Differential regulation of mouse kidney sodium-dependent transporters mRNA by cadmium.

Chronic exposure to cadmium can result in renal glycosuria. Previously, we reported that cadmium reduced the relative abundance of the sodium-glucose cotransporter mRNA (Blumenthal et al., Toxicol. Appl. Pharmacol.149, 49-54, 1998). To investigate this phenomenon further, we isolated full-length cDNA clones encoding both high- and low-affinity sodium-dependent glucose transporters SGLT1 and SGLT2, respectively, from cultured mouse kidney cortical cells. We also amplified a fragment of another putative sodium-glucose cotransporter with homology to the known SAAT1/pSGLT2 or SGLT3 from our cultured cells and named it SGLT3. In order to examine the effect of cadmium on these transporters, primary cultures of mouse kidney cortical cells were exposed to micromolar concentrations of cadmium for 24 h and levels of SGLT1, SGLT2, and SGLT3 mRNA were determined by semiquantitative RT-PCR. Five to 10 microM of cadmium inhibited sodium-dependent uptake of the glucose analog, alpha-methyl D-glucopyranoside and progressively reduced the level of SGLT1. Cadmium also inhibited SGLT2 mRNA by 37%, but no further decline was observed at concentrations of cadmium greater than 5 microM. While cadmium inhibited SGLT1 and SGLT2, it significantly stimulated the expression of SGLT3 by fivefold. These results imply that individual sodium-glucose cotransporter mRNA species are not regulated in a similar fashion. In addition, the isolation of three separate SGLT species from these cultures suggests that, in addition to SGLT1 and SGLT2, glucose reabsorption by renal epithelial cells might involve additional glucose transporters such as SGLT3.

Cadmium decreases SGLT1 messenger RNA in mouse kidney cells.

Mouse renal cortical tubule cells in primary culture exposed to cadmium (Cd2+) develop decreased Na(+)-glucose cotransport activity as measured by uptake of the glucose analogue alpha-methyl-glucoside. RNA was isolated from kidney cell cultures, and after reversed transcription, the DNA was amplified with primers to rat SGLT1 (the high affinity isoform of the sodium glucose cotransporter) and mouse beta-actin. Only one product was identified after amplification with the rat SGLT1 primers, which on sequencing was 96% identical to rat SGLT1. Compared to beta-actin, the intensity of the SGLT1 message declined progressively as CdCl2 concentration in the medium increased from 0 to 10 microM. Similar decreases in SGLT1 mRNA were also observed as media zinc (Zn2+) concentrations rose from 0 to 75 microM or as copper (Cu) concentrations increased from 0 to 150 microM. Exposure to 8 microM Cd as Cd-metallothionein (Cd7-MT) also caused a fall in relative SGLT1 mRNA abundance, and at nearly identical internal Cd concentrations of 40-43 pmol/microgram DNA, both Cd7-MT and CdCl2 reduced SGLT1 mRNA to 33% of control. In general, the fall in SGLT1 mRNA was more rapid than the decline in Na(+)-dependent glucose uptake after cells were exposed to Cd2+. These findings suggest that the effects of Cd2+ and other metals on renal glucose transport are related to decreased expression of SGLT1 message.

Comparative effects of Cd2+ and Cd-metallothionein on cultured kidney tubule cells.

The effects of Ca2(+) and Cd-metallothionein on two cultured cells with proximal tubule characteristics, mouse kidney cortical cells and pig kidney LLC-PK1 cells, have been compared. Cd2+ inhibits Na(+)-glucose cotransport in LLC-PK1 cells and in the process decreases the number of binding sites for [3H]phlorizin, a competitive inhibitor of glucose for the Na(+)-glucose cotransporter. During 24 hr incubation and over a range of concentrations in the two cell types, only Cd2+ inhibited Na(+)-glucose cotransport even when approximately equal concentrations of intracellular Cd resulted from these treatments. Indeed, at low concentrations of Cd-metallothionein in mouse cells, transporter activity was elevated. Extension of incubations to 72 hr in mouse cells led to increased Cd uptake and reduction in cell density with both sources of Cd but only a progressive decline in Na(+)-glucose cotransport activity with Cd2+. Zn-metallothionein was without effect under comparable conditions. Both forms of Cd were accumulated by these cells, with the large majority of the metal ion localizing in metallothionein as a Cd, Zn-protein in LLC-PK1 cells. Under equal exposure conditions, the net uptake of Cd from Cd-metallothionein in the two cell types. It is evident that the mechanisms of toxicity of Cd2+ and Cd-metallothionein as well as their modes of uptake differ in these two cell types.

Effect of serum proteins on sodium-linked transport processes in cultured kidney cells.

The mechanism for increased Na+ retention in the nephrotic syndrome is unknown. To determine if Na+ transport systems in the proximal tubule might be affected by filtered proteins, mouse cortical tubule cells grown in defined medium were exposed to concentrations of bovine serum albumin (BSA) ranging from 0.01 to 0.5%. Activity of the Na(+)-glucose cotransporter, measured as Na(+)-dependent uptake of alpha-methylglucoside, increased progressively to a maximum of 2.3-fold above baseline (P < 0.001; N = 10). The increase in transporter activity was due to an increased Vmax, and the magnitude of the increase was inversely related to the basal cotransporter activity of the cultures. Increased cotransporter activity was detectable 6 h after exposure, was sustained for 24 h after cells were removed from an albumin-free medium, and was prevented by cycloheximide. Heat-treated BSA, fatty-acid and globulin-free BSA, and gamma-globulins were as effective at increasing Na(+)-glucose cotransporter activity as untreated Fraction V BSA. Dextran, dextran-sulfate, and amino acid supplements were ineffective. Neither protease inhibitors nor chloroquine added to an albumin-containing medium prevented increased alpha-methylglucoside uptake. Albumin did not change the rate of fluid-phase endocytosis in the cultured cells. Na(+)-amino acid cotransport and Na(+)-H+ exchange were either decreased or unchanged after BSA exposure. Exposing apical surfaces of cells grown on permeable membranes to BSA led to a greater increase in activity of the Na(+)-glucose cotransporter relative to controls than did exposing the basolateral surface (145 versus 89%; P < 0.05; N = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of Na(+)-glucose cotransport in kidney cortical cells by cadmium and copper: protection by zinc.

Properties of the inhibition of Na(+)-glucose cotransport by Cd2+ in mouse kidney cortical cells have been determined. In no case was any inhibition observed before 3 hr. The extent of inhibition was dependent upon both the concentration of Cd2+ and the length of exposure. Kinetic studies showed that metallothionein mRNA induction by Cd2+ was initiated within 1 hr after incubation with Cd2+ began and peaked by 3-6 hr. Metallothionein protein increased more slowly, beginning at 3 hr and continuing for at least 9 hr. The protein had both Cd2+ and Zn2+ bound to it throughout this period. Nevertheless, a pool of nonmetallothionein Cd2+ appeared after 3 hr, coinciding with the onset of inhibition of Na(+)-glucose cotransport, and increased over the next 9 hr. Pretreatment of cells with Zn2+ protected them from the effects of Cd2+ on Na(+)-glucose cotransport. It delayed the onset of inhibition of transport as well as the extent of inhibition. Detailed analysis of the distribution of Cd2+ and Zn2+ in the soluble fraction of these cells showed that the concentration of non-metallothionein bound Cd2+ was not suppressed by the presence of Zn-metallothionein after the onset of exposure to Cd2+. Incubation of cells with larger concentration of Zn2+ and Cu2+ also inhibited Na(+)-glucose cotransport.

The calcium oxalate crystal growth inhibitor protein produced by mouse kidney cortical cells in culture is osteopontin.

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.

Cadmium inhibits glucose uptake in primary cultures of mouse cortical tubule cells.

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.

Effect of pH on growth of mouse renal cortical tubule cells in primary culture.

We examined the effect of the medium pH on growth of primary cultures of mouse cortical tubule cells grown in defined medium. A significantly higher DNA content was observed within 24 h of lowering medium pH from 7.4 to 6.8 or 7.1 and persisted for the duration of the study. Further studies revealed that either medium acidification or insulin plus prostaglandin E1 nearly doubled uptake of [3H]thymidine in cells deprived of other growth factors for the previous 72-110 h. Moreover, the effects of insulin, prostaglandin E1, and medium acidification on [3H]thymidine uptake of quiescent cells were additive. An alkaline medium pH appeared to have a small but significant effect on cell hypertrophy, since cells exposed to pH 7.4 and 7.7 had a higher protein-to-DNA ratio than cells incubated at a lower pH. Cell pH of monolayers grown on glass slides determined from fluorescence of the carboxyfluorescein analogue 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) was linearly correlated with medium pH, and changes in medium pH resulted in changes in steady-state cell pH of a similar magnitude. Four hours after medium acidification, relative increases in cell Na+ and water content occurred, whereas medium alkalinization led to decreases in cell Na+ and water content. The increases in cell Na+ and cell water content at pH 6.8 could be inhibited by amiloride. We conclude that decreasing the cell pH can be a mitogenic stimulus for renal tubule cells. Medium acidification is accompanied by changes in cell Na+ transport, which may be mediated in part by altered Na+-H+ antiporter activity.

Regulation of pH in rat papillary tubule cells in primary culture.

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 ATP-ase 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 acid-base transport remains uncertain.

Effects of sulfodeoxycholate on rat and rabbit small intestine.

To determine how sulfation alters the biological properties of dihydroxy bile acids, we compared the effects of 3-sulfodeoxycholate (SDC) and deoxycholate (DC) in the rat and rabbit intestine. While 5 mM DC induced water and electrolyte secretion and inhibited glucose absorption in the rat, SDC enhanced jejunal and ileal water and solute absorption. SDC had no effect in the rabbit ileum. In the rat jejunum DC caused mucosal injury and enhanced mucosal permeability while SDC had no effect. In vitro in the rabbit ileum, 10 mM SDC enhanced net sodium flux and decreased net residual flux, while 0.5 mM DC reduced net sodium flux and induced Cl- secretion. Both bile acids increased short-circuit current and potential difference and decreased tissue conductance. During reversed-phase, high-performance liquid chromatography SDC was more polar than DC. Sulfation reduced the ability of DC to destroy large unilamellar liposomes by a factor of 10. Thus, sulfation abolishes the effects of DC on the intestine by enhancing the polarity of this molecule. The enhancement of intestinal solute and water absorption by SDC requires further study.

Endogenous norepinephrine release induced by tyramine modulates intestinal ion transport.

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.

Detection of benzoylecgonine in human urine.

A thin-layer chromatography (TLC) method is described that can be used to detect benzoylecgonine (BE), a metabolite of cocaine, in human urine. It is a two-part procedure that can be integrated into a rapid screening program for drug abuse. The first part of the method utilizes two TLC solvent systems to identify a variety of drugs, including BE. The second part is specific for the cocaine metabolite and can be used as a confirmation method. The procedure is sensitive to 3-4 microgram/ml of BE in urine.

Radioimmunuassay compared to thin-layer and gas--liquid chromatography for detecting methadone in human urine.

Radioimmunoassay was compared to thin-layer and gas--liquid chromatographic methods for detection of methadone in the urine of patients undergoing methadone maintenance therapy as treatment of heroin abuse. With urine samples known to contain methadone, 84% were positive by thin-layer chromatography as compared to 99% positives by the other two methods. This difference is attributed to the difference in sensitivity of the three methods. All three methods gave consistently positive results with urine samples from patients receiving 25 mg of methadone per day or more. With smaller daily doses the percentage of positive results obtained with thin-layer chromatography decreased. Analysis of urine samples not containing methadone showed no incidence of cross reaction of other drugs with the methadone radioimmunoassay. The methadone radioimmunoassay appears to be both sensitive and reliable; however, certain other factors limit its use as a primary screening method.

Methods of identification and confirmation of abusive drugs in human urine.

A thin-layer chromatography (TLC) procedure is described to be used as the initial drug detection method for urine surveillance in a drug abuse treatment program. While the TLC method is sufficiently sensitive, it is prone to false-positive results. For this reason, two other drug detection methods (gas-liquid chromatography and radioimmunoassay) have been incorporated to confirm positive results obtained with TLC. The combined methodologies result in a urine surveillance procedure that is versatile, sensitive and highly reliable.

Comparison of radioimmunoassay with thin-layer chromatographic and gas-liquid chromatographic methods of barbiturate detection in human urine.

A radioimmunoassay (I) for barbiturates was compared with thin-layer chromatographic (II) and gas-liquid chromatographic (III) methods for barbiturate detection in human urine. Timed urine samples were obtained from volunteers who had ingested 100 mg of a barbiturate. I detected barbiturate in all urines tested up to 76 h after the dose, and III in all up to 52 h and in 90% up to 76 h. II detected barbiturates in 90% of all urine samples for only 30 h, after which is reliability declined. Glutethimide interfered with radioimmunoassay of barbiturate, producing false positives. I is sensitive, reliable, and fast, and lends itself to screening large numbers of urine samples for barbiturates. For routine urine surveillance, however, we found I to be less useful than II, which is still the method of choice. I has, however, proved to be an excellent method for confirming results of II.