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

Abstract

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 vitro 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 (less than 15% lactate dehydrogenase release), limited lipid peroxidation (less than 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. gamma-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.

Calpain Mediates Progressive Plasma Membrane Permeability and Proteolysis of Cytoskeleton-Associated Paxillin, Talin, and Vinculin during Renal Cell Death

“…Enhance chemiluminescence kit and autoradiography films were obtained from Amersham Biosciences (Piscataway, NJ). The sources of the remaining chemicals have been reported previously (Rodeheaver et al, 1990;Groves and Schnellmann, 1996) or were from Sigma-Aldrich. All glassware was silanized and autoclaved before use.…”

“…RPTs were isolated and purified as described by Rodeheaver et al (1990) and Groves and Schnellmann (1996) from 1.5-to 2.0-kg female New Zealand White rabbits (Myrtle's Rabbitry, Thompson Station, TN). RPTs were suspended at a concentration of 2 mg/ml in Krebs' incubation buffer containing 1 mM alanine, 5 mM dextrose, 2 mM heptanoate, 4 mM lactate, 5 mM malate, 115 mM NaCl, 15 mM NaHCO 3 , 5 mM KCl, 2 mM NaH 2 PO 4, 1 mM MgSO 4 , 1 mM CaCl 2 , and 10 mM HEPES (pH 7.4, 295 mOsM/kg).…”

Cytoprotective Properties of Novel Nonpeptide Calpain Inhibitors in Renal Cells

“…SJA7019 and SJA7029 were gifts from Dr. Jun Inoue (Senju Pharmaceutical Company, Kobe, Japan). The sources of other chemicals were reported previously (Rodeheaver et al, 1990;Groves and Schnellmann, 1996) or were obtained from Sigma-Aldrich. All glassware was silanized and autoclaved.…”

“…RPTs were isolated and purified according to the method described by Rodeheaver et al (1990) and Groves and Schnellmann (1996) from New Zealand White rabbits (female, 2 kg; Myrtle's Rabbitry, Thompson Station, TN). RPTs were suspended at a concentration of 2 mg/ml in an incubation buffer containing 1 mM alanine, 5 mM dextrose, 2 mM heptanoate, 4 mM lactate, 5 mM malate, 115 mM NaCl, 15 mM NaHCO 3 , 5 mM KCl, 2 mM NaH 2 PO 4, 1 mM MgSO 4 , 1 mM CaCl 2 , and 10 mM HEPES (pH 7.4, 295 mOsm/kg).…”

Relative contribution of OAT and OCT transporters to organic electrolyte transport in rabbit proximal tubule

“…Rabbit RPT were isolated and purified from New Zealand White rabbits (1.3-1.5 kg) as described previously (23,40). The final tubule pellet was resuspended at a protein concentration of 2 mg/ml in the medium used for dissection of single S2 segments.…”