CYTOKINE mRNA EXPRESSION ANALYSIS IN RENAL TRANSPLANTATION – A NEW PROTOCOL BASED ON A NON–INVASIVE SCREENING METHOD IN URINE CELLS FOR ACUTE REJECTION

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TITLE: CYTOKINE mRNA EXPRESSION ANALYSIS IN RENAL TRANSPLANTATION – A NEW PROTOCOL BASED ON A NON–INVASIVE SCREENING METHOD IN URINE CELLS FOR ACUTE REJECTION

Katja Kotsch,¹ Philipp Tretow,¹ Athanasios Vergopoulos,¹ Hans–Dieter Volk,¹ Petra Reinke.²

¹Institute of Medical Immunology, CharitéHumboldt–University, Berlin, Germany; ²Department of Nephrology, CharitéHumboldt–University, Berlin, Germany

Introduction: The diagnosis of acute clinical and subclinical rejection in renal transplantation is usually performed by the invasive procedure of allograft needle biopsy. Recently, Li et al. (N Engl J Med 2001; 344:947–54) described a non–invasive method by analysing gene expression profiles of perforine and granzyme B in urine sediment. Their investigations revealed a strong correlation between acute rejection and upregulation of these genes. We further improved this method by application of a real–time RT–PCR (TaqMan–PCR) allowing very precise quantification of gene expression.

Furthermore, we applied a linear amplification technique (T7 polymerase) to get sufficient amounts of cDNA from 50 ml urine for analysing a broad range of cell markers, cytokines, chemokines and chemokine–receptors that play an important role in infiltration, recruitment and regulation of immune competent cells.

Methods: We collected 3 times/week urine samples from renal–allograft recipients (n = 26) during hospitalization and once per week from outpatients over a time period of 3 months after Tx. The mRNA was isolated and amplified from urinary cells and mRNA expression was measured for CD3, TNF–a, TGF–b, IFN–g, IL–2, IL–10, perforine, granzyme B, FasL, RANTES, CCR1 and the constitutively expressed HPRT gene (house–keeping gene).The results were correlated with the obtained biopsy data from patients with acute rejection.

Results: Within the first 3 months after Tx 5 from 26 patients showed an acute rejection episode. The RT–PCR revealed in these patients a significant upregulation of the chemokine RANTES and CD3 prior to biopsy proven rejection diagnosis.

Conclusion: Messenger RNA expression measurement of defined markers in urinary cells of renal–allograft recipients allows the early non–invasive detection of kidney graft rejection. The further clinical application may lead to a promising stable and reliable alternative technology for rejection diagnosis.