1613 NMR MEASUREMENTS OF INTRACELLULAR Na+ CONCENTRATION IN PROXIMAL TUBULE SUSPENSION OF RAT KIDNEY

Transport of Na+ and consequently of many other substances by the proximal tubules is dependent on the intracellular concentration of sodium (Nai). It is therefore important that a method be developed that permits to measure Nai without disturbing the function and integrity of the cells. Slices of superficial cortex from saline perfused rat kidneys were digested by emerging them in a solution of 0.15% collagenase for 1 hour at room temperature. The slices were then washed and sheared by passing them through PE-190 and PE-50 tubing. The resulting suspension was filtered through a 150 μm sieve and centrifuged at 50 g for 1 min. The pellet obtained was washed several times with chilled Ringer's, 6% BSA-Ringer's and suspended in 6% BSA Ringer's solution. Microscopic examination showed the suspensions to contain ≃95% proximal tubules which were 98-99% viable when tested with trypan blue. Succinate stimulated (5mM) oxygen consumption was 1.83 μl/min per mg protein. The measurement of Nai were done by NMR spectrometry (Varian XL-200, 53 mHz) following incubation of proximal tubules in the aqueous shift reagent dysprossium tripolyphosphate Dy(PPPi)2 for 30 min. NMR observable Nai (n=6) was 34.06 ± 1.75 mM at room temperature (≃25°C) and 16.3 ± 0.58 mM at 37°C (p < .001). Addition at 37°C of 0.1 mM oubain, an inhibitor of Na+-K+-ATPase, raised the concentration to 30.92±2.93 mM (p < .001), whereas nystatin, a channel former, increased Nai concentration to 81.0±2.39 mM (p < .0005). Thus, NMR, a non-invasive method, provides reliable measurements of Nai in proximal tubule cells under a variety of experimental conditions.