Recent studies have shown that cyclooxygenase metabolites may mediate the increased renal blood flow and GFR in remnant kidneys. These studies suggest an increase in cyclooxygenase enzyme activity per se rather than, or in addition to, increased substrate availability since there were still increases in glomerular prostaglandin production when exogenous arachidonic acid was added. There is evidence in nonrenal cells that prostaglandins may modulate matrix production, but the effects of intrarenal prostaglandins have not been determined upon the longer term, structural alterations occurring in the glomerulus and tubules with progressive nephron destruction following loss of functioning renal mass. The purpose of this project is to study the role and regulation of cyclooxygenase metabolites in the biology of progressive nephron destruction in a model of progressive noninflammatory nephron injury following renal ablation. Studies will be performed in complementary in vivo and in vitro models. The in vivo model will be a model of severe renal ablation (5/6 nephrectomy) in the rat. In vitro models will consist of 1) cultured rat mesangial cells that are subjected to the physical (cyclic mechanical stretch) and hormonal manipulations that simulate the glomerular milieu; and 2) proximal tubule cells employed in co-culture with cortical interstitial cells. There are three specific aims to the project. First, studies will examine alterations in abundance, and distribution of cyclooxygenase (prostaglandin G/H synthase) in the ablation model and will correlate with changes in distribution of p35 (lipocortin I), which has been postulated to play an important regulatory role in eicosanoid metabolism. These studies will be correlated with transcriptional and translational regulation of cyclooxygenase in vitro. The second set of studies will examine the role of cyclooxygenase metabolites in the accumulation of mesangial matrix in vivo following renal ablation and in vitro in response to mechanical stretch and/or angiotensin II or TGF-beta. Immunohistochemistry and in situ hybridization will be utilized to describe the time course of appearance of increased and altered mesangial matrix following renal ablation, and will study the affect of CEI and cyclooxygenase or thromboxane synthase inhibitors upon the abnormal matrix accumulation. Studies in cultured mesangial cells will utilize constructs of the alpha1(I) collagen promoter region linked to CAT to determine potential sites of transcriptional regulation in response to mechanical stretch, angiotensin II and/or cyclooxygenase metabolites. The third specific aim is to study cyclooxygenase products in mediation of tubulointerstitial alterations following renal ablation. In addition to in vivo localization of altered extracellular matrix production, in vitro studies will examine the effects of cyclooxygenase metabolites upon growth and collagen production of proximal tubule cells, alterations of cyclooxygenase metabolism and collagen production in cortical interstitial cells cultured from 5/6 nephrectomized rats, and interactions of cortical interstitial cells and tubule cells in the development of the abnormal accumulation of tubulointerstitial extracellular matrix.
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