Estimation of the osmolality of renal tissue fluid has been carried out in many investigations of the renal concentrating and diluting mechanism. The method applied has usually been a variation of the technique of Appelboom et a![1]. This involves equilibration of the tissue with some volume of diluent after boiling to prevent generation of osmotically active particles due to metabolism. The osmolality of the tissue fluid is calculated from the freezing point depression of the diluted fluid and the dilution factor. Corrections are applied for the change in activity coefficients due to dilution, loss of carbon dioxide due to boiling, and incomplete equilibration between tissue and dilution fluid. These techniques have proven practical for many applications but are necessarily limited in accuracy and precision due to the complexity of the procedure and the corrections involved. In this paper a simple, direct method is described for determination of tissue slice osmolality using vapor pressure osmometry. The method has proven to be more accurate and reproducible than previous ones and yields better spatial resolu-tion. Vapor pressure osmometry has been applied previously to the determination of osmolality in the rat brain by Tornheim [21. Rabbits weighing 1500 to 2500 g were used. The rabbits were thirsted for 48 hr and fasted for 18 hr prior to sacrifice. They also received Pitressin tannate in oil (1 U/kg of body wt im) 18 hr prior to sacrifice. The rabbits were sacrificed by decapitation. After a small amount of blood was collected into a beaker containing heparin and water-equilibrated mineral oil, the left kidney was rapidly removed and frozen in liquid nitrogen. The kidney was wrapped in aluminum foil and kept frozen in a beaker above a saline-ice slurry before slice preparation. Urine samples were collected from the bladder. Slices were prepared in a 2 C cold room. A cork borer (7.5 mm diameter), chilled on dry ice, was used to prepare a tissue core along the cortico-papillary axis. The core was wrapped in aluminum foil and kept frozen on dry ice prior to sectioning. Disc-shaped slices of thickness 0.5 to 1.0 mm were prepared along the entire length of the cortico-papillary axis with a chilled straight razor. The slices were sealed in polyethylene film (Glad® wrap) and aluminum foil, and kept frozen on dry ice until the measurement of tissue osmolality was made. Because the renal medulla is cone-shaped and because the boundaries between zones of the kidney are curved, the slices from the inner stripe of the outer medulla and from the inner medulla were not homogeneous. Consequently, only the center portion of these slices was used, trimming them with smaller cork borers (6 mm diameter for the inner stripe, and 4 mm diameter for the inner medulla).

Measurements of tissue slice, plasma, and urine osmolality were made with a vapor pressure osmometer (Wescor, model 5100CXR). The instrument was calibrated using 290 and 1000 mOsm/kg H20 with sodium chloride standards. The procedure used for standards, plasma, and urine was that recommended by the manufacturer. Specifically, a 6.5 mm diameter filter paper disc was placed in the instrument and was saturated with 7 1j. l of the solution to be measured. For measurement of tissue fluid osmolality, the tissue slices were placed directly into the instrument. When a tissue slice was approximately the same size as the filter paper discs (that is, cut with a 7.5 mm diameter cork borer), only the slice was used. When tissue slices were smaller (medullary slices), they were placed on the filter paper discs allowing the tissue fluid to wet the filter paper. This procedure had the effect of equalizing the effective surface area of all …