Differences in enzymatic and mechanical isolated rabbit renal proximal tubules: Comparison in long-term incubation

Suspensions of renal proximal tubules (RPT) are the in vitro model for many biochemical and physiologic investigations. Inasmuch as there are numerous procedures for tubule isolation and the more commonly used enzymatic procedures may disrupt the basement membrane, there is a need for information comparing the influence of various isolation methods on RPT viability and function in long-term suspension. Rabbit RPT isolated a) enzymatically (ENZ) by in vitro collagenase digestion and Percoll size and density purification, and b) mechanically (MECH) by in invitro iron oxide perfusion and purification by sieving and magnetic removal of glomeruli were compared for viability, morphology, and functional stability during long-term suspension. RPT isolated by ENZ and MECH methods had excellent viability (<15% lactate dehydrogenase release), limited lipid peroxidation (<0.2 nmol MDA mg protein^-1), and stable nystatin-stimulated oxygen consumption (QO₂) (38 and 36 nmol. O₂·mg protein^-1·min^-1) throughout 24 h of incubation. Basal QO₂ was higher in ENZ than MECH tubules (27 and 19 nmol O₂·mg protein^-1·min^-1, respectively), and was unchanged over 24 h in each preparation. The higher basal QO₂ in ENZ tubules was ouabain-sensitive, suggesting an increased rate of Na⁺, K⁺-ATPase activity in these tubules. Total glutathione content (oxidized + reduced) in ENZ and MECH tubules increased over the 24-h incubation from 8 to 18 nmol·mg protein^-1· γ-Glutamyltranspeptidase (GGT) activity of the RPT homogenates was equivalent in both preparations and stable over time. The ratio of suspension GGT activity to homogenate GGT activity doubled (0.4 to 0.8) during the incubation period. MECH tubules retained their tubule structure during 24 h of incubation whereas the ENZ tubules had a striking loss of tubular morphology over time. These results show that ENZ- and MECH-isolated renal proximal tubule suspensions exhibit similar biochemical properties in long-term incubations but differ in ouabain-sensitive QO₂ and the retention of tubular morphology. The loss of tubular morphology and the increase in the rate of Na⁺, K⁺-ATPase activity in ENZ tubules may be secondary to the disruption of the tubular basement membrane.