Introduction
Materials and methods
Study design
Sample collection and the experimental procedure
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(1) Sample collection: ① Donor blood sample collection: When donor death was declared, donor peripheral blood samples (10 ml) were collected into a tube containing EDTA. ② Recipient blood sample collection: Recipient peripheral blood samples were collected into tubes (10 ml) containing EDTA before the induction of immunosuppression.
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(2) Flow cytometry experimental procedure: The frequency of Tregs was measured using flow cytometry. The main experimental equipment includes: CTYOMICS FC 500 flow cytometer, lysis solution (Beckman Coulter, Inc., CA, USA); CD4-FITC, CD25-PC5, and CD127-PE antibodies (BD Pharmingen, San Diego, CA, USA); and phosphate-buffered saline (Bwsm, Inc., Beijing, China).
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(3) ELISA experimental procedure: Plasma levels of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), cytotoxic T-lymphocyte-associated protein-4/cluster of differentiation 152 (CTLA-4/CD152), interleukin-35 (IL-35), and high mobility group Box 1 protein (HMGB1) were measured in the groups using commercially available enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions. The main experimental equipment included IL-2, IL-4, IL-6, IL-10, CTLA-4/CD152, IL-35, and HMGB1 ELISA kits (ML Bio Inc., Shanghai, China).
Study end points
Statistical analyses
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(1) Construction of the prediction model: This analysis was performed based on the discovery cohort (n=71) using a multivariate logistic regression analysis. The independent predictors of DGF were identified if their P value was less than 0.05. The regression coefficients of the predictive variables in the logistic regression model were used to establish the nomogram to predict DGF.
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(2) Validation of the prediction model: Model efficacy was verified by analyzing the data from the discovery cohort (internal validation) and the validation cohort (external validation). ① Internal validation: The predictive ability of the prediction model was assessed using a c‑statistic of the receiver operating characteristic curve (ROC), and the 95% Confidence Interval (CI) of the areas under the ROC curves (AUCs) were nonparametrically obtained by bootstrap resampling (1000 bootstraps). The model prediction threshold was compared by performing decision curve analysis (DCA). By drawing a calibration diagram (1000 bootstraps), the performance characteristics of the model are shown graphically. We calculated the Brier score and R-squared values of the model. ② External validation: This analysis was repeated using data from the validation cohort. The validation cohort was divided into ten groups, and the DGF prediction model calibration was evaluated using the Hosmer‑Lemeshow statistic.
Results
Donor, recipient, and transplant characteristics
variable | Discovery cohort (n = 71) | Validation cohort (n = 32) | P |
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Recipient characteristics | |||
Age, yr | 37.97 ± 11.49 | 37.28 ± 13.50 | 0.790 |
Gender | 0.333 | ||
Male | 47(66.2%) | 18(56.3%) | |
Female | 24(33.8%) | 14(43.8%) | |
BMI, kg/m2 | 21.44(18.67–24.36) | 20.10(18.11–23.53) | 0.259 |
Time on dialysis, m | 24(15–60) | 24(12–59.25) | 0.338 |
Dialysis, HD | 51(71.8%) | 19(59.4%) | 0.403 |
Immunosuppression regimen | 0.267 | ||
ATG,Tac, MMF, steroid | 15(21.1%) | 10(31.3%) | |
Basiliximab,Tac, MMF,steroid | 61(85.9%) | 24(75.0%) | |
Pre-Tx Creatinine,mg/dL | 12.05 ± 3.68 | 11.03 ± 3.43 | 0.189 |
history of hypertension | 61(85.9%) | 22(78.6%) | 0.177 |
history of diabetes mellitus | 3(4.2%) | 0(0%) | 0.238 |
Donor characteristics | |||
Age, (yr) | 44(17–48) | 29.5(15–51.25) | 0.471 |
Gender | 0.545 | ||
Male | 57(80.3%) | 24(75.0%) | |
Female | 14(19.7%) | 8(25.0%) | |
BMI, kg/m2 | 22.86(18.81–24.22) | 23.75(18.11–24.80) | 0.526 |
Terminal Creatinine, mg/dL | 0.88(0.67–1.52) | 0.72(0.59–1.46) | 0.279 |
The donor type | 0.155 | ||
DCD | 13(18.3%) | 4(12.5%) | |
DBD | 52(73.2%) | 28(87.5%) | |
DBCD | 6(8.5%) | 0(0%) | |
Donor history of hypertension | 32(45.1%) | 10(31.3%) | 0.187 |
Donor history of diabetes mellitus | 16(22.5%) | 3(9.4%) | 0.111 |
Transplant characteristics | |||
CIT,h | 9(7–11) | 9(7–9.375) | 0.309 |
HLA mismatches | 5(4–5) | 4(4–5) | 0.371 |
variable | Discovery cohort (n = 71) | Validation cohort (n = 32) | ||||
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DGF(n = 23) | IGF(n = 48) | p | DGF(n = 12) | IGF(n = 20) | P | |
Recipient characteristics | ||||||
Age, yr | 39.26 ± 10.767 | 37.35 ± 11.883 | 0.517 | 36.67 ± 13.30 | 37.65 ± 13.95 | 0.846 |
Gender | 0.904 | 0.854 | ||||
Male | 15(65.2%) | 32(66.7%) | 7(58.3%) | 11(55.0%) | ||
Female | 8(34.8%) | 16(33.3%) | 5(41.7%) | 9(45.0%) | ||
BMI, kg/m2 | 22.53 ± 5.01 | 21.49 ± 3.58 | 0.317 | 20.38 ± 4.23 | 20.98 ± 3.20 | 0.654 |
Time on dialysis, m | 36(24–84) | 24(12.5–49.5) | 0.110 | 24(13.75–29.75) | 22(12–60) | 0.876 |
Dialysis,HD | 16(69.6%) | 35(72.9%) | 0.506 | 9(75.0%) | 10(50%) | 0.289 |
Immunosuppression regimen | 0.930 | 0.076 | ||||
ATG,Tac, MMF, steroid | 5(21.7%) | 10(20.8%) | 6(50%) | 4(20.0%) | ||
Basiliximab,Tac, MMF,steroid | 18(78.3%) | 38(79.2%) | 6(50%) | 16(80.0%) | ||
Pre-Tx Creatinine, mg/dL | 12.04 ± 3.53 | 12.05 ± 3.79 | 0.988 | 11.19 ± 3.51 | 10.93 ± 3.48 | 0.841 |
history of hypertension | 17(73.9%) | 44(91.7%) | 0.044 | 8(66.7%) | 16(80.0%) | 0.399 |
history of diabetes mellitus | 0(0%) | 3(6.3%) | 0.221 | 0(0%) | 0(0%) | NA |
Donor characteristics | ||||||
Age, yr | 43(35–47) | 46(17–49.75) | 0.631 | 38.50 ± 18.76 | 27.70 ± 15.87 | 0.092 |
Gender | 0.106 | 0.092 | ||||
Male | 21(91.3%) | 36(75.0%) | 11(91.7%) | 13(65.0%) | ||
Female | 2(8.7%) | 12(25.0%) | 1(8.3%) | 7(35.0%) | ||
BMI, kg/m2 | 23.39(18.37–24.46) | 22.49(19.42–24.22) | 0.671 | 24.62(18.53–25.48) | 21.35(18.03–24.46) | 0.226 |
Terminal Creatinine, mg/dL | 1.52(0.87–4.24) | 0.80(0.67–1.08) | 0.001 | 0.37(0.92–1.74) | 0.67(0.56–0.80) | 0.003 |
The donor type | 0.490 | 0.581 | ||||
DCD | 6(26.1%) | 7(14.6%) | 2(16.7%) | 2(10%) | ||
DBD | 15(65.2%) | 37(77.1%) | 10(83.3%) | 18(90%) | ||
DBCD | 2(8.7%) | 4(8.3%) | NA | NA | ||
Donor history of hypertension | 15(65.2%) | 17(35.4%) | 0.018 | 4(33.3%) | 2(10.0%) | 0.102 |
Donor history of diabetes mellitus | 10(43.5%) | 6(12.5%) | 0.003 | 3(25.0%) | 0(0%) | 0.019 |
Transplant characteristics | ||||||
CIT,h | 10.61 ± 2.82 | 8.36 ± 2.27 | 0.001 | 9.5 ± 1.23 | 7.95 ± 1.34 | 0.006 |
HLA mismatches | 5(4–6) | 5(4–5) | 0.663 | 4(4–5.75) | 4.5(4–5) | 0.637 |
Variables | Discovery cohort (n = 71) | Validation cohort (n = 32) | ||||
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IGF(n = 48) | DGF(n = 23) | p | IGF(n = 20) | DGF(n = 12) | p | |
Donor | ||||||
IL-2(pg/mL) | 74.23 ± 23.96 | 96.42 ± 26.46 | 0.001 | 76.22(63.25–95.64) | 97.70(89.56–113.83) | 0.003 |
IL-4(pg/mL) | 4.55 ± 1.87 | 3.96 ± 2.17 | 0.240 | |||
IL-6(pg/mL) | 6.04 ± 1.55 | 5.37 ± 2.12 | 0.138 | |||
IL-10(pg/mL) | 67.33 ± 31.53 | 73.59 ± 26.19 | 0.412 | |||
CTLA-4;CD152(pg/mL) | 137.91 ± 58.81 | 136 ± 71.36 | 0.909 | |||
IL-35(pg/mL) | 9.08(5.22–10.43) | 9.67(5.79–10.78) | 0.632 | |||
HMGB1(pg/mL) | 5643.15 ± 1493.09 | 5937.63 ± 1968.60 | 0.486 | |||
recipient | ||||||
IL-2(pg/mL) | 99.62 ± 23.45 | 94.87 ± 20.93 | 0.411 | |||
IL-4(pg/mL) | 5.95 ± 1.79 | 6.26 ± 1.19 | 0.454 | |||
IL-6(pg/mL) | 7.65 ± 1.50 | 7.39 ± 1.41 | 0.481 | |||
IL-10(pg/mL) | 92.76 ± 21.57 | 89.61 ± 20.24 | 0.559 | |||
CTLA-4;CD152(pg/mL) | 214.50 ± 54.42 | 213.83 ± 50.69 | 0.961 | |||
IL-35(pg/mL) | 11.01 ± 2.11 | 11.05 ± 2.86 | 0.944 | |||
Treg (%) | 5.10 ± 2.39% | 5.34 ± 2.53% | 0.700 |
Univariate and multivariate analyses and construction of the DGF prediction model
Variables | Univariate analysis | ||
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OR | 95%CI | P | |
Recipient characteristics | |||
Age, yr | 1.015 | (0.971–1.061) | 0.511 |
Gender | 0.938 | (0.329–2.671) | 0.904 |
BMI, kg/m2 | 1.065 | (0.942–1.203) | 0.313 |
Time on dialysis, m | 1.008 | (0.995–1.021) | 0.239 |
Dialysis,HD | 1.295 | (0.534–3.143) | 0.568 |
Immunosuppression regimen | 1.056 | (0.314–3.544) | 0.930 |
Pre-Tx Creatinine, mg/dL | 1.000 | (0.988–1.002) | 0.987 |
history of hypertension | 0.258 | (0.065–1.027) | 0.055 |
history of diabetes mellitus | NA | NA | NA |
Donor characteristics | |||
Age, yr | 1.004 | (0.976–1.032) | 0.787 |
Gender | 3.500 | (0.713–17.176) | 0.123 |
BMI, kg/m2 | 1.022 | (0.890–1.173) | 0.760 |
Terminal Creatinine, mg/dL | 2.279 | (1.395–3.722) | 0.001 |
The donor type | 0.499 | ||
DCD | REF | ||
DBD | 1.714 | (0.228–12.890) | 0.601 |
DBCD | 0.811 | (0.134–4.907) | 0.819 |
Donor history of hypertension | 3.419 | (1.206–9.695) | 0.021 |
Donor history of diabetes mellitus | 5.385 | (1.641–17.664) | 0.005 |
Transplant characteristics | |||
CIT,h | 1.438 | (1.143–1.809) | 0.002 |
HLA mismatches | 1.000 | (0.692–1.444) | 0.998 |
Donor serum markers | |||
IL-2(pg/mL) | 1.036 | (1.013–1.059) | 0.002 |
IL-4(pg/mL) | 0.857 | (0.663–1.107) | 0.238 |
IL-6(pg/mL) | 0.800 | (0.595–1.076) | 0.140 |
IL-10(pg/mL) | 1.007 | (0.990–1.025) | 0.407 |
CTLA-4(pg/mL) | 1.000 | (0.992–1.008) | 0.908 |
IL-35(pg/mL) | 1.002 | (0.834–1.205) | 0.982 |
HMGB1(pg/mL) | 1.000 | (1.000–1.000) | 0.481 |
Recipient serum markers | |||
IL-2(pg/mL) | 0.990 | (0.968–1.013) | 0.990 |
IL-4(pg/mL) | 1.128 | (0.826–1.539) | 0.449 |
IL-6(pg/mL) | 0.881 | (0.623–1.247) | 0.476 |
IL-10(pg/mL) | 0.993 | (0.969–1.017) | 0.553 |
CTLA-4(pg/mL) | 1.000 | (0.990–1.009) | 0.960 |
IL-35(pg/mL) | 1.008 | (0.815–1.245) | 0.943 |
Treg frequency(%) | 1.042 | (0.848–1.281) | 0.695 |
Variables | Multivariate analysis | ||
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OR | 95%CI | P | |
Donor characteristics | |||
Terminal Creatinine, mg/dL | 0.749 | 1.054–4.245 | 0.035 |
Donor history of diabetes mellitus | 1.789 | 1.191–30.048 | 0.030 |
Transplant characteristics | |||
CIT,h | 0.368 | 1.046–1.995 | 0.026 |
Donor serum markers | |||
IL-2(pg/mL) | 0.047 | 1.017–1.080 | 0.003 |
ROC Area (95% CI) | Cut-Off Point | Sensitivity (%) | Specificity (%) | |
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Discovery cohort | ||||
Donor Terminal Creatinine(mg/dL) | 0.753(0.622–0.883) | 1.3178 | 60.9% | 87.5% |
Donor history of diabetes mellitus | 0.655(0.510–0.799) | 0.5 | 43.5% | 87.5% |
CIT(h) | 0.706(0.573–0.839) | 11.25 | 43.5% | 91.7% |
Donor IL-2(pg/mL) | 0.714(0.585–0.843) | 66.415 | 95.7% | 45.8% |
Prediction_system | 0.894(0.798–0.955) | -0.896 | 86.96% | 85.42% |
KDRI | 0.764(0.649–0.857) | 1.13 | 82.61% | 62.50% |
DGFS | 0.720(0.601–0.820) | 0.2767 | 69.57% | 66.67% |
Validation cohort | ||||
Donor Terminal Creatinine(mg/dL) | 0.817(0.640–0.931) | 0.676 | 91.7% | 65% |
Donor history of diabetes mellitus | 0.625(0.437–0.789) | 0 | 25% | 100% |
CIT(h) | 0.769(0.586–0.899) | 8 | 83.33% | 65% |
Donor IL-2(pg/mL) | 0.819(0.643–0.932) | 87.11 | 100% | 55% |
Prediction_system | 0.879(0.715–0.967) | -0.984 | 0.8333 | 0.800 |
KDRI | 0.829(0.655–0.938) | 0.80 | 100% | 60% |
DGFS | 0.667(0.479–0.823) | 0.327 | 66.7% | 80% |