Presented at:
American Transplant Congress, Washington, DC,
June 3, 2003
Am J Transpl 3 Suppl 5: 448, 2003
Noninvasive surrogate for protocol biopsies of renal allografts and a mechanistic guide for the weaning of immunosuppressive therapy
Priya Anantharaman, Darshana Dadhania, Thangamani Muthukumar, Meredith Aull, Surya Sehsan, Choli Hartono, David Serur, Ruchuang Ding, Baogui Li, Manikkam Suthanthiran, Sandip Kapur
Div of Nephrology, Dept of Medicine & Transplantation Medicine, Weill Medical College of Cornell University, New York, NY; Dept of Pharmacy, Weill Medical College of Cornell University, New York, NY; Dept of Pathology, Weill Medical College of Cornell University, New York, NY; The Rogosin Institute, New York Presbyterian Hospital, New York, NY; Dept of Surgery, Weill Medical College of Cornell University, New York, NY
Immunosuppressants have improved short-term outcome but extort a high price in the long-term. Innovative immunosuppression weaning protocols are being explored. Any rational guide for reductions in therapy is urgently needed. Protocol biopsies, though ideal, are complicated by sampling errors and morbidity. We explored the hypothesis that urine mRNA profiling is a surrogate for protocol biopsies. We tested the hypothesis that mRNA levels for cytotoxic attack proteins granzyme B and perforin in urinary cells would be a surrogate for protocol biopsies. We collected 63 urine specimens from 44 renal allografts recipients; 15 were on a steroid free immunosuppression protocol and underwent protocol biopsies at months 1 and 3 post-transplantation. 30 urine specimens were obtained at time of 30 protocol biopsies (normal by Banff 97). 33 urine specimens (29 patients) were from 33 diagnostic biopsies that met Banff 97 acute rejection criteria. We measured mRNA levels (copies/ug of total RNA) of granzyme B and perforin using real-time quantitative PCR and compared urinary mRNA levels at the time of protocol biopsies with those at the time of diagnostic biopsies with acute rejection. Our investigation demonstrated that (1) median mRNA levels of granzyme B in urine at 1 month protocol biopsies (2840 copies) and 3 month protocol biopsies (1910 copies) were significantly lower than median granzyme B mRNA level (88544 copies) in urine at the time of acute rejection (P<0.001). (2) median mRNA levels of perforin at 1 month protocol biopsies (1720 copies) and 3 month protocol biopsies(568 copies) were significantly lower than perforin mRNA levels (38914 copies) in urine at time of acute rejection ( P<0.001); (3) among the urine specimens at time of protocol biopsies, only 2 had mRNA levels higher than the cutoff point (determined by receiver operating characteristic curve analysis) for acute rejection. Our data demonstrating that 28 of 30 protocol biopsies were predicted by urinary cell levels of mRNA for granzyme B or perforin suggest that measurement of mRNA for granzyme B and perforin is an excellent surrogate for protocol biopsies. Furthermore mRNA levels apppear to reflect immunological quiescence - an important consideration for scheduled weaning of immunosuppressants.