Frank Thévenod

Nephrotoxicity and the Proximal Tubule

Cadmium (Cd2+) is a non-essential heavy metal, which is taken up from the environment into the body through pulmonary and enteral pathways. The S1 segment of the kidney proximal tubule (PT) is a major target of chronic Cd2+ toxicity. Renal dysfunction develops in up to 7% of the general population and in its most severe form displays major features of Fanconi syndrome, such as a defective protein, amino acid, glucose, bicarbonate and phosphate reabsorption. The major pathway for Cd2+ uptake by PT cells (PTCs) in vivo is apical endocytosis of Cd2+ complexed to the high-affinity metal-binding protein metallothionein (MT), which may be receptor-mediated. MT is subsequently degraded in endo-lysosomes, and Cd2+ is liberated for translocation into the cytosolic compartment, possibly using transporters for Fe2+, Zn2+ or Cu2+, such as the divalent metal transporter DMT1. Free Cd2+ ions in the extracellular space are translocated across apical and/or basolateral PTC membranes into the cytosol via transporters, whose identity remains unknown. Cytosolic Cd2+ generates reactive oxygen species (ROS), which deplete endogenous radical scavengers. ROS also damage a variety of transport proteins, including the Na+/K+-ATPase, which are subsequently degraded by the proteasome and endo-lysosomal proteases. Cd2+ causes mitochondrial swelling and release of cytochrome c. If these ROS-mediated stress events are not balanced by repair processes, affected cells undergo apoptosis. But Cd2+ also induces the upregulation of cytoprotective stress and metal-scavenging proteins, such as MT. In addition, Cd2+ upregulates the detoxifying pump multidrug resistance P-glycoprotein, which appears to protect PTCs against Cd2+-induced apoptosis. Thus, Cd2+ interferes with various cellular events ranging from mechanisms of induction of programmed cell death to activation of cell survival genes. A better understanding of the cellular mechanisms involved in Cd2+ nephrotoxicity should provide insights into other heavy metal (e.g. Pb2+, Hg2+) nephropathies and various forms of acquired Fanconi syndrome.

Immunolocalization of Anion Exchanger AE2, Na+/H+ Exchangers NHE1 and NHE4, and Vacuolar Type H+-ATPase in Rat Pancreas

We have studied the expression and localization of several H+ and HCO3 – transporters, whose presence in the rat pancreas is still unclear. The Cl–/HCO3 – exchanger AE2, the Na+/H+ exchangers NHE1 and NHE4, and the 31-kD and 70-kD vacuolar H+-ATPase (V-ATPase) subunits were detected by immunoblotting and immunocytochemical techniques. Immunoblotting of plasma membranes with transporter-specific antibodies revealed protein bands at ≈160 kD for AE2, at ≈90 kD and ≈103 kD for NHE1 and NHE4, respectively, and at 31 kD and 70 kD for V-ATPase. NHE1 and NHE4 were further identified by amplification of isoform-specific cDNA using RT-PCR. Immunohistochemistry revealed a basolateral location of AE2, NHE1, and NHE4 in acinar cells. In ducts, NHE1 and NHE4 were basolaterally located but no AE2 expression was detected. V-ATPase was detected in zymogen granules (ZGs) by immunogold labeling, and basolaterally in duct cells by immunohistochemistry. The data indicate that NHE1 and NHE4 are co-expressed in rat pancreatic acini and ducts. Basolateral acinar AE2 could contribute to Cl– uptake and/or pH regulation. V-ATPase may be involved in ZG fusion/exocytosis and ductal HCO3 – secretion. The molecular identity of the ductal Cl–/HCO3 – exchanger remains unclear.

Immunolocalization of anion exchanger AE2 and Na+- HCO 3 − cotransporter in rat parotid and submandibular glands

Salivary glands secrete K(+) and HCO(-)(3) and reabsorb Na(+) and Cl(-), but the identity of transporters involved in HCO(-)(3) transport remains unclear. We investigated localization of Cl(-)/HCO(-)(3) exchanger isoform AE2 and of Na(+)-HCO(-)(3) cotransporter (NBC) in rat parotid gland (PAR) and submandibular gland (SMG) by immunoblot and immunocytochemical techniques. Immunoblotting of PAR and SMG plasma membranes with specific antibodies against mouse kidney AE2 and rat kidney NBC revealed protein bands at approximately 160 and 180 kDa for AE2 and approximately 130 kDa for NBC, as expected for the AE2 full-length protein and consistent with the apparent molecular mass of NBC in several tissues other than kidney. Immunostaining of fixed PAR and SMG tissue sections revealed specific basolateral staining of PAR acinar cells for AE2 and NBC, but in SMG acinar cells only basolateral AE2 labeling was observed. No AE2 expression was detected in any ducts. Striated, intralobular, and main duct cells of both glands showed NBC expression predominantly at basolateral membranes, with some cells being apically stained. In SMG duct cells, NBC staining exhibited a gradient of distribution from basolateral localization in more proximal parts of the ductal tree to apical localization toward distal parts of the ductal tree. Both immunoblotting signals and immunostaining were abolished in preabsorption experiments with the respective antigens. Thus the mechanisms of fluid and anion secretion in salivary acinar cells may be different between PAR and SMG, and, because NBC was detected in acinar and duct cells, it may play a more important role in transport of HCO(-)(3) by rat salivary duct cells than previously believed.

Chloride and Potassium Conductances of Mouse Pancreatic Zymogen Granules Are Inversely Regulated by a 80-kDa mdr1a Gene Product

Cl and cation conductances were characterized in zymogen granules (ZG) isolated from the pancreas of wild-type mice (+/+) or mice with a homozygous disruption of the multidrug resistance P-glycoprotein gene mdr1a (-/-). Cl conductance of ZG was assayed in isotonic KCl buffer by measuring osmotic lysis, which was induced by maximal permeabilization of ZG membranes (ZGM) for K with valinomycin due to influx of K through the artificial pathway and of Cl through endogenous channels. To measure cation conductances, ZG (pH 6.0-6.5) were suspended in buffered isotonic monovalent cation acetate solutions (pH 7.0). The pH gradient was converted into an outside-directed H diffusion potential by maximally increasing H conductance of ZGM with carbonyl cyanide m-chlorophenylhydrazone. Osmotic lysis of ZG was induced by H diffusion potential-driven influx of monovalent cations through endogenous channels and nonionic diffusion of the counterion acetate. ZGM Cl conductances were not different in (-/-) and (+/+) mice (2.6 ± 0.3 hversus 3.1 ± 0.2 h (relative rate constant)). The nonhydrolyzable ATP analog adenosine 5′-(β,-methylene)triphosphate (AMP-PCP) (0.5 mM) activated the Cl conductance both in (+/+) and (-/-) mice. However, activation of Cl conductance by AMP-PCP was reduced in (-/-) mice as compared with (+/+) mice (5.0 ± 0.4 hversus 7.6 ± 0.7 h; p < 0.005). In contrast, ZGM K conductance was increased in (-/-) mice as compared with (+/+) mice (14.2 ± 2.0 hversus 8.5 ± 1.2 h; p < 0.03). In the presence of 0.5 mM AMP-PCP, which completely blocks K conductance but leaves a nonselective cation conductance unaffected, there was no difference between (-/-) and (+/+) mice (5.3 ± 0.7 hversus 3.2 ± 0.5 h). In Western blots of ZGM from wild-type mice, a polyclonal MDR1 specific antibody labeled a protein band of ≈80 kDa. In mdr1a-deficient mice, the intensity of this band was reduced to 39 ± 7% of the wild-type signal. This indicates that a mdr1a gene product of ≈80 kDa enhances the AMP-PCP-activated fraction of mouse ZGM Cl conductance and reduces AMP-PCP-sensitive K conductance.

Immunolocalization of potassium-chloride cotransporter polypeptides in rat exocrine glands

Abstract. Potassium-chloride cotransporters (KCCs) encoded by at least four homologous genes are believed to contribute to cell volume regulation and transepithelial ion transport. We have studied KCC polypeptide expression and immunolocalization of KCCs in rat salivary glands and pancreas. Immunoblot analysis of submandibular, parotid, and pancreas plasma membrane fractions with immunospecific antibodies raised against mouse KCC1 revealed protein bands at caxa0135xa0kDa and caxa0150xa0kDa. Immunocytochemical analysis of fixed salivary and pancreas tissue revealed basolateral KCC1 distribution in rat parotid and pancreatic acinar cells, as well as in parotid, submandibular, and pancreatic duct cells. KCC1 or the polypeptide product(s) of one or more additional KCC genes was also expressed in the basolateral membranes of submandibular acinar cells. Both immunoblot and immunofluorescence signals were abolished in the presence of the peptide antigen. These results establish the presence in rat exocrine glands of KCC1 and likely other KCC polypeptides, and suggest a contribution of KCC polypeptides to transepithelial Cl- transport.

MDR1 in taste buds of rat vallate papilla: functional, immunohistochemical, and biochemical evidence

Multidrug resistance P-glycoprotein (MDR1) is a membrane protein of 150-170 kDa that catalyzes the ATP-driven efflux of hydrophobic xenobiotics, including fluorescent dyes, from cells. Expressed in many epithelial tissues and in the endothelia of the blood-brain barrier, the MDR1 protein provides major routes of detoxification. We found that taste cells of the rat vallate papilla (VP; posterior tongue) had only a slow increase in fluorescence due to uptake of the hydrophobic dye calcein acetoxymethyl ester. However, the development of fluorescence was accelerated two- to threefold by substrates and/or inhibitors of MDR1, such as verapamil, tamoxifen, and cyclosporin A, and by addition of the transport-blocking antibody to MDR1, UIC2. Western blots of vallate tissue rich in taste buds with the MDR1-specific monoclonal antibodies C219 and C494 revealed an immunoreactive protein at approximately 170 kDa. In contrast, the lingual epithelium surrounding the VP showed a much weaker band with these antibodies. Furthermore, using the antibodies C494 and UIC2 with tissue sections, MDR1-like immunoreactivity was found in taste cells. These results show that MDR1 is present and functional in vallate taste cells of the rat. MDR1-related transport may achieve active elimination of xenobiotics from the sensory cells and thereby protect the peripheral taste organs from potentially harmful molecules contained in an animals food.

Fe2+ Block and Permeation of CaV3.1 (α1G) T-Type Calcium Channels: Candidate Mechanism for Non–Transferrin-Mediated Fe2+ Influx

Iron is a biologically essential metal, but excess iron can cause damage to the cardiovascular and nervous systems. We examined the effects of extracellular Fe2+ on permeation and gating of CaV3.1 channels stably transfected in HEK293 cells, by using whole-cell recording. Precautions were taken to maintain iron in the Fe2+ state (e.g., use of extracellular ascorbate). With the use of instantaneous I-V currents (measured after strong depolarization) to isolate the effects on permeation, extracellular Fe2+ rapidly blocked currents with 2 mM extracellular Ca2+ in a voltage-dependent manner, as described by a Woodhull model with KD = 2.5 mM at 0 mV and apparent electrical distance δ = 0.17. Extracellular Fe2+ also shifted activation to more-depolarized voltages (by ∼10 mV with 1.8 mM extracellular Fe2+) somewhat more strongly than did extracellular Ca2+ or Mg2+, which is consistent with a Gouy-Chapman-Stern model with surface charge density σ = 1 e−/98 Å2 and KFe = 4.5 M−1 for extracellular Fe2+. In the absence of extracellular Ca2+ (and with extracellular Na+ replaced by TEA), Fe2+ carried detectable, whole-cell, inward currents at millimolar concentrations (73 ± 7 pA at −60 mV with 10 mM extracellular Fe2+). With a two-site/three-barrier Eyring model for permeation of CaV3.1 channels, we estimated a transport rate for Fe2+ of ∼20 ions/s for each open channel at −60 mV and pH 7.2, with 1 μM extracellular Fe2+ (with 2 mM extracellular Ca2+). Because CaV3.1 channels exhibit a significant “window current” at that voltage (open probability, ∼1%), CaV3.1 channels represent a likely pathway for Fe2+ entry into cells with clinically relevant concentrations of extracellular Fe2+.

Evidence for a 65 kDa sulfonylurea receptor in rat pancreatic zymogen granule membranes

In rat pancreatic zymogen granules (ZG), a K+ selective conductance which can be blocked by ATP has been characterized. Here we show that this pathway can be specifically blocked by glibenclamide. Using a rapid filtration assay, we also found specific binding of [3H]glibenclamide to a low‐affinity site (K d 5.6±1.1 μM) in rat pancreatic zymogen granule membranes (ZGM). In photoaffinity labeling experiments with [3H]glibenclamide, a 65±1.5 kDa polypeptide was specifically labeled. Previously, a ∼65 kDa mdr1 gene product has been demonstrated to be involved in the regulation of the K+ selective conductance of ZG. We conclude that this protein may be a subunit of, or associated with, a ZG KATP channel.

Cd2+ Block and Permeation of CaV3.1 (α1G) T-Type Calcium Channels: Candidate Mechanism for Cd2+ Influx

Cd2+ is an industrial pollutant that can cause cytotoxicity in multiple organs. We examined the effects of extracellular Cd2+ on permeation and gating of Cav3.1 (α1G) channels stably transfected in HEK293 cells, by using whole-cell recording. With the use of instantaneous I-V currents (measured after strong depolarization) to isolate the effects on permeation, Cd2+ rapidly blocked currents with 2 mM Ca2+ in a voltage-dependent manner. The block caused by Cd2+ was relieved at more-hyperpolarized potentials, which suggests that Cd2+ can permeate through the selectivity filter of the channel into the cytosol. In the absence of other permeant ions (Ca2+ and Na+ replaced by N-methyl-d-glucamine), Cd2+ carried sizable inward currents through Cav3.1 channels (210 ± 20 pA at −60 mV with 2 mM Cd2+). Cav3.1 channels have a significant “window current” at that voltage (open probability, ∼1%), which makes them a candidate pathway for Cd2+ entry into cells during Cd2+ exposure. Incubation with radiolabeled 109Cd2+ confirmed uptake of Cd2+ into cells with Cav3.1 channels.

Expression of the electrogenic Na+–HCO3−-cotransporters NBCe1-A and NBCe1-B in rat pancreatic islet cells

It was recently proposed that, in rat pancreatic islets, the production of bicarbonate accounts for the major fraction of the carbon dioxide generated by the oxidative catabolism of nutrient insulin secretagogues. In search of the mechanism(s) supporting the membrane transport of bicarbonate, the possible role of the electrogenic Na+–HCO3−-cotransporters NBCe1-A and NBCe1-B in rat pancreatic islet cells was investigated. Expression of NBCe1-A and NBCe1-B in rat pancreatic islet cells was documented by RT-PCR, western blotting, and immunocytochemistry. The latter procedure suggested a preferential localization of NBCe1-B in insulin-producing cells. Tenidap (3–100xa0μM), previously proposed as an inhibitor of NBCe1-A-mediated cotransport in proximal tubule kidney cells, caused a concentration-related inhibition of glucose-stimulated insulin secretion. It also inhibited 2-ketoisocaproate-induced insulin release and to a relatively lesser extent, the secretory response to l-leucine. Tenidap (50–100xa0μM) also inhibited the metabolism of d-glucose in isolated islets, increased 22Na net uptake by dispersed islet cells, lowered intracellular pH and provoked hyperpolarization of plasma membrane in insulin-producing cells. This study thus reveals the expression of the electrogenic Na+–HCO3−-cotransporters NBCe1-A and NBCe1-B in rat pancreatic islet cells, and is consistent with the participation of such transporters in the process of nutrient-stimulated insulin secretion.