Monitoring of renal transplant recipients by use of complement factors, kidney-specific cell-free DNA, Torque Teno Virus, and HLA epitope matching: Investigation and application of biomarkers in kidney transplantation: From development to clinical and research relevance

Kidney transplantation is the preferred treatment for end-stage kidney disease, offering costeffectiveness and better outcomes than dialysis. The global demand for kidney transplants is rising due to an aging population and increased prevalence of chronic kidney disease. Although short and long-term graft survival rates have improved, efforts to enhance graft survival are crucial, particularly for younger recipients who may need retransplantations.

Kidney transplant recipients require immunosuppressive medication to avoid rejection. However, striking the right balance between excessive and insufficient immunosuppression is a persistent dilemma in kidney transplantation. Insufficient immunosuppression can lead to increased clinical and subclinical alloreactivity, resulting in reduced graft survival. On the other hand, excessive immunosuppression raises the risk of infections, cancer, and medication side effects.

Further improvements are difficult employing our current surveillance strategy. Personalized approaches and precise biomarkers reflecting overall immunosuppression and kidney health are necessary to optimize transplant conditions and improve outcomes.

The dissertation aimed to investigate and elucidate the development, validation, and employment of biomarkers in kidney transplantation. The Studies I-V represents examples of different stages in biomarker development and application.

Study I

Development of a multiplex digital droplet PCR assay to detect kidney-specific cell-free DNA. 

Study I combined the knowledge of the upcoming biomarker of graft injury in transplantation, donor-derived cell-free DNA, with knowledge from cancer research applying differentially methylated regions to detect cell-free DNA from tumors in the circulation. The aim was to develop a multiplex digital droplet PCR assay to detect cell-free DNA originating from the kidney in circulation. Elevated levels of cell-free DNA indicate organ injury, as cell-free DNA released to circulation is a result of inflammation and necrosis. 

By identifying differentially methylated regions in kidney tissue from databases, we identified five regions able to distinguish kidney tissue from other tissue, and the five markers were combined into an assay for digital droplet PCR to measure cell-free DNA originating from the kidney(s). The markers were present in kidney tissue, and a cut-off was determined from the blood of healthy individuals. Applying the cut-off, the assay successfully detected all recently transplanted recipients as well as five out of seven recipients with rejection. Although further validation is required, the test's advantages, such as quick turnaround time, affordability, and applicability in multiorgan recipients and pregnant individuals, make it a promising tool for clinical use.

Study II 

Exploration of complement split products in plasma and urine as biomarkers of kidney graft rejection.

As part of the innate immune system, the complement system plays a crucial role in immune responses and is thought to contribute to allograft injury in kidney transplantation. However, it is largely unknown to which extent complement activation can be measured in recipients with rejection. Therefore, Study II aimed to investigate whether complement activation can be measured in plasma and urine in kidney transplant recipients with rejection. 

We applied a case-control design, comparing 15 kidney transplant recipients with rejection to 15 recipients without rejection (controls) and examined the levels of complement factors C3a, C3dg, C4a, and C5a in plasma and C3dg and sC5b-9 associated C9 neoantigen in urine. Additionally, the dynamics of the complement split products within the rejection group were explored. 

Plasma C3a and C3dg were similar between the groups but significantly increased during cases of primarily T cell mediated rejection (TCMR) compared to a stable phase in the rejection group. C4a and C5a showed no differences between or within the groups. Furthermore, the pretransplant P-C3dg level was correlated with posttransplant levels, and P-C3dg was lower in recipients treated with steroids.

Urine complement split products did not differ between the groups, and the dynamics observed within the groups were likely caused by glomerular injury rather than rejection. 

P-C3dg may be associated with TCMR, and evaluating C3dg as a potential biomarker of alloreactivity in prospectively and consecutively collected samples is feasible. 

Study III 

Posttransplant kinetics of Torque Teno Virus in a Danish cohort of mainly steroid-free kidney transplant recipients.

Torque Teno Virus (TTV) is a non-pathogenic virus highly prevalent in transplant recipients. Elevated TTV levels have been linked to infections, whereas low levels have been associated with rejection. Consequently, TTV has been suggested as a potential biomarker for assessing the overall immunosuppressive state. However, the studies predominantly focus on adults, with none comparing TTV load between adult and pediatric kidney transplant recipients.

In Study III, we aimed to investigate the dynamics of TTV after transplantation in a Danish mainly steroid-free population, to compare the level and kinetics between pediatric and adult kidney transplant recipients, and to explore the influence of baseline characteristics on TTV levels. 

Prospectively and consecutively collected blood samples were analyzed using an in-house PCR to quantify the TTV load. The results showed that TTV levels increased up to three months after transplantation, in accordance with previous studies. The load stabilized around 3.5-4.0 log10 copies/ml, which was lower than previously reported. There were no significant differences in TTV kinetics and levels between adult and pediatric recipients. Nevertheless, age above 59 years and below six was associated with higher TTV levels. Male recipients had higher TTV loads than females, and there was a tendency towards higher TTV levels in recipients who received prednisolone at transplantation. 

The similarity in TTV levels and kinetics between adult and pediatric recipients suggests that utilizing TTV load as a biomarker could also apply to pediatric kidney transplant recipients. In our mainly steroid-free cohort, we observed indications of reduced TTV levels compared to prior studies involving cohorts receiving steroids. These findings, combined with a tendency towards increased TTV levels in steroid-treated recipients, warrant further investigation of the influence of prednisolone on TTV levels.

Study IV 

Association of HLA B and T cell molecular mismatches with HLA antibodies, rejection, and graft survival in pediatric kidney transplantation.

Enhancing HLA matching through in silico methods for predicting HLA epitopes represents a promising approach to improve transplant outcomes.

Study IV investigated whether improved HLA molecular matching, applying HLA-EMMA and PIRCHE-II to predict the epitope mismatches, was associated with better graft survival, reduced rejections, and reduced HLA antibody formation. We retrospectively studied 49 mainly steroid-free pediatric kidney transplant recipients from our center. Logistic regression analyzed the association between the number of molecular mismatches and de novo donor-specific antibodies, HLA sensitization, graft loss, and rejection. 

We did not find significant associations between molecular mismatches and de novo donorspecific antibodies, HLA sensitization, graft loss, or rejection. Despite the small cohort and limited number of events, there was a tendency towards an association between increased PIRCHE-II score and de novo donor-specific antibodies formation, especially on HLA class II. 

Even though the findings do not clearly support a clinical improvement with molecular matching in our cohort, we consider PIRCHE-II an additional tool for selecting among multiple living donors.

Study V 

The impact of reduced immunosuppression on alloimmunity: A retrospective study of pediatric kidney transplant recipients.

Dosages of immunosuppressive medication used in pediatric kidney transplantations are extrapolated from the adult populations, and pediatric clinical trials are sparse. Reductions in immunosuppressive medication are often necessary, possibly increasing the risk of alloimmunity. 

In Study V, we retrospectively studied 49 mainly steroid-free pediatric kidney transplant recipients to describe the changes in immunosuppressive medication, explore the reasons for the alterations, and assess the potential impact on alloimmunity. 

We found that 72% of recipients received reduced immunosuppression after one year, with the median dosage of mycophenolate mofetil being nearly half of the intended. The reductions were primarily due to gastrointestinal side effects, leukopenia, and infections, mostly related to mycophenolate mofetil. The trough level of tacrolimus was well-adjusted. Recipients under six years had a significantly increased rate of infections, while gastrointestinal side effects and leukopenia were evenly distributed among the age groups. 

De novo donor-specific antibodies were applied as surrogate endpoint, and as such, we explored the association with kidney function. We found that eGFR was significantly reduced in recipients with de novo donor-specific antibodies but not in HLA-sensitized recipients, indicating the detrimental effects of de novo donor-specific antibodies.

Despite the reduction in immunosuppressive medication, only four percent had a rejection within the first year, while 11% developed de novo donor-specific antibodies during follow-up. Sixty percent had detectable HLA antibodies pre- or posttransplant. Graft and patient survival rates were comparable to previous reports.

These findings suggest that reducing mycophenolate mofetil while maintaining a sufficient trough tacrolimus level in predominantly steroid-free pediatric kidney transplant recipients does not lead to unacceptable alloimmunity.