Urinary Biomarkers as Predictors of Long-Term Allograft Function After Renal Transplantation

Abstract

There is a recent evidence that urinary biomarkers are useful in the early prediction of delayed graft function (DGF) after renal transplantation and in assessing the severity and prognosis of acute tubular necrosis (1, 2). It is unknown whether urinary biomarkers would predict long-term allograft function after renal transplantation. We evaluated to see whether urinary biomarkers including N-acetyl-β-(d) glucosaminidase (NAG), kidney injury molecule (KIM)-1, and matrix metalloproteinase (MMP)-9 would predict 6- and 12-month allograft function after renal transplantation. NAG is a lysosomal brush border enzyme of proximal tubular cells, KIM-1 is a transmembrane glycoprotein in the proximal tubular cells, and MMP-9 is a gelatinase with proteolytic activity toward basement membrane components. Urinary levels of all three biomarkers are increased in ischemia-reperfusion injury (3–5). Urinary NAG levels may also be increased with proteinuria, calcineurin inhibitor toxicity, and acute rejection. The study protocol was approved by the institutional review board. After obtaining informed consent, 10 mL of urine was collected at hours 0, 6, 12, and 24 and on days 2 and 3 after the transplant surgery from 20 patients who underwent renal transplantation at our institution during a 2-month period (17 deceased donor and 3 live donor transplants). Each urine sample was then placed in a centrifuge at 3300 rpm for 3 min. Samples obtained in evening were placed on ice overnight and were centrifuged on the following morning. The urinary supernatant from centrifugation was frozen at −80°C and assayed in batches for urinary KIM-1, NAG, MMP-9, and creatinine levels. Urinary NAG was measured by colorimetric assay, whereas KIM-1 and MMP-9 were quantified using ELISA. The values were normalized for urinary creatinine concentration. DGF was defined as the requirement for dialysis in the first week after transplantation. Glomerular filtration rate (GFR) at 6- and 12-month posttransplantation time points were calculated using the modification of diet in renal disease equation. Average recipient age was 50.2± 9.8 years, two patients underwent preemptive transplantation, and seven had prior transplants. Other patient characteristics include cold ischemia time 28.2±14.4 hr, panel reactive antibody titer 7.8%±14.9%, human leukocyte antigen match 1.4±1.5, and human leukocyte antigen mismatch 3.9±1.6. Seven patients developed DGF, and five patients experienced acute rejection in the first 30 days posttransplantation. All 20 patients had functional allografts at 6 months, and 2 patients lost allograft function by the end of 12 months. The levels of individual biomarkers at different posttransplantation time points (absolute values not shown) were correlated with 6- and 12-month GFR using Spearman's correlation (ρ) and are shown in Table 1. Receiver operator characteristic curve analysis showed that a threshold urinary KIM-1 value of 9.3 mU/mg urine creatinine on day 2 after transplantation predicted a 12-month GFR less than 45 mL/hr with a sensitivity of 89%, specificity of 73%, positive predictive value of 91%, and negative predictive value of 89% with area under the curve of 0.81. Same parameters for urine NAG value of 20 mU/mg urine creatinine from 24-hr urine sample were 67%, 73%, 67%, 73%, and 0.73 respectively.TABLE 1: Correlation of urinary biomarkers with 6- and 12-mo GFROur data show that early posttransplant urinary NAG and KIM-1 levels showed a significant negative correlation with 6- and 12-month allograft function after renal transplantation. Persistent increase of urinary biomarkers may reflect an ongoing inflammatory cascade triggered by the initial ischemia-reperfusion injury or subclinical acute rejection episodes with subsequent poor long-term allograft function. Small sample size is a limitation of our study. In the future, a panel of urinary biomarkers with specific cutoff values may help to identify a subgroup of high-risk renal transplant recipients who may warrant closer monitoring. Urinary biomarkers may open an exciting new era in transplantation research. ACKNOWLEDGMENTS The authors thank Won Han, M.D., for doing the biomarker assay. Kalathil K. Sureshkumar Richard J. Marcus Division of Nephrology and Hypertension Department of Medicine Allegheny General Hospital Pittsburgh, PA