N-terminal PDZ domain is required for NHERF dimerization.

NHERF, a 55 kDa PDZ-containing protein, binds receptors and ion transporters to mediate signal transduction at the plasma membrane. Recombinant NHERF demonstrated an apparent size of 150 kDa on gel filtration, which could be reduced to approximately 55 kDa by protein denaturing agents, consistent with the formation of NHERF dimers. Biosensor studies established the time- and concentration-dependent dimerization of NHERF. Overlays of recombinant NHERF fragments suggested that NHERF dimerization was principally mediated by the N-terminal PDZ-I domain. In PS120 cells, reversible protein phosphorylation modulated NHERF dimerization and suggested a role for NHERF dimers in hormonal signaling.

Trypsin digestion increases Na(+)-H+ exchange rates in native rabbit brush border membrane.

Experiments were designed to examine the effects of trypsin-mediated proteolysis on the activity of the rabbit renal brush border membrane Na(+)-H+ exchanger. Incubation of brush border membrane vesicles with trypsin resulted in a concentration and time-dependent change in proton gradient-stimulated sodium uptake. Extensive trypsin digestion resulted in a marked decrease in the activity of the transporter. By contrast, limited trypsin digestion resulted in a significant increase in proton gradient-stimulated, amiloride-inhibitable sodium uptake. Sodium-dependent proton efflux was also significantly increased by limited trypsin digestion. The passive permeability of the vesicles to sodium and protons, and the sodium-dependent and sodium-independent uptake of glucose were not affected by limited trypsin digestion. These data indicate that limited trypsin digestion increases the activity of the renal brush border membrane Na(+)-H+ exchanger. It is suggested that the renal Na(+)-H+ exchanger contains one or more inhibitory components or sites which are sensitive to inactivation by limited trypsin-mediated proteolysis.

Lipid methylation, sodium transport and the response to PTH in renal tubules.

The possible role of progressive methylation of phosphatidylethanolamine to phosphatidylcholine on sodium transport was examined in a suspension of rabbit proximal convoluted tubules. The relation between progressive methylation and the action of parathyroid hormone on sodium transport in this nephron segment was also determined. Incubation of the suspended tubules for 10 minutes at 37 degrees C with 200 microM S-adenosyl-L-[3H]-methyl methionine, a methyl donor, revealed a significant rate of de-novo phosphatidylcholine synthesis. The inactive adenosine analogue, 3-deazaadenosine (DZA), had a significant inhibitory effect on lipid methylation. Despite the inhibition of methylation by DZA, the ouabain sensitive component of oxygen consumption, an index of sodium transport, was not affected. PTH significantly inhibited ouabain sensitive oxygen consumption but had no effect on the methylation of phosphatidylethanolamine. Inhibition of methylation by DZA, did not affect the inhibitory effect of PTH on oxygen consumption. These studies demonstrate that in the proximal convoluted tubule of the rabbit the progressive methylation pathway is present and that inhibition of this pathway does not affect sodium transport. In addition, these studies suggest that the inhibitory effect of PTH on sodium transport is not mediated by the methylation pathway.

Secretion of urate in the proximal convoluted tubule of the rat.

In order to examine the transepithelial secretory flux of urate in the rat proximal tubule, simultaneous perfusions of capillaries and lumens were performed. The capillary perfusate contained [2-14C]urate in concentrations of 0.305--2.94 mM. The secretory flux of urate increased as the concentration of urate in the capillary perfusion solution was increased from 0.305 to 1.235 mM but tended toward a plateau at higher concentrations. An apparent Km of 0.41 mM and Vmax of 4.7 pmol . min-1 . mm-1 were calculated from the observed net flux and an estimated passive permeability coefficient of 0.725 pmol . min-1 . mm-1. The addition of probenecid (10(-4) M) to the capillary perfusion solution inhibited urate secretion in a manner consistent with that of a competitive inhibitor. The addition of p-chloromercuribenzoate (10(-4) M) to the capillary perfusion solution inhibited the Vmax but not the Km, suggesting a non-competitive type of inhibition. These data provide the first estimates of the apparent transport constants for urate secretion in the rat proximal tubule determined in vivo. Urate secretion is mediated by a saturable carrier-mediated system. This carrier is not affected by the presence or absence of potassium in the perfusion solution but can be inhibited by the addition of either probenecid or p-shloromercuribenzoate.

Renal transport of oxalate: effects of diuretics, uric acid, and calcium.

Clearance experiments were performed in the rat to examine the effects of diuretics on the renal transport of oxalate. In addition, the effect of infusing either uric acid or calcium on the renal transport of oxalate was examined. During control periods, the fractional excretion of oxalate (FEOx) averaged 118.0 +/- 2.1%. Acute administration of either chlorothiazide, furosemide, or indanyl-oxyacetic acid (MK-196), a new uricosuric diuretic, resulted in a significant decrease in the FEOx in all groups to 104.8 +/- 2.4%, 111.3 +/- 2.1%, and 108.6 +/- 2.7%, respectively. Infusion of uric acid increased urinary uric acid excretion from 2.41 +/- 0.28 to 4.26 +/- 0.03 micrograms/min/g kidney wt (P less than 0.001) and decreased FEOx to 104.0 +/- 2.4% (P less than 0.001 compared to control). Infusion of calcium chloride increased urinary calcium excretion from 0.10 +/- 0.04 to 0.44 +/- 0.06 micrograms/min/g kidney wt (P less than 0.001) but had no effect on the FEOx which averaged 118.3 +/- 8.3% (P = NS compared to control). These studies show that the acute administration of chlorothiazide, furosemide, or MK-196, as well as increasing urinary uric acid excretion by uric acid infusion, are all associated with a decrease in the FEOx. Acutely increasing urinary calcium excretion, however, had no effect on oxalate transport.