Recent evidence suggests that biochemical reactions occurring during reperfusion of ischemic kidneys accentuate ischemic injury. Activated neutrophils (PMN) appear to contribute to this process since 1) perfusates from ischemic kidneys recruit PMN and 2) ischemic kidneys reperfused with PMN by the isolated kidney technique had more severe impairment of renal function than kidneys reperfused without PMN. The major targets of PMN attack in ischemia/reperfusion are vascular tissue and proximal tubular epithelium. Activated PMN release a number of mediators which could be toxic. These include oxygen metabolites, myeloperoxidase, and proteases. The target site at the cellular level of activated PMN has not been clearly defined. Cell membrane components including receptors and signalling enzymes appear to be likely targets because these are the first cell domains to come into contact with these highly reactive compounds. The present Hypothesis is that PMN cause renal dysfunction by attacking these and other key elements in vascular and epithelial cell membranes and that O2 metabolites, myeloperoxidase and proteases contribute to cell injury. However, because of differences in molecular size, charge and substrate specificity, the target of oxidant and protease attack differs.
The Specific Aims are to determine the effects of activated PMN on 1) vascular smooth muscle cells in culture. We will use the following probes of cell injury: a) membrane composition; b) receptor expression; c) basal and agonist stimulated phospholipase C (PLC) activity and calcium signalling; 2) polarized proximal tubular epithelial cells in culture. Probes of cell injury to be studied include a) basolateral membrane composition; b) basolateral receptor expression; c) basal and agonist stimulated adenylate cyclase and PLC activity, paracellular leak and cellular transport. The Methods to be utilized include monolayers of vascular smooth muscle and epithelial cells from rat proximal tubules grown on porous filters. With these methods, target tissue can be co-incubated with PMN and with specific PMN products. Moreover, the basolateral side of proximal tubule cells can be selectively studied. A variety of morphologic, biochemical, and physiologic techniques will be applied. The Significance of these studies is to improve understanding of mechanisms of kidney injury in ischemic renal failure. This new information may also be relevant to other types of renal diseases including glomerulonephritis and nephrotoxic renal failure.
| Linas, S L; Whittenburg, D; Parsons, P E et al. (1995) Ischemia increases neutrophil retention and worsens acute renal failure: role of oxygen metabolites and ICAM 1. Kidney Int 48:1584-91 |
| Linas, S L; Whittenburg, D; Parsons, P E et al. (1992) Mild renal ischemia activates primed neutrophils to cause acute renal failure. Kidney Int 42:610-6 |
