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      Mechanism of Cd-Induced Inhibition of Na-Glucose Cotransporter in Rabbit Proximal Tubule Cells: Roles of Luminal pH and Membrane-Bound Carbonic Anhydrase

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          Background/Aims: We have previously reported that a complex of cadmium-metallothionein (Cd-MT) directly affects the apical Na-glucose cotransporter on the luminal side in proximal tubules, suggesting that Cd-MT is more toxic than CdCl<sub>2</sub> in causing tubulopathy. To find the potential mechanisms, we evaluated the effect of luminal pH alteration and carbonic anhydrase (CA) inhibition on Cd-MT-induced reduction of glucose-dependent transmural voltage in rabbit S2 segments perfused in vitro. Methods: Before and after the addition of Cd-MT (1 µg Cd/ml) to the lumen, the deflections of transmural voltage upon the elimination of glucose from the perfusate (ΔVt<sub>glu</sub>) were measured as a parameter of activity of the Na-glucose cotransporter. Results: During perfusion with a control solution of pH 7.4, the ΔVt<sub>glu</sub> significantly decreased after addition of Cd-MT for 10 min. A reduction in pH to 6.8 significantly shortened the time needed to reduce the ΔVt<sub>glu</sub> to <5 min, whereas an increase of pH to 7.7 significantly prolonged the time to >20 min. Furthermore, simultaneous addition of acetazolamide with control perfusate prevented the reduction. P-Fluorobenzyl-aminobenzolamide (pFB-ABZ), a membrane-impermeable CA inhibitor, added to the lumen also completely prevented the reduction in ΔVt<sub>glu</sub>. In rabbits with chronic Cd exposure, acetazolamide prevented the glucosuria. Conclusion: Cd-MT-induced inhibition of Na-glucose cotransporter activity in the S2 segment strongly depends on luminal pH, and that an increase in pH by inhibition of luminal membrane-bound CA is useful to prevent renal Cd toxicity.

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          Most cited references 13

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          Licorice inhibits corticosteroid 11 beta-dehydrogenase of rat kidney and liver: in vivo and in vitro studies.

          In humans, glycyrrhetinic acid (GE), the active pharmacological ingredient of licorice, produces symptoms resembling those caused by excess mineralocorticoid secretion. We are proposing that 11 beta-dehydrogenase inhibition, and not intrinsic mineralocorticoid activity, is the primary mechanism of licorice induced pseudoaldosteronism. Glycyrrhizic acid (glycyrrhetinic acid glucuronide), when given orally to rats, partially inhibited renal 11 beta-dehydrogenase. In rats treated with dexamethasone before glycyrrhizic acid administration there was similar enzyme inhibition, suggesting that antimineralocorticoid effects of dexamethasone in licorice excess states are not mediated through a direct effect on 11 beta-dehydrogenase activity. Dispersed renal proximal tubular preparations, kidney homogenates, and microsomes readily converted corticosterone to 11-dehydrocorticosterone. GE and its synthetic analog carbenoxolone inhibited the conversion in these systems in a dose-dependent manner. Corticosteroid 11-oxoreductase, which was present in kidney homogenates at a level 10-20% that of 11 beta-dehydrogenase was not inhibited by any of the agents. With homogenate and microsomes, the Ki of GE was about 10(-9)-10(-8) M; with intact tubules, the Ki of GE was about 10(-5)-10(-6) M. It is suggested that a permeability barrier slows the entry of GE into the tubule cells. We conclude that the effects of licorice on corticosteroid metabolism in the kidney are based on its inhibition of 11 beta-dehydrogenase. Our data, supplemented by published evidence, is inconsistent with the conclusion that interaction with mineralocorticoid receptors accounts for the pharmacological effects of GE.
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            Megalin-dependent internalization of cadmium-metallothionein and cytotoxicity in cultured renal proximal tubule cells.

            Chronic cadmium (Cd2+) exposure results in renal proximal tubular cell damage. Delivery of Cd2+ to the kidney occurs mainly as complexes with metallothionein-1 (molecular mass approximately 7 kDa), freely filtered at the glomerulus. For Cd2+ to gain access to the proximal tubule cells, these complexes are thought to be internalized via receptors for small protein ligands, such as megalin and cubilin, followed by release of Cd2+ from metallothionein-1 in endosomal/lysosomal compartments. To investigate the role of megalin in renal cadmium-metallothionein-1 reabsorption, megalin expression and dependence of cadmium-metallothionein-1 internalization and cytotoxicity on megalin were studied in a renal proximal tubular cell model (WKPT-0293 Cl.2 cells). Expression of megalin was detected by reverse transcriptase-polymerase chain reaction and visualized by immunofluorescence both at the cell surface (live staining) and intracellularly (permeabilized cells). Internalization of Alexa Fluor 488-coupled metallothionein-1 was concentration-dependent, saturating at approximately 15 microM. At 14.3 microM, metallothionein-1 uptake could be significantly attenuated by 30.9 +/- 6.6% (n = 4) by 1 muM of the receptor-associated protein (RAP) used as a competitive inhibitor of cadmium-metallothionein-1 binding to megalin and cubilin. Consistently, cytotoxicity of a 24-h treatment with 7.14 muM cadmium-metallothionein-1 was significantly reduced by 41.0 +/- 7.6%, 61.6 +/- 3.4%, and 26.2 +/- 1.8% (n = 4-5 each) by the presence of 1 microM RAP, 400 microg/ml anti-megalin antibody, or 5 microM of the cubilin-specific ligand, apo-transferrin, respectively. Cubilin expression in proximal tubule cells was also confirmed at the mRNA and protein level. The data indicate that renal proximal tubular cadmium-metallothionein-1 uptake and cell death are mediated at least in part by megalin.
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              Megalin mediates renal uptake of heavy metal metallothionein complexes.

              Although several heavy metal toxins are delivered to the kidney on the carrier protein metallothionein (MT), uncertainty as to how MT enters proximal tubular cells limits treatment strategies. Prompted by reports that MT-I interferes with renal uptake of the megalin ligand beta(2)-microglobulin in conscious rats, we tested the hypothesis that megalin binds MT and mediates its uptake. Three lines of evidence suggest that binding of MT to megalin is critical in renal proximal tubular uptake of MT-bound heavy metals. First, MT binds megalin, but not cubilin, in direct surface plasmon resonance studies. Binding of MT occurs at a single site with a K(d) approximately 10(-4) and, as with other megalin ligands, depends on divalent cations. Second, antisera and various known megalin ligands inhibit the uptake of fluorescently labeled MT in model cell systems. Anti-megalin antisera, but not control sera, displace >90% bound MT from rat renal brush-border membranes. Megalin ligands including beta(2)-microglobulin and also recombinant MT fragments compete for uptake by megalin-expressing rat yolk sac BN-16 cells. Third, megalin and fluorescently labeled MT colocalize in BN-16 cells, as shown by fluorescent microscopic techniques. Follow-up surface plasmon resonance and flow cytometry studies using overlapping MT peptides and recombinant MT fragments identify the hinge SCKKSCC region of MT as a critical site for megalin binding. These findings suggest that disruption of the SCKKSCC motif can inhibit proximal tubular MT uptake and thereby eliminate much of the renal accumulation and toxicity of heavy metals such as cadmium, gold, copper, and cisplatinum.

                Author and article information

                Nephron Physiol
                Nephron Physiology
                S. Karger AG
                November 2008
                13 October 2008
                : 110
                : 2
                : p11-p20
                aDepartment of Pharmacology, Jichi Medical University, Shimotsuke, Tochigi, and bDepartment of Nephrology, University of Tsukuba, Tsukuba, Japan; cMedical Service, Veterans Affairs, Puget Sound Health Care System, University of Washington, Seattle, Wash., USA
                161986 Nephron Physiol 2008;110:p11
                © 2008 S. Karger AG, Basel

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                Page count
                Figures: 9, References: 25, Pages: 1
                Original Paper


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