Sclerostin and DKK1: new players in renal bone and vascular disease

http://www.kidney-international.org 2015 International Society of Nephrology mini review and DKK1: new players in renal bone and vascular disease Pieter Evenepoel 1, Patrick D Haese 2 and Vincent Brandenburg 3 1 Laboratory of Nephrology, Department of Immunology and Microbiology, KU Leuven, and Department of Nephrology and Renal Transplantation, University Hospitals Leuven, Leuven, Belgium; 2 Laboratory of Pathophysiology, Department of Biomedical Sciences, Antwerp University, Wilrijk, Belgium and 3 Department of Cardiology, University Hospital RWTH Aachen, Aachen, Germany For more than a decade, the Wnt enin pathway has been the focus of intense basic and clinical research in the bone field because of its importance in skeletal development and maintenance of bone mass. Wnt activation increases bone formation and decreases bone resorption. The Wnt βcatenin signaling pathway is tightly regulated by several inhibitors, among which Dickkopf-related protein 1 (DKK1) and sclerostin have been most comprehensively studied. Mounting evidence indicates that a disturbed Wnt enin signaling is also implicated in the pathogenesis of the chronic kidney disease associated bone and mineral disorder (CKD-MBD) and affects its various components. DKK1 and sclerostin, more specifically, may be involved in the intense cross-talk between the kidneys, vasculature, and bone. Studies exploring clinical correlates of circulating sclerostin and DKK1 levels so far yielded conflicting results. Biological variability and analytical issues account at least partly for this inconsistency. Antibodies neutralizing Wnt inhibitors may be an appealing strategy to prevent or treat CKD-MBD. Caution is however warranted as sclerostin not only opposes mineralization in the bone but possibly also in the vasculature. Additional studies are required to define determinants of Wnt inhibitors in CKD and to evaluate the efficacy and safety of recently introduced pharmaceuticals targeting these inhibitors. Kidney International (2015) 88, 235 240; doi:10.1038/ki.2015.156; published online 17 June 2015 KEYWORDS: bone; cardiovascular disease; DKK1; mineral metabolism; sclerostin Chronic kidney disease (CKD) is a pandemic affecting 410% of the Western population. CKD is associated with high rates of cardiovascular mortality, making it more likely that affected individuals will be prone to cardiovascular morbidity and mortality than they would evolve toward end-stage renal disease requiring renal replacement therapy. CKD also confers an increased fracture risk. 1 Data from a large cohort of patients of the Third National Health and Nutrition Examination Survey in the United States demonstrate that compared with individuals with an estimated glomerular filtration rate of 60 ml/min, patients with an estimated glomerular filtration rate of <60 ml/min had a twofold increased risk of hip fracture. The risk for fractures further increases up to fourfold in dialysis patients as compared with age- and gender-matched non-ckd individuals. 2 CKD patients with fractures moreover have increased rates of death and hospitalization. 3 The CKD mineral bone disorder (CKD-MBD) is a new acronym coined in 2006 in recognition that the skeletal (renal osteodystrophy) and mineral disorders caused by CKD are critical contributors to the high cardiovascular morbidity and mortality and fracture rates. Similar to what is observed in the general population, low bone tissue mineralization inversely associates with pathological cardiovascular calcification in CKD. Despite its vast clinical significance, the precise nature of this reciprocal relationship remains obscure. 4 CKD-MBD develops early in the course of kidney disease and is already clinically detectable in stage 2 CKD. Increasing evidence indicates that circulating Wnt (Wingless) signaling inhibitors may play a crucial role in the pathogenesis of CKD- MBD. This mini-review aims to present an update on the role of Wnt signaling inhibitors in CKD-MBD and to identify current gaps in our knowledge. Correspondence: Pieter Evenepoel, Dienst Nefrologie, Universitair Ziekenhuis Gasthuisberg, Herestraat 49, Leuven B-3000, Belgium. E-mail: pieter.evenepoel@uzleuven.be Received 30 January 2015; revised 16 March 2015; accepted 30 March 2015; published online 17 June 2015 FUNDAMENTALS OF WNT SIGNALING Wnts are a large family of secreted glycoproteins that trigger multiple signaling cascades essential for embryogenic development and tissue generation. The (canonical) Wnt βcatenin pathway emerged as the predominant component of Wnt signaling with profound effects on the skeleton. 5 Essentially, canonical signaling is initiated by the binding of Wnt ligands to the dual receptor complex comprising Kidney International (2015) 88, 235 240 235

mini review P Evenepoel et al.: and DKK1 Canonical pathway DKK-1 Wnt Frizzled Wnt Frizzled Wnt Frizzled LRP5/6 Dsh APC Axin GBP GSK3 KRM DKK-1 LRP5/6 Dsh Rapid endocytosis of LRP5/6 LRP5/6 binds to LRP5/6 Dsh TCF/LEF APC Axin GSK3 P APC Axin GSK3 P Targets TCF/LEF TCF/LEF Transcription of target genes No transcription of target genes No transcription of target genes Wnt: LRP5/6: Dsh: APC: : Wingless-type MMTV (mouse mammary tumor virus) integration site family member Low density lipoprotein receptor related protein 5/6 Dishevelled Adenomatous polyposis coli Beta-caterin TCF: T-cell factor LEF: Lymphoid enhancing factor GSK3: Glycogen synthase kinase 3 GBP: GSK3 binding protein Dkk-1: Dickkopf-related protein-1 KRM: Kremen (kringle containing transmembrane protein) Figure 1 The canonical Wnt enin signaling pathway and its extracellular regulation. (Left panel) Extracellular binding of Wnt to the Frz LRP5/6 receptor complex causes intracellular accumulation of enin that can induce the expression of target genes after translocation to the nucleus. (Middle panel) DKK1 dampens Wnt signaling by forming a tertiary complex with LRP5/6 and the cell surface co-receptor, Kremen-1 (KRM), thereby promoting internalization of the receptor complex. (Right panel) inhibits Wnt-induced signaling by binding to LRP5/6, thereby preventing Wnt to bind to the Frz LRP5/6 receptor complex. frizzled (FZD) protein and either low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6). This results in inactivation of the multiprotein enin destruction complex, thus relieving the central signaling mediator enin from its constitutive proteosomal degradation. β-catenin subsequently accumulates in the cytoplasm and translocates into the nucleus, where it associates with transcription factors to control target gene transcription. Current evidence indicates that Wnt directly affects both the osteoblast and the osteoclast bone cell lineages and also indirectly affects these cells through cross-talk in the bone environment, inducing an overall increase in osteoblastogenesis together with a decrease in osteoclastogenesis, thus resulting in an enhanced bone formation and reduced bone resorption. 6 The Wnt enin signaling pathway is tightly regulated by several antagonists (Figure 1). These inhibitors either directly bind and block Wnt ligands, preventing their interactions with receptors (for example, secreted frizzledrelated protein and Wnt inhibitory factor), or bind to LRP5/6, inducing receptor internalization and/or reducing their availability to Wnt ligands (for example, Dickkopf factors and sclerostin). Among the numerous Wnt inhibitors, Dickkopf-related protein 1 (DKK1) and especially sclerostin have been studied most intensively. DKK1 (26 kd) and sclerostin (28 kd) are small secreted glycoproteins. Their expression occurs in the limb bud during embryogenesis and continues postnatally in the established skeleton within osteoblasts (DKK1) and osteocytes (sclerostin and DKK1). 6 DKK1 dampens Wnt signaling by binding to LRP5/6 and the cell surface co-receptor, Kremen-1, thereby promoting internalization of the receptor complex. inhibits Wnt-induced signaling by binding to LRP5/6. not only alters bone formation and mineralization, but also influences serum concentrations of hormones that regulate mineral accretion, including calcitriol and fibroblast growth factor 23 (FGF23). 7 Rodent genetic modification models and human genetic mutations teach us the role of various receptors, messengers, and modulators of the Wnt signaling pathway in skeletal health and disease. 5,6 Loss-of-function mutations in LRP5/6 decrease bone mineral density (BMD), whereas gain-offunction mutations of LRP5 increase BMD. Increased BMD is observed in individuals with the high bone mass disorder sclerosteosis that results from loss-of-function mutations in the sclerostin encoding gene SOST. Another high bone mass disorder, Van Buchem disease, that has a phenotype similar in many aspects to sclerosteosis is caused by a 52-kb deletion of genomic DNA 35 kb downstream SOST. It has been hypothesized that sclerostin exerts more specific regulatory actions within the Wnt cascade, whereas DKK1 is more a pan-wnt inhibitor encompassing different Wnt pathways. Accordingly, notable differences exist between the two rodent knockout models SOST / and DKK1 /. 236 Kidney International (2015) 88, 235 240

P Evenepoel et al.: and DKK1 mini review Although SOST / mice present a macroscopically rather unremarkable phenotype with the predominant characteristic finding of strong bones, DKK1-null mutant embryos lack head structures anterior of the midbrain that is associated with premature death. 5 The regulation of sclerostin and DKK1 expression in bone is complex and only partly understood. Important regulators include growth factors and hormones that affect bone formation. Calcitonin and bone morphogenetic proteins stimulate, whereas parathyroid hormone (PTH) 8 and estrogens 9 suppress the expression of sclerostin and/or DKK1. The latter most probably explains why sclerostin levels on average are lower in females. 10 12 Circulating Wnt signaling inhibitors may be modulated by glucocorticosteroids, but their impact is not unequivocal. 5 DKK1 and sclerostin are upregulated in diabetes 13 and by mediators of inflammation. 14 DKK1 and sclerostin expression in osteocytes are decreased during skeletal mechanical loading. 6 Signal transduction during mechanical stress resulting in an anabolic response allowing the bone to adapt to changing mechanical requirements is one of the most important functions of osteocytes. Matrix mineralization activates sclerostin expression by yet undefined signaling mechanisms. 5 In human iliac biopsies, sclerostin-negative osteocytes were nearer to bone surfaces, suggesting that sclerostin expression is related to osteocyte and/or bone age. 15 Increased bone age, decreased renal clearance, and decreased mechanical bone loading may also explain why sclerostin levels increase with aging. 16 The expression of sclerostin and DKK1 is not restricted to bone. 5 DKK1 expression has been shown in skin, placenta, prostate, kidney, and platelets, with lower levels of expression observed in endothelium and other tissues. Extraosseous sclerostin expression has been observed in calcifying vascular smooth muscle cells and aortic valves 17,18 and to a limited extent in the kidney and in articular chondrocytes. 5 Wnt enin signaling emerged to be important in vascular (patho)biology as well. 19 Importantly, the effects of Wnt enin signaling in cardiovascular tissue are cell and stage specific. 20 Overall, Wnt enin signaling promotes atherogenesis and vascular calcification. 19,21 Experimental 17,22 and clinical 23 evidence suggests that the expression of Wnt antagonists in calcifying vascular smooth muscle cells, including sclerostin, 17 DKK1, 21,24 26 and secreted frizzledrelated proteins, 22 may represent a counterregulatory mechanism to avoid further progression of ossification. Of note, this defensive response is only observed in late-stage vascular disease. Furthermore, DKK1 promotes endothelial mesenchymal transition and as such increases the pool of mesenchymal progenitors in the vascular smooth muscle cell lineage available for tissue repair. 20 WNT SIGNALING INHIBITORS IN CKD-MBD For more than a decade, the Wnt enin signaling pathway has been the focus of intense research in the bone field because of its importance in skeletal/bone development, bone mass maintenance, and therapeutic potential. In recent years, the nephrology community has also gained interest in this pathway. A better knowledge of this pathway and its inhibitors may open new avenues for the prevention and treatment of CKD-MBD. Determinants and correlates of circulating sclerostin Serum sclerostin levels increase along the progression of CKD to reach levels that are two- to fourfold higher in patients with end-stage renal disease as compared with individuals with normal renal function. 11,27 32 levels may be elevated in response to reduced renal clearance, as has been shown for many other (small) proteins. This hypothesis is refuted by a recent clinical study in 120 patients with CKD stages 1 5 showing increased absolute and fractional urinary excretion of sclerostin with declining kidney function. 29 Alternatively, increased circulating levels in CKD may be the consequence of enhanced production. In the jck mouse, a genetic model of polycystic kidney disease that exhibits progressive renal disease, an increase in bone sclerostin expression was observed already in early stages of disease. 33 The mechanisms responsible for this increased skeletal expression of sclerostin in early CKD are not known. It may be related partly to increased calcitonin exposure. 12 A disturbed phosphate metabolism may also be implicated in its increased expression. 34 In a CKD animal model of adynamic bone disease, high dietary phosphate intake was associated with a high osteocytic expression of SOST. 35 Moreover, a significant and independent positive association has been observed between serum phosphate, FGF23, and sclerostin concentrations in different cohort studies. 11,27,30 Register et al. 36 speculated that high levels of circulating sclerostin might be indicative of impaired receptor binding and therefore reduced local skeletal activity. Finally, as vascular calcification tends to progress with declining kidney function, extraskeletal (vascular) production might increase accordingly and thus contribute to the high circulating levels in patients with advanced CKD. 23 It is of note that in a recent bone biopsy study, 37 increased circulating sclerostin levels in the elderly were not accompanied by an increase in sclerostin mrna levels in bone, fueling the hypothesis that the contribution of extraskeletal sources, including the vasculature, to circulating levels may be relevant. Circulating sclerostin levels rapidly decrease following renal transplantation in parallel to the recovery of renal function 38 (P Evenepoel et al., unpublished data). In line with experimental data showing suppressed SOST expression by PTH, circulating sclerostin levels were observed to correlate negatively with serum PTH levels in both the general population and dialysis patients 28,39,40 and to significantly increase following parathyroidectomy 41 (P Evenepoel et al. unpublished data). Of note, high levels of PTH and sclerostin coexist in CKD. This observation raises the suspicion that sclerostin contributes to the well-known PTH resistance in CKD. Multiple mechanisms have been shown to contribute to PTH resistance in CKD including Kidney International (2015) 88, 235 240 237

mini review P Evenepoel et al.: and DKK1 oxidative modification of the PTH peptide and downregulation of the PTH receptor. Circulating sclerostin levels associate with several indices of bone and vascular disease and with mortality in CKD patients. 10,11,27,28,32,39,40 and bone disease. Similar to what has been observed in the general population, 16,36,42 high circulating sclerostin levels in CKD patients 10,32 correlate with higher BMD and better bone microarchitecture. Acknowledging that sclerostin is an inhibitor of bone formation, this finding is perplexing and awaits a plausible explanation. It may be hypothesized that high sclerostin levels reflect increased osteocyte number and skeletal mass. 16,31 Both serum and bone sclerostin correlate negatively with the histomorphometric parameters of bone turnover and osteoblastic number in dialysis patients. 13,28 In line with this observation, circulating sclerostin levels were found to correlate negatively with biomarkers of bone formation 31,39 and resorption. 31 High sclerostin levels may explain why persistently elevated sclerostin levels in CKD patients with mild hyperparathyroidism could eventually overcome osteitis fibrosa and lead to adynamic bone disease. The exact value of sclerostin as a biomarker of bone turnover remains yet to be established. and vascular disease. In most but not all crosssectional observational studies, circulating sclerostin levels positively associate with cardiovascular calcification, at least in univariate analysis. 23,43 Remarkably, in some cohorts, adjustment for covariates reversed this association. 23 This supports the notion that sclerostin expression represents a counterregulatory mechanism aimed to suppress the progression of vascular calcification. 22 This also aligns with the physiological role of sclerostin, that is, the downregulation of matrix mineralization. Additional longitudinal observational and intervention studies are required for definite confirmation. Data on a possible correlation of sclerostin with arterial stiffness (as assessed by flow-mediated dilatation and pulse wave velocity) are limited and inconclusive. 32,44 and mortality. Studies evaluating the association between circulating sclerostin levels and (cardiovascular) mortality in CKD have yielded inconsistent results, with some investigators reporting high circulating sclerostin levels to associate with better survival 39,40 and other investigators reporting an inverse 12,27 or no 30 association. These conflicting data may be explained by case-mix, use of different assays, and different competing factors used in multivariate analysis. Moreover, the relationship may not be linear, but U-shaped. Additional clinical and experimental studies are required to establish the true nature of the relationship between sclerostin and survival and to elucidate the underlying pathophysiological mechanisms. Determinants and correlates of circulating DKK1 Compared with sclerostin, data about correlations and associations of DKK1 in the setting of CKD-MBD are sparse. Circulating DKK1 levels, in contrast to sclerostin, are only minimally affected by gender, renal function, and age 32,45,46 and do not correlate with BMD and bone histomorphometry parameters. 28,44 Most, but not all, studies report a negative association between circulating DKK1 and vascular calcification, 44 46 similar to what has been observed with sclerostin in the adjusted models. Data on the association between DKK1 and arterial stiffness are inconclusive. 32,44 Kidney Kidney injury DKK1 Adynamic bone Bone antibody Volume Mineralization Osteochondrogenic transdifferentiation Vascular smooth muscle cell Osteoblastlike cell Vascular calcification antibody Mortality Blood vessels? Figure 2 and the bone vascular axis. There is intense cross-talking between the kidneys, the vasculature, and the bone. Chronic kidney disease (CKD) goes along with an increased incidence of vascular calcification and the development of adynamic bone disease. Increased renal production and circulating levels of Dickkopf-related protein 1 (DKK1) in CKD have been associated with osteochondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs), vascular calcification, and renal osteodystrophy. However, it has also been postulated that Wnt inhibitors, in particular sclerostin produced in the vascular wall, may not only have beneficial paracrine effects (retard the progression of vascular calcification) but also, when spilled over to the circulation, induce negative endocrine effects on the skeleton (decreased osteoblastogenesis and increased osteoclastogenesis). Reciprocally, the role of skeletal sclerostin in vascular pathobiology also remains to be defined. Both adynamic bone disease and vascular calcification have been associated with an increased mortality in patients with CKD. 238 Kidney International (2015) 88, 235 240

P Evenepoel et al.: and DKK1 mini review There are no published data on the relationship between circulating DKK1 levels and mortality. Wnt inhibitors and the kidney bone vascular axis Mounting evidence indicates that secreted Wnt antagonists play an important role in the cross-talk between the kidneys, the vasculature, and the bone (Figure 2). 17,22,23,46,47 Fang et al. 47 showed increased renal production of DKK1 and sclerostin and increased circulating DKK1 levels in a mouse model of CKD. Neutralization of DKK1 by a monoclonal antibody prevented osteochondrogenic transdifferentiation of vascular smooth muscle cells, vascular calcification, and renal osteodystrophy. These findings are intriguing and call for additional studies. Special caution is warranted when translating these data to the clinical setting. For example, elevated DKK1 levels and downregulated vascular klotho, both key findings in the abovementioned mouse model, are not universally observed in patients with (early stage) CKD. 32,48 A large number of studies demonstrated a relationship between vascular disease and bone pathology. 4 The coexistence of osteoporosis and vascular calcification has been consistently demonstrated in postmenopausal women and elderly people 49 and also in patients with CKD. 50 This bone vascular axis involves bone-derived proteins that regulate vascular calcification and proteins of vascular origin that regulate bone metabolism. 4 Clinical 23,46 and experimental 17,22 data suggest that Wnt inhibitors may be involved in these bone vascular interactions. An appealing hypothesis is that Wnt inhibitors produced in the vascular wall may not only have beneficial paracrine effects (retard the progression of vascular calcification) but also, when spilled over to the circulation, may induce negative endocrine effects on the skeleton (decreased osteoblastogenesis and increased osteoclastogenesis). Targeting Wnt inhibitors Mounting evidence points to a central role of a disturbed Wnt enin signaling in the pathogenesis of CKD-MBD. This opens perspectives for targeted therapy, including pharmacological neutralization of sclerostin or DKK1 by monoclonal antibodies. Moe et al. 51 observed improved bone properties in an animal model of progressive CKD treated with anti-sclerostin antibodies, however, only when the PTH levels were low. In another animal model of early CKD, Fang et al. 47 demonstrated that the combination of DKK1 neutralization and phosphate binder therapy was sufficient to decrease vascular calcification and to correct renal osteodystrophy. Extrapolation of these exciting experimental data to the clinical setting warrants caution, mainly in view of the clinical and experimental data suggesting that both Wnt inhibitors may attenuate progression of vascular calcification and that the beneficial effects of Wnt inhibitor neutralization may differ depending on the precise type of CKD-MBD (see above). A recent prospective randomized study in 261 postmenopausal women receiving romosozumab (a humanized monoclonal antisclerostin antibody) over 12 months did not report remarkable cardiovascular side effects. 52 Of note, patients with an estimated creatinine clearance below 30 ml/min were excluded from this trial. REMAINING QUESTIONS, FUTURE DIRECTIONS, AND CONCLUSIONS According to the current level of evidence, DKK1 and especially sclerostin may qualify as a novel biomarker of CKD-MBD. However, several gaps in our knowledge must be resolved before sclerostin and DKK1 measurements are incorporated into clinical practice. These relate in part to biological variability and analytical issues. Questions related to the biological variability include: (1) day-to-day, seasonal, and circadian variability; (2) the proportion of locally produced Wnt inhibitor released into the systemic circulation; and (3) impact of the uremic state on biologic activity and endorgan sensitivity. Dialysis and medication (bisphosphonates, denosumab, estrogens, teriparatide, and so on) may affect circulating levels, causing another level of complexity. In addition, analytical variability occurs between different assays as well as between serum and plasma samples. 53 As for other biomarkers such as FGF23 and PTH, these sources of variability may account, at least partly, for the often inconsistent clinical findings reported so far. There is an unmet need for standard operating procedures and crossvalidation of the different assays. In addition, appropriately designed and powered studies are required to confirm the value of Wnt inhibitors as biomarkers of CKD-MBD. Finally, the efficacy and safety, especially with regard to cardiovascular calcification, of pharmaceuticals targeting Wnt inhibitors in CKD need to be investigated. DISCLOSURE PE has been a member of advisory boards of Amgen and Sanofi, and has received grant support from Amgen and Sanofi and speaker s fees from Amgen, Sanofi, and Shire. VB received lecture fees or consulting fees from Abbvie, Amgen, Fresenius, Sanofi, Novartis, Servier, and Synlab. 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