PRECLINICAL MODEL OF CHRONIC RENAL ALLOGRAFT REJECTION

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Advances in immunosuppressive therapy for transplantation have done little to decrease the percentage of allografts which eventually develop chronic rejection (CR). Studies proposed in this application collectively serve as a preclinical bridge with direct relevance to organ transplantation. The experiments are designed within the framework of the hypothesis that in response to oxidative stress associated with superoxide nitric oxide, and their reaction product, peroxynitrite, acidic fibroblast growth factor (FGF-1) mediates a transforming potential during chronic renal allograft rejection by an extracellular pathway. An underlying theme of these studies includes quantitating the production and targeted molecular reactions of reactive nitrogen/oxygen species that inactivate antioxidant defense mechanisms (manganese superoxide dismutase and glutathione) in the kidney allograft to compromise renal structure/function. To establish: (a) a cause-and-effect relationship between oxidant stress, FGF-1, and CR: (b) a diagnostic criteria for monitoring this process; and (c) therapeutic interventional strategies, the administration of neutralizing antibodies against FGF-1 and antioxidants will be evaluated for their ability to modulate the kinetics of developing CR lesions in an established allogeneic rat model of kidney transplantation. The availability of antibodies, nucleic acid probel/amplimers, and established techniques permits the dissection of responding molecular cascades during CR by analyses of mRNA (RT-PCR, hybridization) and protein (Western, ELISA, immunohistochemistry, enzymatic activity) expression in the kidney. Special emphasis will focus on the ability of FGF-1 to modulate growth (PCNA), peroxynitrite-induced apoptosis (TUNEL), and tyrosine nitration in target proteins. The correlation of urinary appearance of FGF-1, nitrite/nitrate, albumin, and nitrotyrosyl proteins with in situ development of pathologic lesions will form the basis of a non-invasive diagnostic assay for monitoring CR during renal transplant dysfunction. While the precise involvement of FGF-1, reactive nitrogen/oxygen species, antioxidants, and nitrotyrosyl proteins are not known, efforts within this application will elucidate detailed characteristics reflective of pathophysiologic processes associated with CR not only in the rat model but also in kidney transplanted primates and human patients undergoing immunosuppressive therapies.