Ammonia production increases in several models of renal hypertrophy in vivo. The present study was designed to determine whether ammonia can directly modulate the growth of renal cells in the absence of a change in extracellular acidity. In serum-free media NH4Cl (0-20 mM) caused JTC cells and a primary culture of rabbit proximal tubule cells to hypertrophy (increase in cell protein content) in a dose-dependent fashion without a change in DNA synthesis. Studies in JTC cells revealed that the cell protein content increased as a result of both an increase in protein synthesis and a decrease in protein degradation. Total cell RNA content and ribosome number increased after NH4Cl exposure and the cell content of the lysosomal enzymes cathepsin B and L decreased. Inhibition of the Na+/H' antiporter with amiloride did not prevent the hypertrophic response induced by NHI-Cl. The results indicate that ammonia is an important modulator of renal cell growth and that hypertrophy can occur in the absence of functioning Na+/H' antiport activity.

A new fluorescent probe, 6,7-(4-methyl)coumaro-[2.2.2] cryptand, has been developed for measuring K+. This compound was made by fusing [2.2.2] cryptand with 4-methylcoumarin through 2 ethoxy bridges at the 6 and 7 positions. The probe has a fluorescent excitation peak at 340 nm and an emission peak at 420 nm. In aqueous solutions, increasing the K+ concentration from 0 to 10 mM causes the fluorescence intensity to increase by 143%. The dissociation constant (Kd) for K+ in aqueous solutions is 1.9 mM. In 100% methanol, the Kd for K+ decreases to 0.012 mM, making it possible to measure K+ concentrations in the micromolar range. Na+, tetramethylammonium, NH4+, Ca2+, and Mg2+ have a minimal affect on the fluorescence of the probe in the absence of K+. The coefficient of variation for the measurement of K+ by use of this new dye is less than 1%. In this report, the synthetic procedure is described and the spectral properties of the probe are characterized. Experiments are described demonstrating the use of this probe 1) in measuring K+ in aqueous solutions from 0 to 10 mM and in a microfluorometric assay to measure K+ from 0.0005 to 0.003 mM and 2) in monitoring K+ transport in rabbit proximal tubule brush-border membrane vesicles.

With dietary phosphate (Pi) restriction, fluidity of renal proximal tubule brush-border membranes (BBM) and Na-dependent Pi transport (Na-Pi) are increased, suggesting that changes in BBM fluidity are critical for adaptation to Pi restriction. To test this hypothesis, the temporal relationship between Na-Pi transport and changes in BBM fluidity was assessed after Pi deprivation in rats. Renal cortex was obtained from rats fed either a 0.03% (-P) or a 0.6% (+P) Pi diet for 4 h or 7 days, and BBM were prepared. Na-Pi uptake by BBM was measured by use of rapid filtration, and BBM fluidity was assessed by use of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). After 4 h on the diets, Na-Pi uptake was 439 +/- 142 (SD) and 984 +/- 184 pmol.mg protein-1.5 s-1 in +P and -P, respectively (P less than 0.01, n, = 8). Na-dependent proline uptake was unchanged. DPH anisotropy and total cholesterol were similar between groups: 0.204 +/- 0.025 and 0.401 +/- 0.047 nmol/mg protein, respectively, in +P and 0.205 +/- 0.015 and 0.392 +/- 0.037 in -P (P greater than 0.05, n = 8-10). After 7 days, Na-Pi uptake was 841 +/- 291 in +P and 2,168 +/- 848 pmol.mg protein-1.5 s-1 in -P, P less than 0.01, n = 8. DPH anisotropy and BBM cholesterol were 0.175 +/- 0.019 and 443 +/- 132 nmol/mg protein, respectively, in +P and 0.162 +/- 0.020 (n = 8) and 341 +/- 128 (n = 3) in -P (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The present study was designed to determine whether rat glomerular mesangial cells possess Cl- -dependent intracellular pH (pHi) regulatory processes. Rat glomerular mesangial cells were grown to confluence on glass coverslips. Intracellular pH (pHi) was measured with BCECF. Steady state pHi in HCO3- containing solutions was 7.08 +/- 0.03 (N = 13). When extracellular Cl- was acutely removed, pHi increased at a rate of 0.57 +/- 0.03 pH/min units (N = 8), P less than 0.001. DIDS (0.5 mM) significantly decreased the rate of increase in pHi to 0.34 +/- 0.04 pH/min, P less than 0.01. Na+ removal and amiloride (1 mM) did not alter the increase in pHi induced by Cl- removal. Steady state pHi in the absence of Cl- was significantly increased above control, 7.39 +/- 0.02 (N = 7), P less than 0.001. Following the acute alkalinization of pHi by CO2 removal, pHi recovered at a rate of 0.07 +/- 0.01 pH/min (N = 9). In the absence of Cl-, the pHi recovery rate was significantly decreased to 0.01 +/- 0.008 pH/min (N = 5), P less than 0.01. DIDS (0.5 mM) significantly decreased the rate of pHi recovery to 0.02 +/- 0.01 pH/min (N = 5), P less than 0.01. Na+ removal and amiloride (1 mM) had no effect on the rate of pHi recovery following acute alkaline loading.(ABSTRACT TRUNCATED AT 250 WORDS)