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Reabsorption of glucose in mouse kidney may involve four SGLTs. Cadmium affects mRNA expression of all four SGLTs in vitro.

Urine contains proteins that inhibit the growth of calcium oxalate (CaOx) crystals and may prevent the formation of kidney stones. We have identified a potent crystal growth inhibitor in the conditioned media from primary cultures of mouse kidney cortical cells. Conditioned media, incubated with the kidney cells for 6-72 h, was assayed for crystal growth inhibition; inhibitory activity increased 15-fold by 24 h. Inhibitory activity was purified from serum-free media containing proteinase inhibitors using anion-exchange and gel-filtration chromatography. A single band of molecular weight 80,000 daltons was seen after SDS-polyacrylamide gel electrophoresis. The sequence of the N-terminal 21 amino acids of this protein matched that of osteopontin (OP), a phosphoprotein initially isolated from bone matrix. Antisera raised to fusion proteins produced by plasmids containing the N-terminal or C-terminal portions of OP cDNA also cross-reacted with the protein purified from cell culture media on western blots. The effect of the purified protein on the growth of CaOx crystals was measured using a constant composition assay. A 50% inhibition of growth occurred at a protein concentration of 0.85 micrograms/ml, and the dissociation constant of the protein with respect to CaOx crystal was 3.7 x 10(-8) M. The concentration of OP in mouse urine, measured using antibodies raised to the purified protein, was approximately 8 micrograms/ml. We conclude that OP is synthesized by kidney cortical tubule cells and functions as a crystal growth inhibitory protein in urine.

To study the effects of endogenous norepinephrine on intestinal ion transport, we tested the actions of an indirect sympathomimetic agent, tyramine, on electrolyte fluxes in the short-circuited rabbit ileum in vitro. Tyramine (10(-5) M) alone had no effect on short-circuit current or Na transport but increased Cl absorption. Tyramine decreased the short-circuit current, stimulated both Na and Cl absorption, and increased tissue conductance when its breakdown by endogenous monoamine oxidase enzymes was inhibited by pretreatment with pargyline (10(-4) M). Pargyline alone had no effect on short-circuit current and NaCl transport. The effect of norepinephrine on NaCl transport was inhibited by the alpha-adrenergic receptor antagonist, phentolamine (10(-7) M). This response was also prevented when animals were chemically sympathectomized with 6-hydroxydopamine. Although sympathectomy decreased measurable tissue norepinephrine by 80%, it did not alter basal short-circuit current, Na and Cl absorption, and the short-circuit current response to glucose-stimulated Na transport and to exogenous norepinephrine. Thus, a pool of norepinephrine in intestinal adrenergic neurons released by tyramine affects intestinal ion transport but does not alter basal ion transport. These data suggest close neuropharmacologic similarities between the adrenergic nervous system in the intestine and other organs.

We studied the effect of cadmium (Cd2+) on transport of alpha-methylglucoside in primary cultures of mouse kidney cortical tubule cells grown in defined medium. When cultured cells were exposed to Cd2+ concentrations from 0 to 6 microM for 24 h, uptake of alpha-methylglucoside was inhibited in a dose-dependent manner by up to 50%. By contrast, acute exposure of the cells to 7 microM Cd2+ for 60 min did not inhibit alpha-methylglucoside uptake. Increasing Cd2+ concentrations progressively decreased the Vmax of Na(+)-dependent glucose cotransport but not the Km for glucose. Cell ATP/ADP ratios of unexposed monolayers and of cells exposed to 4.5 microM Cd2+ for 24 h were 5.0 and 4.9, respectively (n = 3). Intracellular volume, lactate dehydrogenase activity, and cell Na+ and K+ concentrations were unaltered even after 24 h of exposure to 7 microM Cd2+. Untreated and Cd2+-treated monolayers preloaded with alpha-methylglucoside released the sugar analogue into the medium at nearly identical rates, indicating that Cd2+ did not alter cell permeability to glucose. Uptake of the amino acid analogue alpha-(methylamino)isobutyric acid was not affected by prior Cd2+ exposure. Whereas cell DNA content declined in Cd2(+)-exposed plates, both Na(+)-glucose and Na(+)-amino acid cotransport were enhanced at lower cell densities. Protein and DNA synthesis, estimated, respectively, by incorporation of [3H]leucine and [3H]thymidine into acid-insoluble material, were not significantly affected at 6 microM Cd2+. We conclude that after a lag time Cd2+ selectively inhibits renal Na(+)-dependent glucose transport despite an unchanged gradient for Na+ across the cell membrane.

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To investigate the mechanisms responsible for urinary acidification in the terminal nephron, primary cultures of cells isolated from the renal papilla were grown as monolayers in a defined medium. Morphologically, cultured cells were epithelial in type, and similar to collecting duct principal cells. Cell pH measured fluorometrically in monolayers grown on glass slides showed recovery from acid loads in Na+-free media. Recovery was inhibited by cyanide, oligomycin A, and N-ethylmaleimide. Cyanide and oligomycin inhibited recovery less in the presence than in the absence of glucose. When cells were first acid loaded in a Na+-free medium and then exposed to external Na+, pH recovery also took place. This recovery exhibited first-order dependence on Na+ concentration and was inhibited by 5- (N-ethyl-N-isopropyl)amiloride. These studies demonstrate that in culture, collecting duct principal cells possess at least two mechanisms for acid extrusion: a proton ATPase and an Na+-H+ exchanger. The former may be responsible for some component of the urinary acidification observed in the papillary collecting duct in vivo; the role of the latter in acidbase transport remains uncertain.