We profiled 52 urine samples from 52 patients with biopsy specimens indicating acute rejection (26 acute T cellCmediated rejection and 26 acute antibody-mediated rejection) and 32 urine samples from 32 patients with acute tubular injury without acute rejection. (black/other RU 58841 categories), (%)11/15 (42/58)8/18 (31/69)9/23 (28/72)0.50?Cause of end stage kidney disease, (%)??Diabetes mellitus7 (27)4 (15)6 (19)0.90??Hypertension6 (23)7 (27)8 (24)??GN3 (12)5 (20)6 (19)??Others6 (23)4 (15)6 (19)??Unknown4 (15)6 (23)6 (19)?Donor information??Age (yr), mean (SD)43 (21)45 (13)53 (11)0.10??Women, (%)15 (68)10 (45)12 (44)0.20??Racial categories (black/other categories), (%)4/22 (15/85)8/18 (31/69)5/27 (16/84)0.50??Deceased donor organ, (%)10 (38)14 (54)19 (59)0.30?Human leukocyte antigen mismatches, mean (SD)4 (1.2)5 (1.6)4 (1.8)0.10?Cold ischemia time (deceased donors; h), median (IQR)21 (12C29)24 (12C26)28 (18C33)0.50?Induction therapy, (%)??Lymphocyte-depleting Thymoglobulin16 (62)16 (62)27b (84)0.02c??Lymphocyte-nondepleting IL-2 receptor antibody4 (15)8 (30)5 (16)0.01d??None6 (23)2 (8)0 (0)After transplant and before the index allograft biopsy?Delayed graft function, (%)8 (31)5 (19)19 (59)0.01?Maintenance immunosuppression, (%)??Tacrolimus26 (100)26 (100)32 (100)??Mycophenolate26 (100)26 (100)32 (100)??Corticosteroids16 (62)17 (65)7 (22)0.001?AR, (%)2 (8)2 (8)0 (0)?Bacterial urinary tract infections,e (%)9 (35)8 (31)5 (16)0.20?BK virus nephropathy, (%)0 (0)1 (4)0 (0)?Cytomegalovirus disease, (%)0 (0)0 (0)0 (0)At the time of the index allograft biopsyf?Time from transplantation to DLEU2 biopsy (mo), median (IQR)4.4 (0.6C10.5)16.2 (0.5C34.4)1.2 (0.6C2.8)0.03g?Biopsy within 1 mo of transplantation, (%)10 (39)9 (35)12 (38) 0.001?Biopsy between 1 and 12 mo of transplantation, (%)10 (39)3 (12)18 (56)?Biopsy beyond 12 mo of transplantation, (%)6 (23)14 (54)2 (6)?Indication for biopsy (creatinine increase/delayed graft function), (%)25/1 (96/4)23/3 (88/12)24/8 (75/25)0.10?Serum creatinine (mg/dl), median (IQR)3.20 (1.90C4.33)2.62 RU 58841 (2.01C4.29)3.10 (2.46C5.12)0.30?Urine protein-to-creatinine ratio, median (IQR)0.56 (0.3C2.4)1.03 (0.4C2.9)0.31 (0.2C0.9)0.10?Serum tacrolimus trough (ng/ml), median (IQR)5.2 (4.6C8.7)6.5 (4.4C8.9)9.3 (7.8C10.4) 0.001h?Biopsy information??AMR (I/II/III)i1/25/0??ACR (IA/IB/IIA/IIB/III)i5/15/4/2/0??ATI32??Focal necrosis/isometric vacuolization, (%)9/20 (28/63)??Positive staining for complement split product C4d, (%)0 (0)26 (100)0 (0)??Concomitant interstitial fibrosis/tubular atrophy (moderate RU 58841 to severe), (%)3 (12)7 (27)1 (3)0.10?Antibodies to one or more donor-specific human leukocyte antigens??Data available, (%)13 (50)22 (85)25 (78)???MFI of the highest rank donor-specific bead 10003 (23)0 (0)17 (68) 0.001???MFI=1000C30006 (46)2 (9)4 (16)???MFI=3000C10,0003 (23)10 (46)3 (12)???MFI 10,0001 (8)10 (46)1 (4)?Urine specimens??Collected on the day of biopsy, (%)18 (69)18 (69)21 (66)??Collected 1 d before/1 d after biopsy, (%)3/5 (12/19)4/4 (15/15)2/9 (6/28)0.70??Urine volume (ml), median (IQR)45 (28C70)43 (28C55)35 (25C45)0.10??Urinary cell total RNA quantity (value derived by chi-squared test for categorical variables or KruskalCWallis test for continuous variables. bIncludes one patient with alemtuzumab (Campath-1H) induction. cvalue based on chi-squared test of independence for three rows (lymphocyte-depleting induction immunosuppression, lymphocyte-nondepleting induction, and no induction) and three columns (ACR, AMR, and ATI). dvalue based on chi-squared test of independence for two rows (induction immunosuppression and no induction) and three columns (ACR, AMR, and ATI). eDefined as the presence of 105 colony forming units per milliliter of urine. fThree patients (two AMR patients and one ATI patient) had BK virus replication (1 copy of BK virus VP1 mRNA per picogram total RNA from urinary cells) in the urine collected at the time of biopsy. None of the three patients had BK virus nephropathy (negative for SV40 staining in the biopsy tissue). gValue (KruskalCWallis Test)Value (Dunns Test)values were calculated using KruskalCWallis test of no differences among the ACR, AMR, and ATI biopsy groups. After KruskalCWallis test, pairwise comparisons among the three groups were done using the Dunns test. values 0.05 were considered statistically significant. The rationale for the mRNAs selected for inclusion in the mRNA panel is provided in the Supplemental Appendix. The levels of all RU 58841 26 mRNAs and the levels of 18S rRNA were significantly different ([area under the ROC (AUC)=0.88], likelihood ratio test, axis. The AUC is the estimate of the expected value in an independent sample not used for deriving the diagnostic signature. Clinical Benefit of the Six-Gene Urinary Cell Diagnostic Signature We used decision curve analysis (Figure 3) to assess whether the signature was clinically beneficial.12 This analysis depicts the net benefit of the signature at various threshold probabilities (is 20%; this may vary among physicians). Our analysis showed that, across a range of reasonable threshold probabilities from 10% to 50%, the highest net benefit was for the diagnostic signature. The net reduction in avoidable biopsies per 100 patients when using the diagnostic signature is shown RU 58841 in Figure 3, lower panel. Open in a separate window Figure 3. Decision curve analysis to assess the clinical benefit of the six-gene urinary cell diagnostic signature to differentiate AR from ATI. We used the predicted probability for each patient from the 10-fold cross-validation in decision curve analysis to quantify the clinical benefit of the diagnostic signature in terms of the number of unnecessary biopsies that can be avoided in the diagnosis of AR. In the upper panel, the axis represents the net benefit ((true positive count/is the total number of patients, and is the threshold probability. Here, axis, the corresponding value on the axis represents the net reduction.