CHARACTERIZATION OF FRESH MECHANICALLY ISOLATED RAT PROXIMAL TUBULE SUSPENSIONS DURING PROLONGED INCUBATION

The majority of methodologies for tubule isolation involve the use of proteolytic enzymes to release the elemental nephronal regions from th eir connective tissue matrix. Although concerns have long been felt re garding potential structural damage incurred by the use of these enzym es, little has been done to characterize alternative non-enzymatic met hods-with most of that being conducted on rabbit proximal tubules (PTs ). The present work is performed on rat kidneys using the standard mec hanical technique of Brendel and Meezan (1975), which involves perfusi on, loading the renal vasculature with magnetic iron oxide, disrupting the cortex with a series of meshes, removing the glomeruli with a mag net and finally harvesting the rat PTs on a nylon mesh. The PTs were i ncubated in a 50% mixture of Ham's F12: Dulbecco's medium containing o nly penicillin and dextran as supplements. The viability and integrity of these PTs were assessed using morphology, enzyme leakage and oxyge n uptake and were found to be well maintained in excess of 10 hr. It i s now hoped to extend these measurements beyond 10 hr and to compare t hese measurements with those obtained with collagenase isolated PTs.