Trypsin inhibitor type ii s

Plasma membranes from proximal tubule cells were also prepared by differential centrifugation as in [29] (link). The kidneys were placed in an isotonic solution containing 250 mM sucrose, 10 mM Hepes-Tris (pH 7.4), 2 mM EDTA and 0.15 mg/ml trypsin inhibitor type II-S (Sigma-Aldrich) (1 g tissue/4 ml solution). Membranes from different rats (see “number of animals” above) were prepared from the outer region of the cortex (cortex corticis) as described elsewhere [30] (link), where the predominant cell population is proximal tubule cells [31] (link). Controls for enrichment with basolateral membranes (3–4 fold with respect to the total homogenate using (Na⁺+K⁺)ATPase as a marker) and for minimal residual contamination with intracellular membranes and cytosol were as described in [29] (link), [30] (link). No attempt at further enrichment was made in this case, as the (Na⁺+K⁺)ATPase and the ouabain-resistant Na⁺-ATPase are exclusively located in the basolateral membranes of epithelial cells [32] (link), and a low yield of purified basolateral membranes was obtained using the Percoll gradient method with the minimum number of animals recommended by the Committee for Ethics in Animal Experimentation. The plasma membrane fraction was stored under liquid N₂. The protein concentration was also measured by the Folin reagent method [28] (link).