c/em\u003eseen in the vehicle-treated mice was abolished by antibody treatment. The expression of osteoclast genes (K) \u003cem\u003eTnfsf11, \u003c/em\u003e(M) \u003cem\u003eAcp5, \u003c/em\u003e(N)\u003cem\u003e Ctsk, \u003c/em\u003eand (O) \u003cem\u003eMmp9 \u003c/em\u003ewas increased in vehicle-treated CKD mice and this difference in expression was nullified by antibody treatment. (L) \u003cem\u003e\u003cstrong\u003eNfatc1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, was similar\u003c/strong\u003ein CTRL and CKD samples and was not affected by antibody treatment. For the data shown in the violin plots (E - O) the significant effects of CKD, antibody treatment, and CKD – antibody treatment interaction were assessed using two-way ANOVA and overall p values for each variable, and their interactions, are shown beneath each graph. Significant differences between comparable groups were assess using Tukey’s multiple comparison test and are indicated by * P \u0026lt; 0.05, **P \u0026lt; 0.01 or ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"16.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7788058/v1/ee7913841cc374414677bdda.jpg"},{"id":95320696,"identity":"081833b9-2b39-4d8b-ad57-c4fc75fb24fd","added_by":"auto","created_at":"2025-11-06 16:39:58","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":660153,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProteomic profiling of bone marrow. \u003c/strong\u003e(A, B) Volcano plots and (C, D) Venn diagrams illustrate global differential protein expression in the bone marrow of CKD mice and mice treated with antibody. (E) Gene set enrichment analysis revealed that gene sets associated with adipogenesis, osteoclast differentiation and signalling, and bone resorption pathways were enriched in vehicle-treated CKD mice as compared to similarly treated CTRL mice. \u0026nbsp;Antibody treatment reversed these pathway changes in CKD mice. The expression of proteins associated with adipocyte differentiation including (F) FABP4, (G) PLIN1, (H) LEPR, (I) ADIPOQ and (J) LPL and lipolytic protein, (K) LIPE and (L) PNPLA2 were increased in vehicle-treated CKD mice compared to vehicle-treated control mice. This increased expression was decreased by antibody treatment. In addition, CKD increased the expression of (O) NFKB1, (P) NFATC1, (Q) ACP5 and (R) CTSK which are critical for osteoclast differentiation and bone resorption, while antibody treatment normalized (O) NFKB, (P) NFATC and (R) CTSK but not (Q) ACP5. \u0026nbsp;(M and N) SOST and DKK1 expression was similar in vehicle-treated CTRL and CKD mice and increased with antibody treatment. \u0026nbsp;Comparison of the expression of individual protein are shown as violin plots and significant effects of CKD, antibody treatment, and CKD – antibody treatment interaction were assessed using two-way ANOVA and overall p values for each variable, and their interactions, are shown beneath each graph. Significant differences between comparable groups were assessed using Tukey’s multiple comparison test and are indicated by * P \u0026lt; 0.05, **P \u0026lt; 0.01 or ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"17.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7788058/v1/101701c76c3d16c162691046.jpg"},{"id":95530763,"identity":"265c9575-57cd-4b91-887b-12de26012745","added_by":"auto","created_at":"2025-11-10 10:21:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5456664,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7788058/v1/8f229774-8071-4a4d-b099-73434ead1582.pdf"},{"id":95320681,"identity":"cb84489b-d086-4211-83fb-c792f7b4ce5a","added_by":"auto","created_at":"2025-11-06 16:39:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12689,"visible":true,"origin":"","legend":"eviewer access details for Proteomics data","description":"","filename":"RevieweraccessdetailsforProteomicsdata.docx","url":"https://assets-eu.researchsquare.com/files/rs-7788058/v1/1a8b6c4a0447d4d9e2679b9b.docx"},{"id":95320682,"identity":"4065b29c-80f2-4247-87c0-b4b078e08122","added_by":"auto","created_at":"2025-11-06 16:39:57","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":91703,"visible":true,"origin":"","legend":"Suppl Figure 1. Correlation analysis.","description":"","filename":"SupplFigure1forWPromruk.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7788058/v1/070f920988d6f94fda79abb1.pdf"},{"id":95320689,"identity":"b964039e-d84f-40f9-aadf-8a02df6d788a","added_by":"auto","created_at":"2025-11-06 16:39:58","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":146405,"visible":true,"origin":"","legend":"Suppl Table 1","description":"","filename":"SupplTable1PrimersWPromruk.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7788058/v1/08ae1b6f4a336b5ad82b2a69.pdf"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Bispecific antibody against sclerostin and DKK1 improves bone health and reduces bone marrow adipose tissue accumulation in experimental chronic kidney disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe progressive loss of kidney function that occurs in chronic kidney disease (CKD) leads to disturbed mineral metabolism, with CKD patients commonly presenting with hyperphosphatemia, hypercalcemia, hyperparathyroidism and increased fibroblastic growth factor-23 (FGF23) levels [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. These systemic changes are the primary indicators for the diagnosis of CKD\u0026ndash;mineral bone disorder (CKD\u0026ndash;MBD), which develops in the early stages of CKD. Disease progression results in a myriad of complications including vascular calcification and a wide spectrum of bone disorders characterised by abnormalities in bone quantity and quality, and generally referred to as renal osteodystrophy (ROD) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The prevalence of osteoporosis in CKD patients ranges from 18\u0026ndash;32% and fracture incidence of patients with end-stage kidney disease is 8-fold higher than that of the general population [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA histopathological classification system based on abnormalities in bone turnover, volume and mineralisation is used to diagnose ROD and categorise it into various diseases states. These include high turnover with either fibrosis (osteitis fibrosa) or abnormal mineralisation (mixed disease), and low turnover with either normal (adynamic bone disease) or abnormal mineralisation (osteomalacia) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The stimuli for increased bone remodelling are unclear, but enhanced osteoclastic bone resorption in response to elevated parathyroid hormone (PTH) is associated with a net loss of bone mass despite an accelerating bone turnover [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Rapid remodelling also results in bone that is both immature and poorly mineralised [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Nevertheless, while high-turnover bone lesions induced by secondary hyperparathyroidism are common in late-stage CKD, alternative causative mechanisms are likely as very low serum PTH levels correlate with adynamic bone disease and an increased fracture rate [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This is clinically important, as accumulating evidence suggests that low bone turnover disease is the dominant form of ROD in the early stages of CKD, making it an ideal period to develop targeted therapeutic interventions [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe factors contributing to the development of low bone turnover disease are unclear but may include elevated levels of phosphate, FGF23 and/or protein-bound uremic toxins [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Uremic toxins can induce skeletal resistance to PTH and oxidative stress and mitochondrial dysfunction in osteoblasts/osteocytes [\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Circulating concentrations of sclerostin, a Wnt/β-catenin antagonist, are also increased in CKD patients and animal models, and these changes, which are inversely correlated to estimated glomerular filtration rate in CKD patients, are increased during adynamic bone disease and prior to rises in circulating PTH and FGF-23 [\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The relationship between CKD and another Wnt/β-catenin inhibitor, dickkopf-related protein 1 (DKK1), is less clear, with studies reporting unchanged [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] decreased [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] or increased [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] serum levels.\u003c/p\u003e\u003cp\u003eDisturbed Wnt/β-catenin signalling may contribute to the low bone mass characteristic of ROD. Although Sost-deficient (\u003cem\u003eSost\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) mice have increased bone mass, they have limited protection against nephrectomy-induced cortical and trabecular bone loss [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]; however, the high bone mass of \u003cem\u003eSost\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice at baseline may confound the interpretation of these data. Therefore, approaches using a sclerostin-neutralising antibody (Scl-Ab) such as romosozumab, which is widely recognised to increase bone mineral density (BMD) and reduce fractures in healthy men and postmenopausal women, may be more informative and offer potential therapeutic options to prevent bone loss in CKD [\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eStudies on CKD patients are limited. Two studies reported that romosozumab improved BMD at the lumbar spine, total hip, and femoral neck and reduced the relative risk of new vertebral fractures in postmenopausal women with osteoporosis and mild-moderate CKD [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, in a pre-clinical rat model of progressive ROD, administration of a Scl-Ab provided limited protection to the architecture of the long bones and no improvement in their biomechanical properties [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Interestingly, DKK1 serum levels are raised with prolonged romosozumab treatment and also in patients with sclerosteosis and van Buchem disease [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Similarly, \u003cem\u003eSost\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice and mice administered sclerostin antibody also present with a compensatory increase in the expression of DKK1 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] raising the possibility that elevated DKK1 may attenuate the anabolic effects of sclerostin inhibition and negate major improvement in skeletal health in Scl-Ab-treated mice [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn addition to bone loss, CKD can also lead to bone marrow adipose tissue (BMAT) accumulation in animal models [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and humans [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The mechanism(s) responsible are unclear but may also involve Wnt/β-catenin signalling, which can inhibit adipogenesis and adipocyte-specific gene expression in white adipose tissue [\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Congruently, Scl-Ab treatment reduced BMAT accumulation in ovariectomised rabbits [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] and irradiated [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], diabetic [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] and rosiglitazone-treated [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] mice; however, the changes were modest, suggesting that additional mechanisms may be involved and/or a compensatory increase in DKK1 may blunt the neutralising effects of the Scl-Ab.\u003c/p\u003e\u003cp\u003eThe increased DKK1 expression in response to Scl-Ab treatment may limit the ability of the antibody to protect bone health in CKD mice and decrease BMAT accumulation. Therefore, in this study we tested whether a rodent bispecific antibody (rbsAb) against sclerostin and DKK1 can improve bone health and decrease BMAT accumulation in an experimental model of CKD.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eDisease profile is similar in vehicle- and rbsAb-treated CKD mice\u003c/h2\u003e\u003cp\u003eWe first confirmed the development of the CKD-MBD phenotype in the experimental mice. Dietary adenine-supplementation led to a loss of bodyweight within the first week and by end of the study the CKD mice were ~\u0026thinsp;40% lighter than control (CTRL) mice. This was reflected in a decreased mass of the inguinal and gonadal white adipose tissue depots (iWAT and gWAT, respectively) of the vehicle-treated CKD mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA - C). The CKD mice also presented with hyperphosphatemia and increased BUN, creatinine and calcium (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD - F). Similar effects were observed between CTRL and CKD mice administered the rbsAb. The rbsAb treatment also modestly decreased iWAT and gWAT masses and increased serum phosphate in CTRL mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C, G). Plasma PTH levels were similar in vehicle-treated CTRL and CKD mice but were raised in both CTRL and CKD mice with the rbsAb administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePlasma DKK1 and sclerostin levels are elevated in CKD mice\u003c/h3\u003e\n\u003cp\u003eThe ability of the rbsAb to reduce circulating levels of sclerostin and DKK1 was next investigated. In vehicle-treated mice, plasma levels of sclerostin were higher in CKD compared to CTRL mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Sclerostin concentrations in rbsAb-treated mice were extremely high, reaching supraphysiological concentrations and with no evidence of antibody-mediated neutralisation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). This is likely due to the antibody-sclerostin complex having cross-reactivity with the sclerostin ELISA, as reported previously [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In vehicle-treated mice, CKD increased median DKK1 concentrations by almost 3-fold. However, the unadjusted P value for this relatively large effect (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.0297)\u003c/em\u003e was no longer below the significance threshold after adjusting for multiple comparisons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). Moreover, treatment with the rbsAb strongly and significantly decreased DKK1 concentrations in both CTRL and CKD mice to levels that were indistinguishable from each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). This demonstrates effective DKK1 neutralisation by the rbsAb. Plasma sclerostin and DKK1 levels of vehicle treated CTRL and CKD mice were not correlated to plasma PTH levels (Suppl Figs.\u0026nbsp;1A, B).\u003c/p\u003e\n\u003ch3\u003eRbsAb treatment prevents tibial bone loss in CKD mice\u003c/h3\u003e\n\u003cp\u003eGiven that CKD causes bone loss [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], we next tested if rbsAb treatment was able to prevent bone loss in the tibia and vertebrae of CKD mice. Trabecular structure and BMD of the tibia were compromised in vehicle- but not rbsAb-treated CKD mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA \u0026ndash; F). Moreover, while rbsAb administration increased trabecular BMD, bone volume fraction, thickness and number in the tibia of CTRL mice, these rbsAb effects were even greater in the CKD mice. This resulted in higher values for these skeletal properties in rbsAb-treated CKD mice compared to similarly treated CTRL mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB - E). The decrease in trabecular separation in rbsAb-treated CTRL and CKD mice showed a similar effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). In cortical bone of vehicle-treated CKD mice, bone area, thickness, periosteal perimeter and polar moment of inertia were reduced whereas medullary area, endosteal perimeter and porosity were increased when compared to the tibia of vehicle-treated CTRL mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG - N). RbsAb treatment increased bone area, thickness, periosteal perimeter, porosity and polar moment of inertia but reduced medullary area and endosteal perimeter along the tibial length in both CTRL and CKD mice. The cortical bone changes in response to rbsAb treatment were similar in CTRL and CKD mice and accordingly the magnitude of differences noted between vehicle-treated CTRL and CKD mice were maintained in the rbsAb-treated mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG - N). This suggests that, within long bones, CKD alters the skeletal effects of sclerostin and DKK1 in a bone-type-specific manner. Furthermore, the influence of CKD and/or rbsAb treatment on cortical architecture appeared to be site dependent. For example, differences in cortical porosity were greater in the proximal tibia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eN) whereas more marked differences in cortical thickness were noted in the distal tibia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Neither plasma sclerostin or plasma DKK1 had any correlation with trabecular BV/TV (Suppl Figs.\u0026nbsp;1C, D).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn L4 vertebrae, CKD also had significant effects on trabecular and cortical BMD and bone architecture (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-H). In vehicle-treated mice, only cortical thickness was decreased by CKD, whereas the effects of CKD were more pronounced in rbsAb-treated mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-H). RbsAb administration increased trabecular BMD, bone volume fraction, thickness and number whereas trabecular separation was unaffected (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u0026ndash; E). Similarly, cortical BMD, volume and thickness were also increased in response to the rbsAb (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF \u0026ndash; H). However, the anabolic effects of the rbsAb on vertebral trabecular and cortical bone differed between CTRL and CKD mice: for trabecular bone, the rbsAb had a stronger anabolic effect in CKD than in CTRL mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B, D, E), while the rbsAb effects on cortical bone were stronger in CTRL than in CKD mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF - H). This bone-type specific response, within the vertebrae, to rbsAb treatment is similar to that observed in the tibia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eRbsAb treatment improves tibiae and vertebrae biomechanical properties of CKD mice\u003c/h3\u003e\n\u003cp\u003eAs bone structure and geometry influence biomechanical properties, we next examined the response of the femur and L4 vertebrae to mechanical loading. Femur stiffness, maximum load and work to fracture were all lower in vehicle-treated CKD mice when compared to their respective CTRLs (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, J, L). All biomechanical properties apart from post-yield displacement were increased in rbsAb-treated CTRL and CKD mice and, in all cases, the values from the rbsAb-treated CKD mice remained lower than similarly treated CTRL mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI - M) and reflect the structural changes observed in the cortical bone of the tibia (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG \u0026ndash; M). In contrast, compression loading of L4 vertebrae revealed that stiffness, maximum load, yield load and work to fracture were similar in vertebrae from vehicle-treated CTRL and CKD mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eN - Q) and reflect their similar trabecular and cortical architecture (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u0026ndash; H). Similarity in biomechanical properties was also observed in vertebrae from rbsAb-administered CTRL and CKD mice, albeit all were increased when compared to their respective vehicle-treated mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eN - Q).\u003c/p\u003e\u003cp\u003e\u003cb\u003eGene and proteomic profiling of cortical bone reveals rbsAb-mediated prevention of CKD-induced dysregulation of osteoblast differentiation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further understand the cellular events responsible for bone loss in CKD mice and its prevention by rbsAb administration, we performed gene expression and proteomic analyses of cortical bone. The expression of osteoblast/osteocyte genes was similar in tibial cortical bone of vehicle-treated CTRL and CKD mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA - J), while the effects of rbsAb treatment were variable: it increased the expression of \u003cem\u003eSost\u003c/em\u003e in both CTRL and CKD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI); increased \u003cem\u003eSp7, Bglap, Alpl, Col1a1\u003c/em\u003e and \u003cem\u003eDkk1\u003c/em\u003e in tibiae of CTRL mice but not CKD mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB \u0026ndash; E, J); and increased \u003cem\u003eTnfrsf11b\u003c/em\u003e expression in CKD mice only (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe proteomic analysis by LC-MS/MS revealed that 199 proteins were down regulated and 189 proteins were upregulated in bone from vehicle-treated CKD mice compared to vehicle-treated CTRL mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, C). Moreover, in CKD mice, the expression of 123 proteins was downregulated and 336 proteins were upregulated in bone by the administration of rbsAb compared with vehicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, D). To identify the osteoblast/osteocyte-related pathways implicated in the aetiology of ROD, we performed functional enrichment analysis. From the top 8 ontology terms, the osteoblast differentiation pathway was depleted, whereas both bone resorption and bone remodelling pathways were enriched in vehicle-treated CKD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). The enrichment analysis of CKD mice further indicated that the rbsAb was able to prevent the defective osteoblast differentiation and excessive bone resorption and bone remodelling which is consistent with the increased cortical bone mass observed in the rbsAb-treated mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG \u0026ndash; L). Given that the osteoblast differentiation pathway was notably affected by CKD and rbsAb treatment, we further assessed the expression patterns of some of the significantly altered proteins associated with this pathway (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF \u0026ndash; M). The expression of COL1A1 and TNAP, were both down regulated in vehicle-treated CKD mice but the osteoblast transcription factor, SP7 was unchanged (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF \u0026ndash; H). With regard to Wnt signalling, the expression of LRP5, LRP6 and sclerostin, but not CTNNB1 and DKK1, was decreased in vehicle-treated CKD mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI \u0026ndash; M). The rbsAb increased TNAP, SOST and DKK1 expression which is in accord with the gene expression results (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, I, J).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003e\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cb\u003eRbsAb treatment prevents BMAT accumulation in CKD mice\u003c/b\u003e\u003c/div\u003e\u003cp\u003eWe next quantified BMAT along the tibial length as visualised by microCT imaging of osmium-stained bones to determine if the accumulation of BMAT that occurs in CKD animal models and patients was prevented by rbsAb treatment [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Total BMAT area, or BMAT area normalised to bone marrow area, of vehicle-treated CKD mice was increased compared to vehicle-treated CTRL mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA - C). RbsAb treatment reduced BMAT accumulation along the entire length of both CTRL and CKD tibiae (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA - C). Moreover, rbsAb treatment of CKD mice decreased the relative BMAT volume to levels similar to those observed in vehicle-treated CTRL mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Correlation analysis revealed an inverse relationship between rBMAT and trabecular BV/TV, which is in agreement with previous studies [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Bone marrow adiposity can be decreased by PTH [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], but no correlation was found between plasma PTH and rBMAT (Suppl Fig.\u0026nbsp;1E); this is in broad agreement with prior observations [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Similarly, neither plasma sclerostin nor plasma DKK1 had any correlation with rBMAT (Suppl Figs.\u0026nbsp;1F, G).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eBone marrow adipocyte and osteoclast gene and protein expression are dysregulated in CKD but normalised by rbsAb treatment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe next investigated the cellular and molecular mechanisms responsible for the accumulation of BMAT in CKD and its prevention by the rbsAb. To do so, we assessed the expression of key adipocyte transcription factors and phenotype-specific genes and proteins in bone marrow from CTRL and CKD mice, both with and without rbsAb treatment. In comparison to vehicle-treated CTRL mice, the expression of \u003cem\u003ePparg2\u003c/em\u003e and \u003cem\u003eAdipoq\u003c/em\u003e, but not \u003cem\u003eCebpa\u003c/em\u003e, \u003cem\u003eFabp4\u003c/em\u003e or the lipase-encoding genes, \u003cem\u003ePnpla2\u003c/em\u003e and \u003cem\u003eLipe\u003c/em\u003e, was increased in vehicle-treated CKD mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE - J). RbsAb treatment down regulated \u003cem\u003ePparg2\u003c/em\u003e and \u003cem\u003eAdipoq\u003c/em\u003e expression in CKD mice, resulting in similar levels to those in rbsAb -treated CTRL mice; this is consistent with the effects on relative marrow adiposity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). \u003cem\u003eFabp4\u003c/em\u003e expression was also down regulated by antibody treatment of both CTRL and CKD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Sclerostin can promote the differentiation of bone marrow stem cells into osteoclasts; thus, we also quantified the expression of osteoclast transcription factors and mature osteoclast genes. The expression of the master transcription regulator of osteoclast differentiation, \u003cem\u003eNfatc1\u003c/em\u003e, was similar in CTRL and CKD samples and was not affected by rbsAb treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL). In contrast, the expression of \u003cem\u003eCtsk, Mmp9, Acp5\u003c/em\u003e and \u003cem\u003eTnfsf11\u003c/em\u003e was increased by CKD in vehicle-treated mice and rbsAb treatment reduced Tnfsf11 and Ctsk expression in CKD samples resulting in similar expression levels between CTRL and CKD mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK - O).\u003c/p\u003e\u003cp\u003eWe further interrogated these bone marrow effects using proteomics. The volcano plots (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B) and Venn diagrams (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D) illustrate the global proteomic alterations observed under CKD conditions and the modulatory effects of rbsAb treatment in CKD mice. Functional enrichment analysis of the top 6 ontology terms disclosed that, under vehicle treatment, adipogenesis, osteoclast differentiation and signalling, and bone resorption pathways were enriched in CKD vs CTRL mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). These enriched pathways were depleted by rbsAb treatment of CKD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE) and together these data are in accord with the ability of the rbsAb to protect CKD mice from bone loss and BMAT accumulation (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), respectively. Adipocyte proteins were then analysed, which illustrated upregulation of FABP4, PLIN1, LEPR, ADIPOQ, LPL, LIPE and PNPLA2 in CKD mice. These increases were significantly prevented by rbsAb treatment, thereby cancelling or minimising the difference in their expression levels between CTRL and CKD mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF-L). In addition, proteins associated with osteoclast differentiation and bone resorption were upregulated in vehicle-treated CKD vs CTRL mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eO \u0026ndash; R). This CKD effect was generally prevented by rbsAb administration, resulting in expression levels of NFKB, NFATC and CTSK that were similar between rbsAb -treated CTRL and CKD mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eO \u0026ndash; R). SOST and DKK1 expression were similar in vehicle-treated CTRL and CKD mice but, in agreement with the bone gene expression data (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ, K), the expression of both β-catenin inhibitors was increased by rbsAb treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eM, N).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTherapies for the treatment of ROD are currently focussed on the use of calcitriol and calcimimetics to lower circulating levels of PTH and reduce the adverse skeletal effects of secondary hyperparathyroidism [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. This approach is recommended prior to consideration of other more targeted interventions but as elevated PTH levels are a poor indicator of low bone turnover disease there is a requirement for alternative treatments that can improve bone health regardless of PTH status [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Furthermore, CKD often coexists with osteoporosis in the ageing population, resulting in an increased fracture risk in patients with these combined comorbidities [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. While there are some recognised limitations and caveats, many established therapeutic approaches to osteoporosis can improve bone health in CKD patients [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. In patients with mild to-moderate CKD, bisphosphonates raise BMD and reduce fractures but their administration to patients with pre-existing very low bone turnover is a concern, as is their accumulation in CKD patients with impaired renal clearance [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Other anti-osteoporosis drugs such as denosumab, teriparatide and abaloparatide increase BMD and reduce fracture risk in patients with mild to moderate CKD. However, PTH analogues may aggravate existing hyperparathyroidism and denosumab, like bisphosphonates, can induce hypocalcemia [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan additionalcitationids=\"CR67\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAn attractive alternative approach may involve the neutralisation of sclerostin; an inhibitor of bone formation and recognised to be elevated in bone [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and serum [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] of CKD patients. Romosozumab is a humanised antibody that targets sclerostin resulting in the transient activation of bone formation and inhibition of bone resorption [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Romosozumab can reduce fracture risk in CKD but evidence is limited to a recent post hoc analysis of patients from the phase 3 clinical trials, FRAME and ARCH [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Preclinical studies have also reported that romosozumab can improve bone health in murine models of diabetes and CKD [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. A limitation of targeting sclerostin alone to promote bone formation is the up-regulation of DKK1 expression, which may dampen the bone formation response to sclerostin neutralisation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Therefore, one of the aims of this present study was to examine the ability of a bispecific antibody to sclerostin and DKK1 to improve bone health in a mouse model of CKD.\u003c/p\u003e\u003cp\u003eIt has been widely reported that the structure and biomechanical properties of long bones are compromised in CKD rodents [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR73\" citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. A role for secondary hyperparathyroidism is often implicated in the progression of ROD but structural and mechanical integrity defects are also found in the tibia of this study despite normal levels of circulating PTH. However, in contrast to previous animal studies in which PTH levels were elevated we do not observe structural or biomechanical abnormalities in the vertebrae of the CKD mice [\u003cspan additionalcitationids=\"CR76\" citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. The gene expression and functional pathway analysis suggest that rbsAb treatment promotes osteoblast differentiation and inhibits bone resorption and this consistent with the rbsAb profound effects on both cortical geometry and trabecular architecture of the vertebrae and long bones of CTRL and CKD mice. The efficacy, of the rbsAb to improve biomechanical and structural properties of the tibia of CKD mice is in stark contrast to that observed in a rat model of CKD where the effects of a Scl-Ab on the structure and biomechanical properties of vertebrae and long bones were limited and only observed in low PTH conditions [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. This superior skeletal response in CKD mice to the neutralisation of sclerostin and DKK1 rather than sclerostin alone is consistent with previous studies where a bispecific antibody targeting sclerostin and DKK1 was more effective than monotherapy treatment in increasing BMD, biomechanical properties and bone repair activity in rats and nonhuman primates [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan additionalcitationids=\"CR79\" citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Also, a 3:1 mixture of Scl-Ab and DKK1-Ab promotes the formation of 2\u0026ndash;3 times more trabecular bone than an equivalent dose of Scl-Ab alone despite DKK1-Ab having no consistent osteoanabolic effects [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan additionalcitationids=\"CR82\" citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. The increased cortical porosity in the rbsAb treated mice was however unexpected as previous studies have indicated that when corrected for bone volume, cortical porosity in Sost\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice was similar to wild-type mice [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study we also aimed to verify if the rbsAb was able to reduce the amount of BMAT that is known to accumulate in CKD patients and animal models [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Though Wnt/β-catenin signalling is known to block expression of C/EBPα and PPARγ in white adipocytes and the loss of Wnt/β-catenin signalling causes a cell fate shift of pre-osteoblasts from osteoblasts to adipocytes, less is known about the regulatory effects of this pathway on bone marrow adipocytes (BMad) [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. Sclerostin levels positively associate with higher vertebral marrow fat in men and in cell culture studies, adipogenesis is promoted in sclerostin challenged bone marrow mesenchymal stromal cells [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. Decreased BMAT accumulation in \u003cem\u003eSost\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice provides further evidence that BMad accumulation in CKD could be a consequence of increased sclerostin levels [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. Nevertheless, romosozumab has no effect on bone marrow adiposity of the iliac crest in postmenopausal osteoporotic women and the ability of Scl-Ab to decrease BMAT in irradiated or rosiglitazone treated mice was modest and limited to specific regions of the long bones [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. These subtle, depot specific effects of Scl-Ab on BMad contrast with the results of this present study. Gene and protein expression data suggest an inhibition of bone marrow adipogenesis with rbsAb treatment and this is consistent with the reduced BMAT accumulation along the entire tibia of rbsAb -treated CTRL and CKD mice. The decrease in total BMAT in rbsAb -treated mice is similar to that reported in Scl-Ab-treated normal adult rats [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e] but this reduction may in part be a consequence of an increase in trabecular bone volume and a corresponding reduction in marrow space in which adipocytes can inhabit. However, when normalised to marrow area, BMAT is still decreased in rbsAb -treated mice and suggests that an increase in Wnt/β-catenin signalling has a direct negative effect on BMad, independent of bone changes. When normalised to marrow area a reduction in BMAT was not observed in Scl-Ab-treated rats [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e] and suggests that the ability of the Scl-Ab alone to restore normal amounts of BMAT is impeded by a compensatory increase in the expression of DKK1.\u003c/p\u003e\u003cp\u003eWnt pathway inhibition increases gradually as kidney function declines and is considered an early event in the pathogenesis of CKD [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Nevertheless, the relationship between blood sclerostin and PTH levels in CKD patients and animal models is unclear. A positive, negative or, as found in this present study, no correlation between circulating levels of sclerostin and PTH have been reported [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Although PTH is known to supress \u003cem\u003eSost\u003c/em\u003e and \u003cem\u003eDkk1\u003c/em\u003e expression by bone cells [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e, \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e], a regulatory role for PTH in this study is unclear as circulating PTH levels in the CKD mice is normal. Possibly, the chronic elevation of circulating sclerostin and DKK1 in CKD overwhelms the ability of PTH to regulate their circulating levels leading to PTH resistance and the promotion/aggravation of adynamic bone disease as well as an inability to decrease marrow adipogenesis [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNevertheless, an explanation for the higher circulating levels of sclerostin and DKK1 in CKD remains unclear [\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Reduced renal excretion is unlikely [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and others have reported immunohistochemistry data indicating increased osteocyte expression of sclerostin in CKD [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Alternatively, the increased expression of sclerostin and DKK1 may involve TGF-β, which is elevated in serum and bone in CKD and promotes osteocyte sclerostin expression [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e, \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e]. Furthermore, sclerostin levels are positively correlated with inflammation markers, phosphate and uremic toxins whereas FGF23 can induce DKK1 and inhibit osteoblast Wnt/β-catenin signalling via a soluble Klotho/MAPK\u0026ndash;mediated process [\u003cspan additionalcitationids=\"CR97\" citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e]. The upregulation of \u003cem\u003eSost\u003c/em\u003e and \u003cem\u003eDkk1\u003c/em\u003e expression and their encoded proteins in bone of rbsAb -treated mice may be a consequence of lowered circulating levels of the proteins although for sclerostin we were unable to confirm this due to possible ELISA cross-reactivity with the bispecific antibody [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. A similar increase in osteocyte \u003cem\u003eSost\u003c/em\u003e expression has been observed previously in Scl-Ab-treated CKD rats [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile the rbsAb improves bone health and decreases BMad accumulation in an experimental model of CKD, it is important to recognise that there may be limitations to this possible therapeutic approach. Cardiovascular adverse events have been reported with romosozumab in some (ARCH and BRIDGE) but not all (FRAME) clinical trials [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e]. The presence of sclerostin [\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e, \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e] and DKK1 [\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e] in cardiovascular tissue may protect against vascular calcification and while theoretically romosozumab inhibition may aggravate the progression of vascular calcification in CKD there is no evidence of aortic mineralisation in rats or cynomolgus monkeys treated long-term with romosozumab [\u003cspan additionalcitationids=\"CR104\" citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e]. Furthermore, neutralisation of DKK1 prevents vascular calcification in mice with renal insufficiency [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and to the best of our knowledge, cardiovascular events have not been reported in \u003cem\u003eSost\u003c/em\u003e deficient mice or individuals with sclerosteosis or van Buchem disease [\u003cspan additionalcitationids=\"CR107\" citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e]. Nevertheless, further animal and clinical studies focused on potential vascular effects in the setting of sclerostin and/or DKK1 blockade are required to evaluate the effect of prolonged treatment on cardiovascular health.\u003c/p\u003e\u003cp\u003eIn conclusion, mice fed an adenine enriched diet present with BMAT accumulation, trabecular and cortical bone loss and impaired biomechanical properties. A bispecific antibody to sclerostin and DKK1 was able to improve bone structure and biomechanical properties of bone and suppress BMAT accumulation. These results highlight a novel therapeutic strategy to enhance bone health in patients with CKD and pave the way for future translational applications in ROD management.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eMice\u003c/h2\u003e\u003cp\u003eTo induce CKD, eight-week-old male C57BL/6JCrl mice (Charles River Laboratories, Margate, UK) were fed a diet supplemented with 0.2% adenine for 6-weeks (Envigo, Bicester, UK). Each week the adenine diet was offered for 5-days and replaced by a normal diet for 2 days. This modification of our previous protocol was based on the studies of Lair and colleagues and introduced to induce CKD but avoid pathological weight loss [\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e, \u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e]. The CTRL mice received the same diet without adenine (Envigo). The bispecific antibody against sclerostin and DKK1 (Angitia Biopharmaceuticals, Guangzhou, China) or vehicle (PBS) was administered (30 mg/kg body weight) by subcutaneous injection to CTRL and CKD mice once a week for 6 weeks (n\u0026thinsp;=\u0026thinsp;10/group). Body weights were obtained from mice twice weekly until sacrifice at 14-weeks of age. All mouse studies were approved by the University of Edinburgh Animal Welfare and Ethical Review Board and were conducted under a project license granted by the UK Home Office. Animal studies were conducted and are reported in line with the ARRIVE guidelines.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ePlasma biochemistry\u003c/h2\u003e\u003cp\u003eAll mice were sacrificed after 6-weeks treatment and blood was collected by cardiac puncture under terminal anaesthesia. Plasma creatinine, blood urea nitrogen (BUN), phosphate and calcium were quantified using a biochemistry analyser (Beckman Coulter AU480). Intact PTH (QuidelOrtho, San Diego, USA), sclerostin and DKK1 (R\u0026amp;D Systems, Abbington, UK) were measured by ELISA according to the manufacturers\u0026rsquo; instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eMicro computed tomography (microCT)\u003c/h2\u003e\u003cp\u003eThe changes in trabecular and cortical bone structure and BMD of L4 vertebrae (unfixed) and left tibia (fixed in 10% formaldehyde for 24 hours) were assessed by microCT (NeoScan N80, Mechelen, Belgium). Briefly, the bones were scanned with an isotropic voxel size of 5 \u0026micro;m (60 kV, 167 \u0026micro;A and 0.5 mm aluminium filter, 0.6\u0026deg; rotation angle) and the scans were reconstructed using the NRecon 1.7.3.0 program (Bruker, Kontich, Belgium) to remove artefacts, including beam-hardening and ring artefacts. CTAn software 1.15.4.0 (Skyscan, Kontich, Belgium) was used to evaluate bone histomorphometric parameters. To generate three-dimensional (3D) images, the scans were reconstructed and 3D images were created using the Neoscan80 software package (NeoScan).\u003c/p\u003e\u003cp\u003eFor L4 vertebrae, a 300-slice subset through the middle of the vertebrae\u0026rsquo;s body was analysed and the following trabecular [bone mineral density (BMD; g/cm\u003csup\u003e2\u003c/sup\u003e), bone volume/tissue volume (BV/TV; %), thickness (Tb. Th; mm), number (Tb. N; 1/mm) and separation (Tb. Sp; mm)] and cortical [BMD; g/cm\u003csup\u003e2\u003c/sup\u003e, bone volume (BV; mm\u003csup\u003e3\u003c/sup\u003e) and Th (mm)] parameters were calculated [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Each tibia was aligned along its longitudinal axis and the trabecular volume of interest (VOI) in the proximal metaphysis was a 1000-\u0026micro;m section of the metaphysis, 250 \u0026micro;m subjacent to the growth plate. The same trabecular parameters measured in the L4 vertebrae were quantified in the tibial reconstructions. For cortical analysis of the tibia, the proximal and distal portions were digitally cropped to exclude the epiphysis, growth plates and trabecular bone from the analysis [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Bone area (B.Ar; mm\u003csup\u003e2\u003c/sup\u003e), thickness (Th; mm), polar moment of inertia (\u003cem\u003eJ\u003c/em\u003e; mm\u003csup\u003e4\u003c/sup\u003e), medullary area (Med.Ar; mm\u003csup\u003e2\u003c/sup\u003e), periosteal perimeter (P.Pm; mm), endosteal perimeter (E.Pm; mm) and porosity (%) were determined. Hydroxyapatite phantoms of known densities (0.25 and 0.75 g/cm\u003csup\u003e3\u003c/sup\u003e) were scanned and reconstructed under identical conditions as the experimental samples to allow the calculation of BMD. R studio was used to create the line graph of the cortical bone parameters along the tibia length [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eQuantification of BMAT\u003c/h2\u003e\u003cp\u003eAfter initial microCT scanning, the bone marrow adipocytes within the left tibiae were stained with osmium tetroxide as previously described [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In brief, the decalcified bones were incubated with 1% osmium tetroxide for 48h, washed and stored in Sorensens\u0026rsquo; buffer at 4\u0026deg;C. The osmium-stained bones were re-scanned by microCT and total BMAT area was calculated as well as being normalised to the size of the bone marrow cavity with trabecular bone was excluded. BMAT volume was quantified in two distinct anatomical regions: the growth plate to tibia/fibula junction (GP-T/F J), which contains regulated BMAT (rBMAT); and the tibia/fibula junction to the end of distal bone (T/F J-End), which contains constitutive BMAT (cBMAT) [\u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e]. R studio was used to create the line graph of BMAT accumulation along the tibia length.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eBiomechanical testing of tibia and vertebrae\u003c/h2\u003e\u003cp\u003eThe L4 vertebrae and right femora were stored, unfixed, at \u0026minus;\u0026thinsp;20\u0026deg;C in water and their biomechanical properties were evaluated by a LS5 Lloyds materials testing machine with NEXYGEN Plus software (Ametek, Leicester, UK). For the 3-point bending, the femora were positioned horizontally on custom supports and a 100N load cell was applied perpendicular to the mid-diaphysis at a speed of 10 mm/min. For compression loading, the vertebral body was isolated from the spinal processes and prepared with flat and parallel ends using a polishing wheel and finally bonded to a fixed bottom plate with cyanoacrylate glue. A 500N load cell at a speed of 10 mm/min, compressed the vertebra. Each femur and vertebra were tested to fracture and data recorded after every 0.2 N change in load. The load\u0026ndash;displacement curve for each bone was analysed, and stiffness, maximum load, yield load, work to fracture and post-yield displacement were calculated [\u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e112\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eRT-qPCR\u003c/h2\u003e\u003cp\u003eThe proximal and distal ends of the right tibiae were removed and the bone marrow was flushed out by centrifugation. Both the tibial shaft and the bone marrow were snap frozen in liquid nitrogen and stored at -80\u003csup\u003e0\u003c/sup\u003eC. The tissue was homogenised by a Rotor-Stator Homogenizer and RNA was extracted using a Qiagen RNeasy Mini kit (Qiagen, Manchester, UK) and quantified by nanodrop spectrophotometry (Thermo Fisher Scientific, Loughborough, UK). RNA quality was evaluated by the 260/280 nm ratio. After reverse transcription, gene expression was quantified using the SYBER green method and an Agilent Aria 2.1 real-time qPCR system (Agilent Technologies, Cheadle, UK). Target gene expression was normalised to a housekeeping gene (\u003cem\u003ePpia\u003c/em\u003e) and analysed using the ΔΔCt method. Oligonucleotide primers (Supp. table 1) were obtained from Sigma-Aldrich (Gillingham, UK) and Thermo Fisher Scientific.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eProteomics\u003c/h2\u003e\u003cp\u003eBoth proximal and distal ends of the right tibia were removed and bone morrow isolated by centrifugation. Cortical bone and bone marrow tissue were homogenised by a Rotor-Stator Homogenizer in a 100 \u0026micro;l of lysis buffer containing 5% sodium deoxycholate, 100 mM Tris-HCl (pH 8.5), 1 mg/mL chloroacetamide, and 1.5 mg/mL tris(2-carboxyethyl)phosphine. The lysates were subsequently heated at 95\u0026deg;C for 15 minutes. Protein capture and digestion from bone and bone marrow lysates were performed using an automated KingFisher Flex system (Thermo Fisher Scientific) [\u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e113\u003c/span\u003e]. Briefly, proteins were captured using MagReSyn HILIC magnetic microspheres (ReSyn Biosciences, Pretoria, South Africa). Protein-bound beads were then digested with 0.5 \u0026micro;g of MS-grade trypsin (Thermo Fisher Scientific) in 50 mM triethylammonium bicarbonate buffer (Sigma-Aldrich) at 37\u0026deg;C. The tryptic peptides were sequentially washed with 95% (v/v) acetonitrile (ACN) and 70% ethanol (EtOH). Digestion was terminated by acidification with 2% formic acid. Peptides were subsequently desalted using a C18-based desalting procedure, eluted with 0.1% trifluoroacetic acid (TFA) in 50% ACN, and dried by speed vacuum. Thereafter, dried peptides were reconstituted in 0.1% TFA for subsequent mass spectrometry analysis.\u003c/p\u003e\u003cp\u003eOne microgram of desalted peptides was loaded onto 25 cm Aurora columns (IonOptiks, Australia) using an RSLC nano \u003cem\u003e\u0026micro;\u003c/em\u003eHPLC system coupled to a Fusion Lumos mass spectrometer. Peptide separation was achieved with a 70-min linear gradient ranging from 5% to 30% acetonitrile in 0.5% acetic acid. The mass spectrometer was operated in data-independent acquisition (DIA) mode, collecting MS scans from 350\u0026ndash;1650 Da at 120k resolution, followed by MS/MS acquisition across 45 windows with 0.5 Da overlap (200\u0026ndash;2000 Da range) at 30k resolution and a normalized collision energy (NCE) of 28. The raw data were processed using DIA-NN 2.0 software with spectral matching performed against the Mus musculus UniProt protein database [\u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e114\u003c/span\u003e]. Normalization was conducted based on the total peptide abundance across LC-MS runs. GSEA was done using GSEA software (version 4.4.3, Broad institute, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM or as violin plots, as indicated in the figure legends, with individual data points representing each biological replicate. Statistical analysis was performed using a two-way analysis of variance (ANOVA) to determine the effect of the rbsAb treatment and CKD status on bone and BMAT alterations. Correlations between individual parameters were performed using Spearman correlation. As the plasma DKK1 levels were not normally distributed the data were analysed using the Mann\u0026ndash;Whitney test with Bonferroni correction for multiple comparisons. Statistical analysis was implemented using GraphPad Prism software (GraphPad Software, Inc., USA) and R studio (for cortical bone and BMAT analysis) and statistical significance was shown as; * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001. For differential expression of proteomic profile, the data were processed using Perseus software [\u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e115\u003c/span\u003e]. Log₂ transformation was applied to the data, and missing values were imputed based on a normal distribution. Pairwise comparisons between groups were conducted using a two-sample t-test. Proteins exhibiting a p-value of less than 0.05 and a fold-change greater than 1.5 were considered significantly altered between the compared groups.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe proteomics data including raw files and search results have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD069117.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interest\u003c/h2\u003e\n\u003cp\u003eHZK and XL are employees of Angitia Biopharmaceuticals (Angitia Incorporated Limited). All other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eConceptualisation: WP, CF, WPC, KAS, LAS, HZK, XL Methodology: WP, SNJ, CC, AVK, HZK, XL. Formal analysis: WP, CF, CC, AVK, WPC, KS, LAS. Writing\u0026mdash;original draft: WP and CF. Writing\u0026mdash;review and editing: all authors. Visualisation: WP, CC. Supervision: KAS, LAS, WPC and CF. Funding acquisition: WP and CF. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe thank Heather Warnock and the staff of the Biological Research Facility (BRF) at the University of Edinburgh for providing invaluable animal support, Colin Wood and the staff at Easter Bush Pathology, Royal (Dick) School of Veterinary Studies, University of Edinburgh for conducting mouse serum biochemistries. We acknowledge financial support from Chulabhorn Royal Academy to WP and the Biotechnology and Biological Sciences Research Council (BBSRC) for supporting LAS via a Discovery Fellowship (BB/X009904/1) and for Institute Strategic Programme Grant Funding (BBS/E/RL/230001C) to CF and SNJ. We also acknowledge the Medical Research Council for funding to KAS (MR/V033506/1 and MR/R022240/2) and WPC (MR/M021394/1 and MR/S010505/1). For the purpose of open access, the authors have applied a CC-BY public copyright license to any Author Accepted Manuscript version arising from this submission.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMoe, S., et al., \u003cem\u003eDefinition, evaluation, and classification of renal osteodystrophy: a position statement from Kidney Disease: Improving Global Outcomes (KDIGO)\u003c/em\u003e. Kidney Int, 2006. 69(11): p. 1945\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFang, Y., et al., \u003cem\u003eEarly chronic kidney disease-mineral bone disorder stimulates vascular calcification\u003c/em\u003e. Kidney Int, 2014. 85(1): p. 142\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGraciolli, F.G., et al., \u003cem\u003eThe complexity of chronic kidney disease-mineral and bone disorder across stages of chronic kidney disease\u003c/em\u003e. Kidney Int, 2017. 91(6): p. 1436\u0026ndash;1446.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbdalbary, M., et al., \u003cem\u003eManagement of osteoporosis in patients with chronic kidney disease\u003c/em\u003e. Osteoporos Int, 2022. 33(11): p. 2259\u0026ndash;2274.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOtt, S.M., \u003cem\u003eHistomorphometric measurements of bone turnover, mineralization, and volume\u003c/em\u003e. Clin J Am Soc Nephrol, 2008. 3 Suppl 3(Suppl 3): p. S151-6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJadoul, M., et al., \u003cem\u003eIncidence and risk factors for hip or other bone fractures among hemodialysis patients in the Dialysis Outcomes and Practice Patterns Study\u003c/em\u003e. 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Calcif Tissue Int, 2023. 113(4): p. 449\u0026ndash;468.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBatth, T.S., et al., \u003cem\u003eProtein Aggregation Capture on Microparticles Enables Multipurpose Proteomics Sample Preparation\u003c/em\u003e. Mol Cell Proteomics, 2019. 18(5): p. 1027\u0026ndash;1035.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDemichev, V., et al., \u003cem\u003eDIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput\u003c/em\u003e. Nat Methods, 2020. 17(1): p. 41\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTyanova, S., et al., \u003cem\u003eThe Perseus computational platform for comprehensive analysis of (prote)omics data\u003c/em\u003e. Nat Methods, 2016. 13(9): p. 731\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bone-research","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"boneres","sideBox":"Learn more about [Bone Research](http://www.nature.com/boneres/)","snPcode":"41413","submissionUrl":"https://mts-boneres.nature.com/cgi-bin/main.plex","title":"Bone Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7788058/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7788058/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChronic kidney disease (CKD) leads to bone loss and bone marrow adipose tissue (BMAT) accumulation. Sclerostin and dickkopf-1 (DKK1) are two inhibitors of Wnt signalling, which suppress bone formation, promote bone marrow adipogenesis, and are elevated in CKD. However, therapies targeting sclerostin have shown limited efficacy in improving bone health in CKD animal models. Herein, we explored whether dual inhibition of sclerostin and DKK1 via a rodent bispecific antibody (rbsAb) could prevent bone loss and suppress BMAT accumulation in a CKD mouse model. CKD was induced using an adenine-supplemented diet in male mice, with CKD and control mice treated weekly for 6-weeks with vehicle or 30 mg/kg body weight of rbsAb. Circulating sclerostin and DKK1 were ~\u0026thinsp;2- and ~\u0026thinsp;3-fold higher, respectively, in CKD mice compared to controls. Proteomic profiling by LC-MS/MS and functional enrichment analysis suggested that in CKD mice, adipogenesis, osteoclast differentiation and bone resorption were increased whereas osteoblast differentiation was inhibited. These changes were prevented by antibody treatment. MicroCT revealed that long bones of CKD mice were characterised by lower bone mineral density, trabecular and cortical bone, and impaired biomechanical properties, but their vertebrae were unaffected. RbsAb treatment prevented cortical and trabecular bone loss and restored biomechanical properties. BMAT, as visualised by microCT imaging of osmium-stained bones, was elevated in CKD but reduced to control levels by rbsAb treatment. In conclusion, dual inhibition of sclerostin and DKK1 improved bone integrity and suppressed BMAT in experimental CKD, suggesting a promising therapeutic avenue for renal osteodystrophy.\u003c/p\u003e","manuscriptTitle":"Bispecific antibody against sclerostin and DKK1 improves bone health and reduces bone marrow adipose tissue accumulation in experimental chronic kidney disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-06 16:39:53","doi":"10.21203/rs.3.rs-7788058/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-12-04T08:23:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-12-02T18:00:48+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-11-24T02:57:51+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-11-21T15:08:31+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-11-12T18:27:23+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-29T18:15:10+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-27T11:50:59+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-10-27T08:56:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-06T10:12:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-06T05:12:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bone Research","date":"2025-10-06T05:12:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bone-research","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"boneres","sideBox":"Learn more about [Bone Research](http://www.nature.com/boneres/)","snPcode":"41413","submissionUrl":"https://mts-boneres.nature.com/cgi-bin/main.plex","title":"Bone Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"12b7ef19-c4fb-4ea9-8889-e7c6b488ada9","owner":[],"postedDate":"November 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":55822890,"name":"Biological sciences/Physiology/Bone quality and biomechanics"},{"id":55822891,"name":"Health sciences/Diseases/Endocrine system and metabolic diseases/Metabolic syndrome"}],"tags":[],"updatedAt":"2026-05-08T09:12:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-06 16:39:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7788058","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7788058","identity":"rs-7788058","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}