MYCOPHENOLATE MOFETIL BUT NOT THE TYPE OF CALCINEURIN INHIBITOR (CYCLOSPORINE VS TACROLIMUS) INFLUENCES THE INTRAGRAFT MRNA EXPRESSION OF CYTOKINES IN HUMAN KIDNEY ALLOGRAFT BIOPSIES BY IN SITU RT-PCR ANALYSIS.

Abstract

P992 Aims: The aim of the study was to assess the molecular background of anti-allograft response by the detection of low-abundance mRNA of cytokines. Methods: Our analysis included 56 renal needle core biopsies from kidney transplant recipients who received their grafts from cadaveric donors. Biopsies were performed in case of graft dysfunction and were assessed on the basis of histopathological evaluation using Banff classification. Recipients were immunosuppressed with one of calcineurin inhibitors (CNI): cyclosporine (CsA) or tacrolimus (TAC) + prednisone + azathioprine or mycopnenolate mofetil (MMF). Tubular and glomerular expression of IL-2, IL-6, IL-10, IFN-γ, TGF-β1 and PDGF-B mRNA were assessed using semiquantitative evaluation of the RT-PCR in situ. The principle was to perform RT-PCR amplification of target mRNA in situ on paraffine tissue sections. Following the amplification DNA copies were hybridized with digoxigenin-labelled nucleotides. This procedure resulted in a light microscopy visualization of granular precipitates at the sites of investigated mRNA chains. Cytokine mRNA expression was quantified by calculating the mean number of positive points per area unit in all glomeruli and 30 randomly chosen tubules by two independent investigators, blinded to the status of the biopsy. Constitutive gene expression was examined in renal specimens obtained from living donor kidney biopsies taken during harvesting of the organ, before procurement. Results: We found that biopsies from patients treated with CNI + MMF were different from those treated with CNI + azathioprine. The tubular expressions of mRNA for IL-6 and TGF- β1 in biopsies with acute rejection (n=34) obtained from patients treated with MMF (n=12) were significantly lower than in biopsies obtained from patients treated with azathioprine (n=22) (p<0.02). Moreover 80% of the recipients in the MMF-treated group had serum creatinine levels less than 2 mg/dL one year after biopsy whereas in the azathioprine-treated group only 25% of the recipients had preserved renal function in the same time (p<0.05). There was no significant difference between the groups treated or not with corticosteroids. Conclusions: Our study did not confirmed previous thesis, that CsA and TAC differ in their ability to stimulate overproduction of TGF-β. The pattern of examined cytokines gene expression did not differ between patients under different CNI treatment. These results indicate strong inhibition of humoral response and downregulation of fibrosis in these recipients that may protect from the development of chronic rejection. Our results support the usefulness of MMF in the long-lasting care of renal transplant recipients.