More than the Eyes Can See: Predicting Albuminuria and Rejection Using Urinary Cell Gene Signatures of Histologically Normal Kidney Allograft Biopsies

Background: It is well-established that molecular alterations often precede histological changes. Thus, molecular signatures of histologically normal biopsies have the potential to predict future outcomes. Methods: To test the hypothesis that urinary cell gene signatures could predict future adverse events even when biopsies do not show acute Banff lesions, we quantified, using customized RT-qPCR assays, copy numbers of CD3E mRNA, CXCL10 mRNA and 18S rRNA (urinary cell 3-gene signature) and BKV-VP1 mRNA in urines matched to histologically normal kidney allograft biopsies. Urine albumin-to-creatinine ratio (UACR) at 12 months post-biopsy was retrieved from electronic medical records. Statistical analysis was performed to determine whether transcript abundance predicts future development of adverse outcomes. Results: UACR higher than 300 mg/g (macroalbuminuria) at 12 months post-biopsy was observed in 41 of 133 patients (31%). The median urine albumin excretion was 1136 mgs in those with macroalbuminuria at 12 months post-biopsy and 34 mgs in the those without (Mann-Whitney P<0.0001). Median Banff acute lesion scores for g, ptc, i, and t were zero in those who did or did not manifest macroalbuminuria, and all biopsies were negative for SV40 immunostaining. The urinary cell 3-gene signature score was higher in those who subsequently manifested macroalbuminuria than in those who did not (median score: -1.19 vs. -1.55; P=0.03). Also, a 3-gene score above -1.213 (TCMR diagnostic cutpoint) was more frequent in those who developed macroalbuminuria than in those who did not (Fischer’s exact test P=0.02). The 3-gene score was higher in urines with BKV-VP1 mRNA copies above the BKVN diagnostic threshold compared to urines below the threshold (median 3-gene score: -0.325 vs. -1.475, P=0.003). Future acute rejection episode was more frequent in those with BKV-VP1 mRNA copies above vs. below the threshold (Fisher’s Exact test P=0.04) Conclusion: Our findings demonstrate that urinary cell molecular signatures predict future outcomes even in the absence of Banff lesions or SV40 positivity. These results support the integration of molecular diagnostics into post-transplant surveillance to enhance early risk stratification and intervention.