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 their connective tissue matrix. Although concerns have long been felt regarding potential structural damage incurred by the use of these enzymes, little has been done to characterize alternative non-enzymatic methods-with most of that being conducted on rabbit proximal tubules (PTs). The present work is performed on rat kidneys using the standard mechanical technique of Brendel & Meezan (1975), which involves perfusion, loading the renal vasculature with magnetic iron oxide, disrupting the cortex with a series of meshes, removing the glomeruli with a magnet and finally harvesting the rat PTs on a nylon mesh. The PTs were incubated in a 50% mixture of Ham's F12: Dulbecco's medium containing only penicillin and dextran as supplements. The viability and integrity of these PTs were assessed using morphology, enzyme leakage and oxygen uptake and were found to be well maintained in excess of 10 hr. It is now hoped to extend these measurements beyond 10 hr and to compare these measurements with those obtained with collagenase isolated PTs.