Renal allograft outcomes have improved with modern immunosuppression,1–3 which has contributed to improved long-term histology and graft survival.4,5 Nevertheless, graft loss remains clinically relevant with an up to 3-fold increased risk of death, immunological sensitization that may hamper retransplantation, lower quality of life and increased costs.6,7 Furthermore, the causes of graft loss are mostly identifiable and primarily immune-mediated which suggests potential reversibility with timely therapeutic intervention.8–11 Therefore, early prognostic markers could improve long-term outcomes by stratifying high-risk patients to more intensive posttransplant surveillance and/or personalized immunosuppression.3
Chemokine (C-C motif) ligand 2 (CCL2) is a C-C receptor 2 (CCR2) chemokine produced by renal tubular and glomerular epithelial cells, and infiltrating leukocytes. CCL2 is a chemoattractant for monocytes/macrophages, T lymphocytes, and natural killer cells,12,13 and may help to generate memory CD8+ T lymphocytes.14 In a multicenter adult renal transplant cohort, we demonstrated that 6-month urinary CCL2 is an independent predictor of 24-month interstitial fibrosis and tubular atrophy (IF/TA), and IF/TA and inflammation,15,16 an important histological surrogate that strongly correlates with graft loss.17-20 Furthermore, we demonstrated that 6-month urinary CCL2 was an early predictor for death-censored graft loss, independent of early acute rejection and donor-specific antibody (DSA).21
CXCR3 is a chemokine receptor expressed by activated T cells and natural killer cells and binds to CXCL9 (monokine-induced by interferon-γ) and CXCL10 (interferon-γ-induced protein of 10 kDa).22 CXCL9 and CXCL10 are secreted by infiltrating inflammatory cells, renal tubular and mesangial cells, and involved in leukocyte recruitment to rejecting allografts.22-24 The CTOT-01 study demonstrated that elevated 6-month urinary CXCL9 correlates with graft dysfunction and acute rejection between 6 and 24 months posttransplant,25 and we recently validated these findings for 6-month urinary CXCL10 in an independent cohort.26
The aim of this study was to validate 6-month urinary CCL2 as a noninvasive predictor of long-term allograft outcomes in an independent prospective cohort and compare its performance to 6-month urinary CXCL10.
MATERIAL AND METHODS
This study was performed with University of Basel institutional ethics approval, and all patients gave informed consent. Since March 2003, midstream urines were collected from renal allograft recipients immediately before indication or surveillance biopsies. All consecutively transplanted patients between October 2005 and March 2009 were considered for inclusion (n = 228). Inclusion criteria were a 6-month surveillance biopsy with a corresponding urine specimen and minimum 5-year clinical follow-up. Forty-three patients were excluded for: no urine sample (n = 37), death (n = 1), and graft loss (n = 4) within the first 6 months posttransplant, and lost to follow-up (n = 1). The final study population consisted 185 of (81%) of 228 patients, and outcomes were determined as of March 31, 2014 (Figure 1).
The primary outcome was a composite including 1 or more of allograft loss, renal functional decline and biopsy proven rejection after 6 months:
- (i) Death-censored graft loss after 6 months posttransplant;
- (ii) Decline in renal function: greater than 20% decrease in estimated glomerular filtration rate (eGFR) calculated by Modification of Diet in Renal Disease equation (MDRD) eGFR between 6 months and last follow-up;
- (iii) Biopsy-proven late rejection after 6 months posttransplant: T cell–mediated acute rejection including borderline changes (t1); antibody-mediated acute rejection, either C4d negative or positive; or mixed rejection phenotype according to the Banff classification.27,28
For patients with more than 1 event, the primary outcome was determined as a time-to-first event.
We previously demonstrated that elevated 6-month urinary CCL2 was an independent predictor for graft loss, but not graft dysfunction or death with function.21 Therefore, these associations were also evaluated:
- (i) Normal graft function (n = 109) defined as 20% or less decline MDRD eGFR from 6 months to last follow-up, and protein/creatinine ratio of 50 mg/mmol or less at last follow-up.
- (ii) Graft dysfunction (n = 57) defined as greater than 20% decline MDRD eGFR from 6 months to last follow-up, and/or protein/creatinine ratio greater than 50 mg/mmol, and/or late biopsy-proven rejection.
- (iii) Death with graft function (n = 11).
- (iv) Graft loss after 6 months posttransplant (n = 8).
Initial immunosuppression was selected based on the presence/absence of pretransplant DSA; defined by single-antigen flow beads, AB0-blood group compatibility and HLA matching as described elsewhere29 and is detailed in Table 1. Normal immunological risk was defined as no DSA or ABO incompatible transplant. High immunological risk patients received induction therapy with polyclonal antithymocyte globulin and/or intravenous immunoglobulin. Subclinical rejection, including tubulitis t1, was treated with steroids and increasing baseline immunosuppression. Delayed graft function was defined as the need for 2 or more hemodialysis sessions during the first week after transplantation (n = 43/185).
Renal Allograft Biopsies
Clinically indicated allograft biopsies were performed when serum creatinine increased greater than 20% from baseline. Since 2001, surveillance biopsies have been performed at 3 and 6 months posttransplant. All allograft biopsies (2 cores, 16-gauge needle) were evaluated by light microscopy, immunofluorescence (C4d, HLA-DR), and immunohistochemistry (SV40 large T-antigen) and scored according to the Banff classification.27,28
Urine Sample Collection
Same-day clean catch urines were collected before performing the biopsy. Urines were centrifuged at 1750g for 10 minutes, and the supernatants aliquoted and stored at −80°C without any additives for future analysis.
Urine Protein Analyses
Measurements of total protein (benzethonium chloride method) and creatinine (enzymatic method) were performed on a Modula clinical chemistry analyzer (Roche Diagnostics, Roche, Switzerland). Urinary α1-microglobulin (α1m) was determined by nephelometry (Beckman-Coulter Nephelometry System, Brea, CA). Measurements of total protein and urinary α1-microglobulin were performed immediately after collection of urine samples.
Urinary CCL2 and CXCL10 Measurement
Urinary CCL2 and CXCL10 were retrospectively measured on stored urines by Enzyme-linked Immunosorbent Assay (ELISA) as previously described.15,30,31 CCL2 and CXCL10 ELISA were performed with commercially available antibodies (Peprotech, Rocky Hill, NJ) on a microplate reader (Biotek Synergy 4 & GEN 5 Software, Fisher Scientific) with good reproducibility. The CCL2 and CXCL10 intra-assay/inter-assay coefficients of variation were 2.2%/3.5% and 3.1%/11%, respectively, and the detection limit 1.95 pg/mL for both assays. Urine proteins are reported relative to urine creatinine to correct for dilution (ie, ng protein/mmol creatinine). Urinary CCL2 per creatinine ratio (CCL2:Cr) in healthy volunteers is 17.2 ±1.8 ng/mmol32 and urinary CXCL10 per creatinine ratio (CXCL10:Cr) in healthy volunteers is 0.3 ± 0.3 ng/mmol (unpublished data).
We used JMP Pro software version 11.0 (SAS Institute Inc., Cary, NC) for statistical analyses. For categorical data, Fisher exact test or Pearson χ2 test was used and data presented as counts and percentages. Parametric continuous data were analyzed by Student t tests. Nonparametric continuous data were summarized as median (interquartile range [IQR]) unless stated otherwise and analyzed by the Wilcoxon rank sum or Kruskal-Wallis rank sum tests. Significant results in the Kruskal-Wallis rank sum test were further analyzed with pairwise nonparametric tests. Multivariate Cox proportional hazards regression analysis was performed to assess independent predictors for the primary composite endpoint. Due to the risk of over-fitting the model, only variables significant on univariate analysis (defined as P < 0.10) were chosen as explanatory variables for the multivariate model. Event-free graft survival was analyzed by the Kaplan-Meier method and groups were compared using the log-rank test. A 2-tailed P value less than 0.05 was considered to indicate statistical significance.
The study population consisted of 185 of 228 patients transplanted from October 2005 to March 2009, with a minimum follow-up of 5 years (range, 5.0-8.5) (Figure 1). Fifty-two patients (28%) reached the primary outcome, at a median 6.0 years (IQR, 5.0-6.5). Eight patients had death-censored graft loss, 36 had graft functional decline, and 19 had biopsy-proven late rejection (Figure 1). During follow-up, 11 of 17 patients died with stable graft function at a median 3.0 years (IQR, 2.0-3.5) and the reasons were: sepsis (n = 4), cardiovascular (n = 2), malignancy (n = 1), suicide (n = 2), and unknown (n = 2). Six patients reached the primary outcome before they died.
In patients who reached the primary outcome, there were more HLA mismatches (P = 0.04) and fewer normal immunological risk patients (P = 0.03). Pretransplant DSA status and the use of thymoglobulin induction therapy were not different in patients reaching the primary outcome (P = 0.14 and P = 0.16, respectively, for comparison between the groups). Six-month allograft function measured by serum creatinine, MDRD eGFR, and total proteinuria were not different between groups. The primary outcome group showed increased urinary α1-microglobulin compared to the stable posttransplant group (P = 0.03). Finally, early subclinical and clinical rejection within the first 6 months posttransplant did not differ between groups (P ≥ 0.13, Table 1).
To ensure that the study population represented the entire transplant population, we compared it with patients excluded from the analysis (n = 43, Figure 1). The patient characteristics were well balanced with the exception of recipient age, which was slightly higher in the excluded group (median, 60 vs 55 years; P = 0.01; Table S1, SDC, http://links.lww.com/TP/B299).
Elevated 6-Month Urinary CCL2 Is Associated With Worse Long-term Outcomes
Six-month urinary CCL2:Cr was significantly elevated in the primary outcome group compared with those patients who did not reach the primary outcome (median urinary CCL2:Cr, 38.6 ng/mmol [IQR, 19.7-72.5] vs 25.9 ng/mmol [IQR, 16.1-45.8], P = 0.009; Table 1, Figure 2A). A sensitivity analysis was performed excluding 11 of 17 patients who died with stable graft function, and this did not change the findings (data not shown).
We next determined the 6-month urinary CCL2:Cr levels in patients with normal graft function, graft dysfunction, death with graft function and graft loss. Consistent with the previous study,21 patients with graft loss had the highest 6-month urinary CCL2:Cr compared with others (median, 66.5 ng/mmol [IQR, 35.9-98] vs ≤33.8 ng/mmol [IQR, 15.9-61.3], P overall = 0.004), whereas patients with death with graft function had similarly low urinary CCL2:Cr as those with normal graft function (median, 26.6 vs 24.1 ng/mmol; P = 0.97) (Figure 2B). Interestingly, the graft dysfunction group represented an intermediate phenotype with CCL2:Cr levels higher than normal graft function patients (P = 0.02), but lower than those with graft loss (P = 0.04; Figure 2B).
Prognostic Characteristics of Urinary Chemokines
Next, we evaluated the individual prognostic performance of urinary CCL2 for prediction of the primary endpoint and compared it with 6-month urinary CXCL10. Six-month urinary CCL2:Cr demonstrated an area under the curve of 0.62 (P = 0.001), and CXCL10:Cr had an area under the curve of 0.63 (P = 0.03). The optimal cutoffs for urinary CCL2:Cr and CXCL10:Cr were 70.0 and 0.70 ng/mmol, respectively; and these were used to classify patients as “high” and “low” subgroups for further analyses. At these cutoffs, CCL2:Cr demonstrated a high specificity (0.94) and negative predictive value (0.77) for late graft outcomes, whereas the sensitivity and positive predictive value were lower at 0.27 and 0.64, respectively. Similarly, the sensitivity/specificity and positive predictive value/negative predictive value of urine CXCL10:Cr was 0.79/0.47 and 0.37/0.85, respectively (Table S2, SDC, http://links.lww.com/TP/B299). Urinary CCL2:Cr and CXCL10:Cr were only modestly correlated (Spearman r = 0.35; P < 0.0001).
Urinary Chemokines Are Independent Predictors of Long-term Outcome
Total HLA mismatch was the only baseline univariate predictor of the primary outcome (P = 0.03), whereas pretransplant DSA and donor age demonstrated borderline significance at best (P = 0.05). Notably, high 6-month urinary CCL2:Cr and CXCL10:Cr were both strong univariate predictors of the primary outcome (P = 0.0002 and P = 0.0005, respectively), whereas other clinical features like early acute rejection, BKV viremia, total proteinuria, and MDRD eGFR were not significant (P ≥ 0.05, Table 2). In the multivariate model, the only independent predictors of the primary endpoint were urinary CCL2:Cr (HR, 2.86; 95% CI, 1.33-5.73; P = 0.009) and urinary CXCL10:Cr (HR, 2.35; 95% CI, 1.23-4.88; P = 0.009), whereas total HLA mismatch demonstrated borderline significance at best (P = 0.07) (Table 2).
Urinary Chemokines Stratify Patients Into Low-Risk Versus High-Risk Subgroups
Kaplan-Meier analysis on patients stratified by urinary CCL2:Cr levels demonstrated that low urinary CCL2:Cr correlated with higher event-free patient survival compared with patients with high urinary CCL2:Cr (P < 0.0001) (Figure 3). Patients with low urinary CCL2:Cr of 70.0 ng/mmol or less had 88% endpoint-free 5-year survival compared with 50% with high CCL2:Cr greater than 70.0 ng/mmol (P < 0.0001). We recently demonstrated that low urinary CXCL10:Cr (<0.70 ng/mmol) is also associated with freedom from the primary endpoint.26 Thus, we proceeded to evaluate their combined 6-month performance by stratifying the population into low CCL2/low CXCL10 (n = 69), intermediate (either CCL2 or CXCL10 elevated, n = 98), and high CCL2/high CXCL10 (n = 18) groups. The time-to-event analysis demonstrated that the high CCL2/high CXCL10 group had the worst endpoint-free survival compared with the low CCL2/low CXCL10 (P < 0.0001) and the intermediate group (P = 0.0009), respectively (Figure 4).
Urinary CCL2 and CXCL10 Are Associated with Different Allograft Outcomes
In a supplementary analysis, urinary CXCL10:Cr, but not CCL2:Cr, was associated with late rejection (n = 19 with late rejection vs n = 166 without; urinary CXCL10:Cr, P = 0.006; urinary CCL2:Cr, P = 0.5), whereas CCL2:Cr was more strongly associated with graft loss (n = 8 with graft loss vs n=177 without; CCL2:Cr, P = 0.006; CXCL10:Cr, P = 0.02) (Figure 5). Interestingly, urinary CXCL10:Cr levels were borderline elevated (P = 0.05) in high immunological risk (ie, DSA, ABO incompatible) patients (n = 42) which was largely driven by patients with pretransplant DSA (n = 33 with pretransplant DSA vs n = 143 with normal risk; P = 0.009). Conversely, urinary CCL2:Cr levels remained the same in high and normal immunological risk groups, which is consistent with the previous study demonstrating that 6-month urinary CCL2:Cr predicts graft loss independent of de novo DSA.21 Both urinary CCL2:Cr and CXCL10:Cr were associated with graft dysfunction (n = 57 with graft dysfunction vs n = 120 with either normal graft function or death with graft function; CCL2:Cr, P = 0.02; CXCL10:Cr, P = 0.03).
This prospective, unselected adult renal transplant cohort study independently validates 6-month urinary CCL2:Cr as an early prognostic marker for the composite outcome of renal function decline, biopsy-proven late rejection, and graft loss in patients receiving modern immunosuppression. A novel, stepwise increase in urinary CCL2:Cr between normal function, graft dysfunction, and graft loss was identified; and we further demonstrated that low 6-month urinary CCL2:Cr significantly correlated with event-free graft survival. These results extend upon our earlier findings that 6-month urinary CCL2:Cr is an independent predictor for the histological surrogates of 24-month IF/TA and IF/TA plus inflammation,15,16 the latter of which is significantly correlated with poor graft outcomes. While the prognostic characteristics of 6-month urinary CXCL10 and CCL2 for prediction of long-term outcomes were only moderate, this study demonstrated that 6-month urinary CCL2:Cr and CXCL10:Cr were the only independent predictors for the primary endpoint. Indeed, both urinary CCL2 and CXCL10 outperformed important clinical risk factors of graft outcome like proteinuria, eGFR, allograft rejection, and pretransplant DSA. Interestingly, patients with high urinary levels of both CCL2 and CXCL10 had the worst outcomes by Kaplan-Meier analysis.
Urinary levels of CCL2 and CXCL10 are increased in subclinical and clinical renal allograft rejection.31,33-37 CXCL10 is an interferon-γ dependent CXCR3 chemokine that recruits activated T cells to rejecting allografts and upregulates proinflammatory cytokine production,22-24 whereas CCL2 is a chemoattractant for monocyte/macrophages.12,13 M1 macrophages are activated by interferon-γ–dependent pathways and demonstrate a proinflammatory phenotype associated with worsening renal injury.38,39 Furthermore, macrophages can undergo an in situ phenotypic switch from M1 to M2 after renal tubular injury to promote repair/fibrosis,38,40 and this may be mediated via a CCL2/CCR2-dependent pathway.41
The consistent association of early urinary CCL2:Cr with poor long-term outcomes15,16,21 strongly supports a role for CCL2 and monocytes/macrophages in the pathogenesis of IF/TA, graft dysfunction, and loss. Indeed, CD68+ macrophage infiltration in 1-year surveillance biopsies with IF/TA and inflammation correlated with increased graft dysfunction and loss,18 and, more recently, CD68+ CD206+ (M2) macrophage infiltration in 1-year surveillance biopsies correlated with IF/TA severity and graft dysfunction at 3 years posttransplant.42 Furthermore, biopsies with M2 macrophage infiltration demonstrated alloimmune activation via upregulation of CCL2 and interferon-γ response genes (CXCL9, CXCL10), even in grafts in which the degree of inflammation was below the Banff threshold for rejection.42 These data suggest that macrophage infiltration in late surveillance biopsies is a negative prognostic feature, and we speculate that the association of elevated CCL2 with poor allograft outcome may be related in part to its role in mediating macrophage injury responses. We also speculate that this injury may be potentiated by interferon-γ–dependent chemokines (ie, CXCL10) causing persistent subclinical alloimmune inflammation as the time-to-event analysis demonstrated that renal outcomes were worse in patients in whom levels of both CCL2 and CXCL10 were elevated (ie, high overall early chemokine burden). A model of the relationships between elevated urinary CCL2 and CXCL10 with different pathways to graft loss is presented in Figure 6.
There are some limitations to this study. First, this is a single-center study; and although we studied an unselected, consecutive patient population, the data may not be generalizable to underrepresented ethnicities (eg, African-Americans). Second, this is a retrospective analysis of a prospective cohort and thus, it is not possible to obtain the missing 6-month urine samples. Third, de novo DSA data in this cohort is lacking. However, we have previously demonstrated that 6-month urinary CCL2 predicts death-censored graft loss independent of de novo DSA.21 This is further supported by the observed lack of association of pretransplant DSA with the primary outcome. Finally, the relative contributions of macrophage as compared to other pathophysiological pathways in the development of graft dysfunction and loss are unknown and cannot be determined by this observational study.
Taken together, this study confirms in an independent, prospective cohort that 6-month urinary CCL2 is a noninvasive, early predictor of renal allograft outcomes. The time-to-event analysis demonstrates that measuring both chemokines at 6 months provides important prognostic information, and this is further supported by the supplementary analysis which suggests that the chemokines reflect different pathways leading to poor allograft outcomes (eg, CXCL10: acute rejection, DSA26,43 and CCL2: macrophage activation with progression to IF/TA and graft loss).15,21 Thus, urinary CCL2 and CXCL10 may be used for the timely identification of patients at increased risk of graft loss when the injury may still be reversible. Such patients could undergo more intensive posttransplant monitoring, avoid drug minimization/withdrawal protocols, and be targeted for clinical trials to improve long-term graft outcomes. Early prognostic surrogate markers also have important implications for the broader transplantation community. Modern immunosuppression has decreased the incidence of FDA-approved endpoints (ie, graft loss, rejection), challenging the feasibility of adequately powered clinical trials. Thus, to develop new therapies, a critical unmet need in transplantation is the development of validated surrogate markers for phase 3 registration clinical trials,44 in which urine chemokines may play a crucial role. Notably, urinary CCL2 and CXCL10 are robust, reproducible, simple, and cost-effective markers that are easily measured by ELISA. Finally, the effectiveness of chemokine-guided posttransplant surveillance strategies needs to be prospectively evaluated in clinical trials.
The authors thank the staff of the University Hospital Basel Renal Transplant Unit and the histocompatibility laboratory for collection and processing of urine samples.
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