Isolation of rat kidney cortical tubules enriched in proximal and distal segments

Abstract

Sedimentation through a discontinuous Ficoll gradient was used to segregate proximal and distal segments of the rat kidney cortex obtained by collagenase treatment and shearing forces. By morphological and biochemical criteria, fraction A was enriched in distal segments and fraction B in proximal segments. The results, expressed as ratios of values in fraction A/fraction B, were as follows: 2.6 (distal tubular length); 0.70 (protein/DNA); 0.50 (alkaline phosphatase activity/DNA); 1.63 (hexokinase activity/DNA); 3.0 (vasopressin-activated adenylate cyclase); 2.0 (isoproterenol-activated adenylate cyclase); and 1.02 (parathyroid hormone-activated adenylate cyclase). Based on the relative abundances of proximal and distal segments (measured in terms of tubular lenths), the segmental distribution of the hormone effects was computed. Vasopressin (20 mU/ml) augmented distal adenylate cyclase activity by 450% and had no effect on proximal segments. The effects of isoproterenol (10-6 M) were predominantly distal: 240%, distal and 40%, proximal. No discrimination was obtained in the action of parathyroid hormone (1 IU/ml): 420% in both segments. Viability of the tubular segments was documented by the measurements of respiration (QO2), which was constant for 6 h in both fractions (.apprx. 1 .mu.l .cntdot. h-1 .cntdot. .mu.g DNA-1 at 28.degree. C). Partial separation of proximal and distal segments of the rat kidney cortex exist at a preparative scale. The described technique extends available methods for studies on biochemical pathways in the nephron.