Wnt/β-catenin pathway proteins in end-stage renal disease

Background. Wnt-pathway proteins play a vital role in kidney development and defects in the Wnt-pathway are associated with kidney disorders. However, the knowledge on the role of Wnt/β-catenin pathway proteins in end-stage renal disease (ESRD) is limited. Aim of the study. To delineate the association of ESRD and Wnt-proteins including the agonist R-spondin 1, the transducer β-catenin and the antagonists Dickkopf-related protein 1 (DKK1) and sclerostin. Methods. Serum Wnt-pathway proteins levels were measured by ELISA, while other biochemicals were measured spectrophotometrically in 60 ESRD patients and 30 normal controls. Results. DKK1 and sclerostin were signicantly higher in ESRD than in controls, and β-catenin and the catenin + R-Sponin-1 / DKK1 + sclerostin ratio, reecting the ratio of agonist and transducer / antagonists (AT/ANTA), were signicantly lower in ESRD. Logistic regression analysis showed that ESRD was signicantly predicted by increased levels of DDK1 and sclerostin and lowered β-catenin (p<0.001). eGFR was signicantly associated with DKK1 and sclerostin (inversely), β-catenin (positively) and the AT/ANTA ratio (r=0.468, p<0.001). DKK1 levels were signicantly and positively correlated with urea, creatinine, and copper. DKK1 and sclerostin were inversely associated with hemoglobin and packet cell volume. Catenin was signicantly negatively associated with copper, urea and creatinine. Conclusion. Wnt/β-catenin pathway proteins show signicant alterations in ESRD, indicating signicantly increased levels of antagonists and, therefore, attenuated Wnt/β-catenin pathway activity. The latter is associated with lowered eGFR and increased serum copper levels. Wnt/β-catenin pathway proteins are possible drug targets to treat ESRD or its consequences. the study groups. ESRD patients have a signicantly lower BMI and employment ratio than healthy controls. Our data showed results that are typical of the ESRD status, namely a signicant increase in serum urea and creatinine and all patients show an eGFR indicative of ESRD. Serum copper was signicantly increased, and serum zinc was signicantly decreased in ESRD patients as compared with controls. 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 brosis 20-22, 46, 57, 58 . Our ndings and previous ndings 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.


Introduction
Acute kidney injury (AKI) is characterized by a reduction in kidney function, including decreased glomerular ltration rate (GFR) and subsequent kidney failure 1 . Kidney failure is considered when GFR is <15 ml/min per 1.73 m 2 body surface area and this condition may lead to end-stage renal disease (ESRD) 2 . ESRD is de ned as irreversible decline in the kidney function, which is severe enough to be fatal in the absence of dialysis or kidney transplantation 3 . The adjusted prevalence of ESRD increased to a new high of 2,242 cases per million people in 2018 and at the end of 2018, there were 554,038 (70.7%) patients undergoing dialysis and 229,887 (29.3%) patients with a functioning kidney transplant 4 . ESRD is accompanied by many biochemical changes including in lipid and vitamins 5 , cytokines 6,7 , trace elements including copper and zinc, 8,9 and oxidative stress biomarkers 10,11 . However, the knowledge on the Wnt/β-catenin pathway in ESRD is limited. speci c hypothesis was that ESRD is accompanied by increased DKK1 and sclerostin and lowered βcatenin and R-spondin 1.

Subjects
Sixty ESRD patients (30 male and 30 female) aged (15-55 years) participated in the current study.
All of them had a previous AKI which progressed into renal failure and all were on continuous dialysis. The patients were recruited at the Dialysis Unit at Al-Hakeem General Hospital and Al-Sader Medical City in Najaf Governorate-Iraq during the period October 2020-December 2020. The assessment of patients was carried out by full medical history which considers the presence of any systemic disease. Patients with diabetes, liver diseases and heart diseases were excluded from the study. Serum C-Reactive Protein (CRP) concentrations were <6 mg/L (assayed using agglutination test) in all participants. The test was carried out to eliminate overt in ammation which causes changes in acute phase reactants. The patients were diagnosed by a senior physician according to the 10th revision of the International Statistical Classi cation of Diseases and Related Health Problems (2021 ICD-10-CM Diagnosis Code N18.6). All patients were on a continuous treatment with folic acid or iron and folate formula (Fefol ® ) in addition to calcium carbonate, Epoetin alfa (Eprex ® ), and heparin. The control group consisted of 30 healthy subjects (15 males and 15 females) without apparent physical illnesses. Their sex and age were matched with those of the patients. Written informed consent was obtained from the patients or their rst-degree relatives. The protocol was approved by the Iraqi institutional review board (IRB) of the University of Kufa (543/2020), Kufa, Iraq. All participants provided written informed consent.

Measurements
Venous blood samples were taken from the participants in the morning between 8.00 a.m. and 9.00 a.m. after 12 hours fasting. Venous blood samples were collected into plain tubes. Samples were aliquoted and stored at −80 °C before assay. Serum was sampled before the hemodialysis session to assay all parameters. After separation, the sera were distributed into three new Eppendorf ® tubes for further analysis. Serum sclerostin (SOST), R-Spondin 1 (RSPO), β-catenin (CAT), and DKK1 concentrations were measured using ELISA kits supplied by Melsin Medical Co., Ltd., Jilin, China. The interassay CV% of all the Wnt-pathway proteins were <15%. All measured concentrations of sclerostin (sensitivity < 0.1 ng/ml), R-spondin1 (sensitivity < 1 pg/mL), β-catenin (sensitivity < 1 pg/mL), and DKK1 (sensitivity < 1 ng/mL) were greater than the sensitivity of the assays. Consequently, we computed 3 z unit weighted composite scores, namely zDKK+zSOST (re ecting inhibitors of the Wnt pathway), zCAT+zRSPO (re ecting agonists / stimulators of the pathway), and z(zCAT+zRSPO) -z(zDKK+zSOST) (Wnt AT/ANTA ratio) re ecting net positive effects on the Wnt pathway). Hematological parameters were measured using a ve-part differential Mindray BC-5000 hematology analyzer (Mindray Medical Electronics Co., Shenzhen, China). Serum copper and zinc were measured spectrophotometrically using kits supplied by Spectrum Diagnostics Co. (Cairo, Egypt). Glucose, albumin, urea, and creatinine were measured spectrophotometrically by ready for use kits supplied by Biolabo ® (Maizy, France Kolmogorov-Smirnov test was used to examine the normality of distribution. Analysis of variance (ANOVA) was used to examine the between-group differences in scale variables. Statistical associations between categorical variables were evaluated by analysis of contingency tables (χ 2 -test). Pearson's correlation coe cients (r) or Spearman's correlation coe cients (ρ, rho) were calculated to assess correlations between biomarkers. We transformed biomarkers into to Ln transformation to normalize the distribution as assessed using the Kolmogorov-Smirnov test. We employed multivariate generalized linear model (GLM) analysis to check the relationship among the biomarkers and the diagnosis (ESRD versus controls) while controlling for background variables including age, BMI, tobacco use disorder (TUD), and sex. Binary logistic regression analysis was used to delineate the most important explanatory variables that predict ESRD versus control as reference group. We used multiple regression analysis to assess the most signi cant Wnt proteins that are associated with eGFR. Power analysis showed that using an effect size of 0.3, alpha=0.05, power=0.8 and 2 groups the total sample size should be 90. All statistical analyses were performed using SPSS Statistics version 25 (2017), IBM-USA.

Demographic data and Clinical data
The socio-demographic and clinical data of ESRD patients and healthy controls are presented in Table 1. There were no signi cant differences in age, sex ratio, marital status, TUD, family history, and rural/urban ratio between the study groups. ESRD patients have a signi cantly lower BMI and employment ratio than healthy controls. Our data showed results that are typical of the ESRD status, namely a signi cant increase in serum urea and creatinine and all patients show an eGFR indicative of ESRD. Serum copper was signi cantly increased, and serum zinc was signi cantly decreased in ESRD patients as compared with controls. Table 2 shows the results of the Wnt-pathway proteins and the three composite scores in both diagnostic groups while adjusting for age and sex. We found that there was a signi cant increase in DKK1 and sclerostin in ESRD patients as compared with healthy controls. We found a signi cant decrease in β-catenin, zCAT+zRSPO, and the Wnt AT/ANTA ratio in ESRD patients as compared with controls while the zDKK+zSOST composite score was signi cantly increased. Figure 1 shows the Wntpathway protein pro le (as z scores in a clustered bar graph) in ESRD patients versus healthy controls. Table 3 shows the results of binary logistic regression analyses examining the best Wnt-pathway predictors of ESRD versus controls using an automatic stepwise method with biomarkers as explanatory variables while allowing for the effects of age, sex, BMI, and TUD. The regression analysis showed that ESRD was best predicted by increased levels of zDDK+zSOST combined with catenin (χ2=25.233, df=3, p<0.001, Nagelkerke=0.494) with an accuracy of 70.0%, sensitivity 68.3% and speci city 73.3%. Also, the Wnt AT/ANTA ratio was a signi cant predictor of ESRD (χ 2 =27.526, df=1, p<0.001, Nagelkerke R 2 =0.366).

Intercorrelation matrix
In the total study group, there was a positive correlation between DKK1 and hemoglobin, PCV, urea, creatinine, eGFR (all inversely) and copper (positively). R-spondin-1 was signi cantly correlated with βcatenin. Sclerostin showed a signi cant positive correlation with blood urea, and negative correlations with hemoglobin, PCV, and eGFR. β-catenin was signi cantly and positively correlated with eGFR and negatively with urea, creatinine and copper. The Wnt AT/ANTA ratio showed a signi cant correlation with hemoglobin, PCV, and eGFR (positively), and urea, creatinine, and copper (inversely). Table 5 shows different stepwise multiple regression analyses with eGFR and the Wnt-pathway proteins as dependent variables and other measured biomarkers as explanatory variables while allowing for the effects of age, sex, BMI, and TUD. Regression #1 shows that 27.7% of the variance in the eGFR was explained by the regression on β-catenin and the composite score zDKK+zSOST. Figure 2 shows the partial regression plot of eGFR on the composite score re ecting antagonist activities. Regressions #2 shows that 6.0% in DKK1 can be explained by urea. Regression #3 shows that 10.4% of the variance in sclerostin was explained by hemoglobin concentrations. eGFR, copper, and PCV explained together 25.6% of the variance in β-catenin (regression #4). Regression #5 shows that 24.4% of the variance in the Wnt AT/ANTA ratio was explained by the combined effects of creatinine and copper. Figure 3 shows the partial regression of this composite score on serum copper concentrations.

Discussion
The rst major nding 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 signi cantly lowered in ESRD. Previous ndings 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  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 repeatcontaining 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 brosis 20-22, 46, 57, 58 . Our ndings and previous ndings 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 nding of this study is the signi cant correlation between Wnt-pathway protein levels and the biochemical biomarkers of ESRD. Most importantly, eGFR is signi cantly 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 in ammation 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 con icting 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 in ammatory 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 calci cation 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 calci cation 67,68 . Also, another study reported that the increased sclerostin levels in CKD are associated with vascular lesions, in ammation, uremia and increased mortality 69 .
Another major nding of this study is that ESRD patients show increased copper and lowered zinc levels as compared with healthy controls and that there is a signi cant 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][71][72][73] , but not all studies 74 . The results indicate that hemodialysis patients are at increased risk of zinc de ciency 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., zebra sh), 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 rm 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.

Conclusions
In ESRD, the Wnt/β-catenin pathway may be inhibited because β-catenin levels are lowered and the levels of the antagonists sclerostin and DKK1 are increased. The Wnt activator/antagonist ratio is signi cantly and positively associated with eGFR, and inversely with urea, creatinine, and serum copper.
Targeting the Wnt-pathway is a new potential drug target to treat CKD/ESRD or its consequences. There is an urgent need to study the mechanism whereby increased copper levels may impact Wnt-pathway proteins and functions.   Wnt AT/ANTA ratio: agonist + transducer / antagonist ratio; *p<0.05 (2-tailed); **p<0.01 (2-tailed)