Over the past year, we have accomplished the following:? 1. In our established clinical protocol, 03-DK-0132, The Expression of Connective Tissue Growth Factor and Other Mediators in the Pathogenesis of Chronic Allograft Nephropathy, and we have enrolled a total of 134 patients. We found that serum and urine CTGF levels are elevated in kidney transplant recipients compared to healthy individuals and in this limited data set, urine levels correlate to biopsy pathology, with the highest in patients with CAN. The results of these studies were published in the Am Journal of Transplantation 2006. We have an approved MTA and are currently analyzing a test set of samples with our collaborator Dr. Gregory Shultz. This data set includes over 900 urine and serum samples, with associated transplant biopsies and clinical data. Results will be entered into our database, Teleresults, and correlated to demographic, transplant, and other medical variables.? 2. To further investigate the mechanism of CTGF in renal fibrosis, we have developed a transgenic construct containing the CTGF gene under control of a proximal tubular epithelial cell specific promoter. Inhibition of CTGF has not been successful utilizing siRNA or antibody approaches and the KO is congenitally lethal. We are constructing a site directed CTGF KO within the kidney as well as a conditionally expressed KO.? 3. Mouse kidney transplants that develop CAN also have significant macrophage infiltration within the interstitium. Associated with this infiltration is a marked upregulation of macrophage activation transcripts, and genes associated with epithelial-mesenchymal transformation. In vitro mechanistic studies indicate that activated macrophages may stimulate expression of EMT markers on tubular epithelial cells when placed in co-culture, and that this is due to a soluble factor, that is not TGF-beta dependent. These kidney allografts also have marked expression of peritubular capillary C4d expression indicating a component of antibody mediated injury that may be enhanced by macrophage infiltration. We are examining the relationship between alloantibody, macrophage activation, and epithelial cell injury in this model to find ? 4. As the potency of immunosuppression has risen over the decade, so has graft loss due to BK polyomavirus nephropathy. We have identified the transcriptional profile of BK PVN and determined that while it is remarkably similar to acute rejection, markers of T cell cytotoxicity are dramatically elevated out of proportion to rejection. Moreover, more so than acute rejection which is tightly linked to CAN, PVN biopsies show transcriptional evidence of fibrogenesis. We are the first group to report transcriptional events in PVN and the presence of a pro-fibrotic milieu is also novel. With collaboration with both NIH IC and extramural investigators, we are identifying whether the profibrotic impact of infection is related to the immune response to virus or to an intrinsic response of tubular epithelial cells infected by virus.? 5. Using our real time PCR low density array as a platform, we continue to investigate, at a molecular level, the gene transcript profile of fibrogenesis, as noted in #4. We are exploring these molecules and other immune factors in transplant glomerulopathy, a pathologic entity associated with rapid rate of graft loss and high levels of proteinuria.? 6. Using an antibody microarray platform in collaboration with Dr. Srivastava at USUHS, we are analyzing the proteomic signature in both urine and blood in normal individuals, as well as in recipients with stable kidney transplant function and normal biopsies, as well as in those with acute cellular rejection, and chronic allograft nephropathy. Our preliminary results indicate that urine samples may more closely indicated intrarenal injury and that CAN recipients demonstrate marked upregulation and downregulation of a numberof signaling pathways compared to both stable function and acute rejection samples. We are further evaluating samples collected prior to the biopsy diagnosis to establish a time course of activity. We are identifying several signaling pathways to target in in vitro studies and in the mouse transplant model.? 7. In order to investigate the role of the dopamine D5 receptor, we have now transplanted a total of 89 kidneys for our collaborators Dr. Pedro Jose and Dr. David Sibley, between mutant D5KO and WT mice. These studies appear to indicate that there are non-renal mechanisms of blood pressure regulation, specifically mediated by this receptor. Despite alterations in baseline blood pressures, there is no difference in histology.
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