23Na NMR spectroscopy of proximal tubule suspensions

Abstract

Intracellular sodium concentrations in proximal tubule suspensions of rat kidney were measured by NMR spectroscopy. A simple method for the preparation of proximal tubule suspension is described. Examination by light microscopy revealed these preparations to contain 93.6 ± 0.6% (N = 5) proximal tubules, and electron microscopy demonstrated that the tubules were open. When incubated with trypan blue for five min, only 2% of tubules picked-up the dye. The basal oxygen consumption rate was 0.42 ± 0.01 μl min-1 mg protein-1 (N = 6). Addition of succinate (5 mM) resulted in a fivefold increase in the rate of oxygen consumption. The 23Na spectra were obtained in proximal tubules incubated for 30 min in the aqueous shift reagent dysprosium tripolyphosphate Dy(PPP(i))27-. The NMR observable sodium concentration was 34.1 ± 1.8 mM at room temperature and 16.3 ± 0.6 mM (P < 0.001) at 37°C. Addition of ouabain (10-4 M) at 37°C resulted in an increase in intracellular sodium to 30.9 ± 2.9 mM (P < 0.001), while nystatin increased the concentration of sodium to 72.0 ± 9.1 mM (P < 0.001), compared to basal concentration. Thus NMR permits the measurement of intracellular concentration of sodium in proximal tubules under basal conditions and to monitor, in the same preparation, the changes that occur under various experimental conditions without interfering with the morphologic integrity of the cells.