Quantitative detection of immune activation transcripts as a diagnostic tool in kidney transplantation

Abstract

Procedures to diagnose renal allograft rejection depend upon detection of graft dysfunction and the presence of a mononuclear leukocytic infiltrate; however, the presence of a modest cellular infiltrate is often not conclusive and can be detected in non-rejecting grafts. We have pursued a molecular approach utilizing reverse transcription (RT)-PCR to test the diagnostic accuracy of multiple immune activation gene analysis as means to diagnose renal allograft rejection. The magnitude of intragraft gene expression of 15 immune activation genes was quantified by competitive RT-PCR in 60 renal allograft core biopsies obtained for surveillance or to diagnose the etiology of graft dysfunction. Results were compared with a clinicopathological analysis based upon the histological diagnosis (Banff criteria) and the response to antirejection treatment. During acute renal allograft rejection intragraft expression of the interleukin (IL)-7 (P < 0.001), IL-10 (P < 0.0001), IL-15 (P < 0.0001), Fas ligand (P < 0.0001), perforin (P < 0.0001), and granzyme B (P < 0.0015), but not IL-2, interferon gamma, or IL-4, genes is significantly heightened. Amplified RANTES and IL-8 gene transcripts are sensitive but nonspecific markers of rejection. A simultaneous RT-PCR evaluation of perforin, granzyme B, and Fas ligand identifies acute rejection, including cases with mild infiltration, with extraordinary sensitivity (100%) and specificity (100%). Effective antirejection therapy results in a rapid down-regulation of gene expression. The combined analysis of Fas ligand, perforin, and granzyme B gene expression by quantitative RT-PCR provides a reliable tool for diagnosis and follow-up of acute renal allograft rejection. Its accuracy and a potential rapid application within few hours suggest its use in the clinical management of renal transplant patients.

Figure 1

Sequences of oligonucleotide primers and competitive templates (CTs) used for the quantitation of 15 genes evaluated. Deletions and insertions are indicated by black and white portions of the bars, respectively. The mutated competitors are coamplified with wild-type cDNA in PCR, and amplified wild-type and competitive template sequences are readily identified as distinct bands by agarose gel electrophoresis.

Figure 2

Quantitative analysis of IL-2, IL-7, IL-15, P, GB, and FasL gene expression in 38 transplant core biopsies taken to aid in the differential diagnosis of graft dysfunction. Biopsies were also obtained from 2 donor kidneys prior to reperfusion. Lines indicate sequential biopsies taken during the course of rejection before and after treatment (ACR, acute cellular rejection; NR, nonrejecting kidneys with acute tubular necrosis or cyclosporine cytotoxicity; CR, chronic rejection; INF REC, infectious complications and recurrence of primary disease; and VASC, vascular complications).