Kidney transplantation remains the preferred treatment for patients with end stage kidney disease due to a better patient survival compared to dialysis. Despite good short-term results, ensuring a long-lasting graft function is an unsolved problem. To date, with serum creatinine, eGFR and albuminuria, only a few parameters are available in everyday clinical practice to monitor graft function. Even in the KDIGO guidelines, these parameters are named as the main monitoring tool (1). However, these parameters have not yet succeeded in ensuring graft survival. In literature, different biomarkers are being discussed in order to ensure a better risk assessment.

Park et al. showed that an eGFR decline of >–10% in a period of 3–12 months was associated with a greater risk of graft failure (2).

The main cause of late allograft loss is the development of interstitial fibrosis and tubular atrophy (IF/TA) (3). These histological changes describe the final stages of different processes (CNI- toxicity, etc.) and can be detected through biopsy (4).

The “iBox,” a prediction score by the group of Loupy et al., aims to ensure a better graft monitoring and thus enable a patient-tailored diagnostic and therapy after transplantation (5). Trailin et al. were able to show that high levels of interleukin 2 in urine are associated with worsened eGFR (6). Kielar et al. showed that elevated neutrophil gelatinase-associated lipocalin (NGAL) levels in the urine were associated with an eGFR loss after transplantation (7). However, sufficient biomarkers to obtain robust and validated information about long-term allograft function and to identify patients at risk, are still lacking today.

Dickkopf 3 (DKK3) has been identified as a biomarker of kidney function in animal and clinical studies. These studies have been based on patients with acute kidney injury (AKI) or chronic kidney disease (CKD). In the context of AKI, DKK3 is currently seen as a prediction score for the development of a kidney failure (8).

DKK3, a profibrotic glycoprotein, belonging to the Dickkopf family consists of five proteins (DKK1-4, DKK like protein 1) that influence the Wnt signaling pathway through inhibition or activation. The Wnt signal pathway is an important signal transduction pathway in embryogenesis (9, 10). It is also relevant in tumor diseases (e.g., familial adenomatous polyposis) (11). In the context of kidney diseases, multiple functions are assigned, e.g., it is potentially associated with the development of ADPKD (10). DKK3 activates the canonical Wnt/b-catenin signaling pathway which induces gene expression (12).

Frederico et al. found that Dickkopf plays a role in embryonic development and was found in mesenchymal progenitor cells and mesenchymal cells. It is normally not detectable in adult cells (13). After kidney damage, DKK3 is expressed in the tubular epithelial cells and causes a profibrotic T-cell response (13). It can therefore be detected in the urine.

Studies on patients with CKD have shown that high Dickkopf values are associated with the increased incidence of tubulointerstitial fibrosis. Frederico et al. were able to show that DKK3 deficient mice showed less pronounced tubular atrophy and an improved kidney. This effect could also be demonstrated by an antibody-mediated blockade of DKK3 (13). Another study showed that higher Dickkopf values were associated with impaired kidney function and patients with high DKK3 values showed more tubulointerstitial fibrosis. The authors conclude that Dickkopf can be used as a biomarker for patients with a rapid eGFR loss over time, regardless of the underlying kidney disease (14).

The role of Dickkopf 3 in the context of kidney transplantation is completely unclear. The aim of our study was to analyze DKK3 in the urine of transplanted patients, which represents the first investigation realized in such a cohort. For this purpose, a highly standardized cohort of kidney transplant recipients was investigated. In addition to kidney function, represented by creatinine, eGFR and albuminuria, both donor and recipient specific influencing factors (e.g., age, PRA level) were analyzed. The goal of our study was to determine if a correlation between the DKK3 values and graft function over the observation period of 3 years exists and if DKK3 could be further developed as a non-invasive marker to identify patients at high risk for a deterioration in transplant function.

In our study, we examined the influence of Dickkopf 3 on graft function in kidney transplant recipients. We were able to show that DKK3 correlates with resulting graft function, represented by creatinine, eGFR and albuminuria, over an observation period of 36 months. DKK3 can even predict kidney function as illustrated by the association of 3 and 12 months DKK3 values and subsequent allograft function. Furthermore, changes in DKK3 values from month 3 to 12 (≥25%) were associated with a significantly deteriorated graft function, being illustrated by tremendous differences in creatinine, eGFR, and albuminuria values. Our study is the first investigation of DKK3 referring to transplantation medicine.

Regarding the function of DKK3, studies have shown that DKK3 is secreted only by stressed tubular epithelial cells in the adult kidney. Using two animal models, an adenine-induced nephropathy and a model of an unilateral ureter obstruction, Gröne et al. showed by usage of a DKK3 knockout that DKK3 deficiency leads to a marked reduction in tubular damage and renal fibrosis. DKK3 deficiency triggered an antifibrotic T cell response and reduced activity of the WNT–β-catenin signaling pathway. These results could also be reproduced by an antibody-mediated blockade of DKK3. DKK3 therefore appears to be an important mediator of renal fibrosis and thus of deterioration in renal function (9).

Schunk et al. were able to show that patients after cardiac surgery and increased DKK3 scores (>471 pg/ml) had an increased risk for developing AKI (22). A comparable observation could be reproduced in our analysis. However, our data are more closely linked with chronic changes. Patients with a DKK3 dynamic of more than 25% showed a deteriorated graft function and also more albuminuria than patients with a smaller change in DKK3. Especially the changes in albuminuria, being 7–12 times higher in patients with intensified DKK3 increase, do not only link DKK3 expression levels with allograft function, but also with arising structural damage.

Zewinger et al. were able to show that high levels of DKK3 are associated with impaired function in patients with CKD. DKK3 could be seen as a predictor for an eGFR loss independent of the underlying disease. This study showed that high DKK3 values can function as a prognostic parameter regardless of the accompanying albuminuria (14). Similar results were showen by Sanchez-Alamo. By determining DKK3 in the urine, patients with a high risk of deterioration in kidney function could be identified, regardless of the underlying disease (23). A correlation between DKK3 and creatinine as well as the eGFR was also found in our work. In contrast to Zewinger, however, a significant influence of DKK3 could also be found for albuminuria.

We were able to see a trend toward lower DKK3 values after T-cell depleting induction therapy in comparison to an immunomodulatory therapy with basiliximab. Regarding the impact of immunosuppressives on the development of DKK3, no further data are available. But in literature, the influence of DKK3 on T- lymphocytes is discussed. As already mentioned, DKK3 seems to trigger a profibrotic T cell response. Federico et al. were also able to show that after an antibody-mediated blockade of DKK3, an increased presence of protective T cells (IFNγ-producing Th1 and Tregs) can be demonstrated (13). Taking this into account, the evidence of lower DKK3 values under a T-cell depleting therapy seems understandable. Further investigations on the influence of immunosuppressives would be useful to further evaluate specific “anti-DKK3 and therefore presumable anti-fibrotic immunosuppressive protocols.”

In our cohort, we were able to recognize a total of 18 rejections over the entire observation period, both T cell-mediated and antibody-mediated rejections. Most of them occurred within the first 14 days. The analysis of the DKK3 values between patients with a rejection compared to patients without a rejection showed no statistically significant difference. However, it should be noted here that there is a relevant difference in the number of cases in the two groups as a possible confounding factor.

Our study is the first analyzing Dickkopf 3 after kidney transplantation. Nevertheless, it is a monocentric study with a limited case number. There are currently no special biomarkers postTx available to estimate the individual risk for a deterioration in graft function. DKK3 can be easily integrated into everyday clinical practice thanks to its detection in urine. Similar to Zewinger in his study, we were also able to see a clear influence of DKK3 on graft function (14). The use of DKK3 as a predictor of graft function should therefore be considered and proofed in a multi-center clinical trial. Animal studies showed that an anti-DKK3 antibody could inhibit the development of fibrosis in mice. DKK3 thus also represents a possible therapeutic target. It should be noted critically that defined cut-off values for DKK3, from which a clinical consequence must result, are still missing. Looking at our study, the determination of DKK3 at time points 3 and 12 months after transplantation could be a helpful new screening parameter in the follow-up. Nevertheless, long-term analyzes and prospective multicenter studies would be necessary in order to address the still open questions and to deepen our findings made in a single-center study.

Whereas, in the context of chronic kidney disease, Dickkopf 3 has been recognized as a marker to identify patients at risk for a progressive loss of kidney function, to date, the impact of DKK3 after transplantation has not yet been analyzed.

In our study on kidney transplant recipients, DKK3 not only could precisely predict subsequent allograft function but an increase in DKK3 values within the first year after transplantation was associated with a deterioration in allograft function.

With the presented data, DKK3 can be considered as a new indicator of impaired graft function after transplantation. However, further prospective and interventional studies are needed to verify our findings.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

The studies involving human participants were reviewed and approved by Ethical Committee of the University of Regensburg. The patients/participants provided their written informed consent to participate in this study.

AS, BB, and TB: concept/design. AS, PF, and TB: data collection. AS, KM, FZ, and TB: statistics. AS, LS, and TB: data analysis/interpretation. AS, LS, BB, and TB: drafting article. AS, LS, KM, FZ, PF, BB, and TB: critical revision of article and approval of article. All authors contributed to the article and approved the submitted version.

This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project number 387509280, SFB 1350 Project B6 to TB and BB.

The 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.

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