The first major finding of the present study is that ESRD is accompanied by increased serum concentrations of DKK1 and sclerostin as compared with controls and that the levels of β-catenin were significantly lowered in ESRD. Previous findings showed that patients with CKD have higher sclerostin levels than controls, with values progressively increasing across the CKD stages 48, 49. Serum sclerostin levels start to increase from CKD stage III and progressively increase as CKD progresses to ESRD 31, 50. It is worthy of note that urinary sclerostin excretion increased with declining eGFR, suggesting that increased serum sclerostin in CKD are not due to decreased renal elimination 30. In another study, serum sclerostin, but not DKK1, was increased in the more advanced stages of CKD 48. After renal transplantation, serum concentrations of sclerostin paralleled improvements in renal functions 51. Fang et al. (2014) showed increased renal production of DKK1 and sclerostin and increased circulating DKK1 levels in a mouse model of CKD 52.
Inhibition of the Wnt pathway leads to degradation of β-catenin and reduction in its cytoplasmic levels 53. The mechanistic explanation is that after binding of the inhibitors to the receptors, axin recruits casein kinase 1 to the multiprotein complex (β-catenin-axin-adenomatous polyposis coli (APC)-glycogen synthase kinase (GSK)-3β), causing priming of β-catenin and initiation of the β-catenin phosphorylation cascade performed by GSK-3β. Phosphorylated β-catenin is then recognized by β-transducin repeat-containing protein (β-TrCP) and degraded by the ubiquitin proteasome system, reducing the level of cytosolic β-catenin 27, 54.
Mounting evidence indicates that secreted Wnt antagonists play an important role in the cross-talk between the kidneys, the vasculature, and the bone 52, 55. Wnt signaling is involved in almost every aspect of embryonic growth and also regulates homeostatic self-renewal in a variety of adult tissues 56. Findings show that in CKD, aberrant activation of the Wnt/β-catenin pathway is associated with proteinuria, renal function decline and kidney fibrosis 20-22, 46, 57, 58. Our findings and previous findings that activation of the Wnt/β-catenin pathway modulates tubular repair and regeneration after acute kidney injury 24 indicate that activation of this pathway may be a new drug target to treat AKI-related ESRD.
The second major finding of this study is the significant correlation between Wnt-pathway protein levels and the biochemical biomarkers of ESRD. Most importantly, eGFR is significantly and inversely associated with DKK1 and sclerostin, and positively with β-catenin and a composite score indicating the ratio between stimulators/antagonists of the Wnt-pathway. These results extend those of a previous study showing a negative association between GFR and sclerostin 32, 59. In CKD patients, sclerostin was associated with indicants of inflammation and vascular damage 31, 50 and negatively correlated with histomorphometric parameters of bone turnover and osteoblast numbers 60. The high sclerostin concentrations in patients receiving dialysis may be explained mainly by increased accumulation due to renal disorders 31.
Previous studies investigating the association between DKK1 and CKD yielded conflicting results with some reports observing no changes 32, 61 or increased DKK1 levels 52. In CKD stage 5D, increased DKK1 levels were associated with higher calcium, CRP, and blood platelets and lower PTH 48. Of note, in our study, serum levels of DKK1 and sclerostin were unrelated pointing to different origin and different regulatory mechanisms 48. Nevertheless, extraskeletal production of DKK1 may impact sclerostin functions as both factors bind to LRP5/6 with consequent inhibition of Wnt signaling 32.
Changes in these Wnt antagonists may have dire consequences on cardio-vascular disease and cause lowered blood supply to the kidneys which may further impact CKD/ESRD. Thus, DKK1 may contribute to atherosclerosis 62 and, additionally, play a role in the inflammatory interactions between platelets and endothelial cells 63. Increased sclerostin levels are associated with adverse CKD outcomes including cardiovascular diseases 64 and all-cause mortality and cardiovascular events 65. In long-term haemodialysis patients, circulating sclerostin, but not DKK1, was inversely associated with aortic calcification and future cardiovascular events 66. Furthermore, CKD patients with sclerostin levels exceeding 0.748 ng/ml show an increased risk to impaired renal functions and vascular and coronary artery calcification 67, 68. Also, another study reported that the increased sclerostin levels in CKD are associated with vascular lesions, inflammation, uremia and increased mortality 69.
Another major finding of this study is that ESRD patients show increased copper and lowered zinc levels as compared with healthy controls and that there is a significant inverse association between copper levels with β-catenin and the Wnt AT/ANTA ratio. Changes in those trace elements in CKD/ESRD were described in many 70-73, but not all studies 74. The results indicate that hemodialysis patients are at increased risk of zinc deficiency 75, 76, which is linked to delayed wound healing, decreased immune functions, and increased infection susceptibility 77. One determinant of increased plasma copper concentrations is perhaps serum creatinine 78. It is interesting to note that patients with Wilson’s disease, which is characterized by abnormal copper metabolism, show an abnormal β-catenin signaling pathway 79. In other species (e.g., zebrafish), copper may suppress the Wnt signaling pathway 80.
Limitations of the study
The results of our study should be discussed with respect to its limitations. First, we performed a case-control study and, therefore, no firm causal inferences may be established. Second, it would have been more conclusive if we would have examined all receptors and agonists of Wnt-pathway in ESRD patients in a larger sample size.