Soluble Co-Signaling Molecules Predict Long-Term Graft Outcome in Kidney-Transplanted Patients

<div><p>Co-signaling molecules are responsible for full T-cell activation after solid organ transplantation. Their increased expression can lead to the release of a soluble form that can modulate the immune response post-transplantation. We analyzed the presence of co-signaling molecules (sCD30, sCD40, sCD137, sCTLA-4, sCD80, sCD28, sCD40L, sPD-1, and sPD-L1) in serum from kidney-transplanted patients (n = 59) obtained at different times (before transplantation, and 15 days, 3 months and 1 year post-transplantation) and their contribution to graft outcome was evaluated using principal component analysis. Before transplantation, high levels of soluble co-signaling molecules (mainly sCD30, sCD137 and sCD40) were detected in all patients. These molecules were modulated soon after receiving an allograft but never attained similar levels to those of healthy controls. A signature based on the determination of six soluble co-stimulatory (sCD30, sCD40, sCD137 and sCD40L) and co-inhibitory (sPD-1 and sPD-L1) molecules at 3 months post-transplantation allowed a group of patients to be identified (27.12%) with a worse long-term graft outcome. Patients with high levels of soluble molecules showed a progressive and gradual deterioration of kidney function (increased creatinine and proteinuria levels and decreased estimated glomerular filtration rate) over time and a higher risk of graft loss at 6 years post-transplantation than patients with low levels of these molecules (62.55% <i>versus</i> 5.14%, p<0.001). Thus, our data show an aberrant expression of soluble co-signaling molecules in kidney-transplanted patients whose quantification at 3 months post-transplantation might be a useful biomarker of immune status and help to predict long-term graft evolution.</p></div

Changes of the creatinine, estimated glomerular filtration rate (eGFR) and proteinuria levels over time in kidney-transplanted patients grouped by the levels of soluble co-signaling molecules.

<p>Patients were clustered according to the levels of soluble co-signaling molecules quantified at 3 months post-transplantation in high co-signaling and low co-signaling groups. The levels of creatinine, eGFR and proteinuria did not differ between these two groups at time the soluble molecules were quantified. At 5 years post-transplantation, patients in the high co-signaling group showed an increase in creatinine levels and a decrease in the eGFR, whilst the characteristics of the patients in the low co-signaling group were unchanged. Changes in the proteinuria levels between both groups were observed by the second year post-transplantation.</p

Clinical characteristics related to disease severity with respect to the rs4819554 genotype distribution.

<p>Clinical characteristics related to disease severity with respect to the rs4819554 genotype distribution.</p

Long-term graft survival based on the determination of soluble co-signaling molecules.

<p>Patients with high levels of soluble co-signaling molecules determined at 3 months post-transplantation have a higher risk of graft failure than those with low levels (low co-signaling group) (p = 0.002). The Nelson-Aalen estimator was used to analyze the incidence of graft loss due to the existence of competing events (patients died with a functioning graft during the follow-up period). The number of patients analyzed each year, taking into account those with graft loss and those who died with a functioning graft are presented at the bottom.</p

Creatinine, estimated glomerular filtration rate and proteinuria levels of both clusters of patients over time.

<p>Evolution of the kidney function in both clusters of patients determined by the levels of co-signaling molecules at 3 months post-transplantation. Medians were compared using Mann-Whitney U test and values of p <0.05 are shown in bold.</p><p>Creatinine, estimated glomerular filtration rate and proteinuria levels of both clusters of patients over time.</p

Demographic characteristics of kidney-transplanted patients.

<p>Continuous variables are summarized as mean ± standard deviation (interquartile range). Categorical variables are shown as n (%). ESRD, end-stage renal disease; mTOR, mammalian target of rapamycin, DSA, donor-specific antibody.</p><p>Demographic characteristics of kidney-transplanted patients.</p

Modulation of soluble co-signaling molecules in kidney-transplanted patients over time.

<p>The levels of the soluble co-signaling molecules CD30, CD40, CD137, CD40L, PD-1 and PD-L1 were assayed by ELISA in serum samples of healthy controls (n = 25) and kidney-transplanted patients (n = 59) obtained at different times: just before transplantation, and 15 days, 3 months and 1 year after transplantation. Data are shown as box-plots, in which the horizontal line within each box represents the median, the bottom and top of each box represent the 25^th and 75^th percentiles, the bars represent the 10^th and 90^th percentiles and circles indicate outliers. Unpaired and paired Wilcoxon tests were used to compare distributions between independent and dependent groups, respectively. * indicates statistically significant differences between healthy controls and kidney-transplanted patient samples, and † indicates statistically significant differences between patients samples obtained at different pre- and post-transplantation times.</p

Effect of soluble co-signaling molecules determined at three months post-transplantation in the long-term (at 5 years post-transplantation) kidney graft evolution.

<p>The chi-square test was used to compare distributions among groups. * p <0.001.</p><p>Effect of soluble co-signaling molecules determined at three months post-transplantation in the long-term (at 5 years post-transplantation) kidney graft evolution.</p

Clinical characteristics related to disease severity with respect to the rs4819554 genotype distribution.

<p>Clinical characteristics related to disease severity with respect to the rs4819554 genotype distribution.</p

Effect of the <i>EPHX2</i> 3’ UTR A/G polymorphism (rs1042032) of the recipient on serum creatinine concentrations throughout the one-year follow-up.

<p>*p<0.05 vs. AA/AG group.</p