Role of the SLC22A17/lipocalin-2 receptor in renal endocytosis of proteins/metalloproteins: a focus on iron- and cadmium-binding proteins.

The transmembrane protein SLC22A17 [or the neutrophil gelatinase-associated lipocalin/lipocalin-2 (LCN2)/24p3 receptor] is an atypical member of the SLC22 family of organic anion and cation transporters: it does not carry typical substrates of SLC22 transporters but mediates receptor-mediated endocytosis (RME) of LCN2. One important task of the kidney is the prevention of urinary loss of proteins filtered by the glomerulus by bulk reabsorption of multiple ligands via megalin:cubilin:amnionless-mediated endocytosis in the proximal tubule (PT). Accordingly, overflow, glomerular, or PT damage, as in Fanconi syndrome, results in proteinuria. Strikingly, up to 20% of filtered proteins escape the PT under physiological conditions and are reabsorbed by the distal nephron. The renal distal tubule and collecting duct express SLC22A17, which mediates RME of filtered proteins that evade the PT but with limited capacity to prevent proteinuria under pathological conditions. The kidney also prevents excretion of filtered essential and nonessential transition metals, such as iron or cadmium, respectively, that are largely bound to proteins with high affinity, e.g., LCN2, transferrin, or metallothionein, or low affinity, e.g., microglobulins or albumin. Hence, increased uptake of transition metals may cause nephrotoxicity. Here, we assess the literature on SLC22A17 structure, topology, tissue distribution, regulation, and assumed functions, emphasizing renal SLC22A17, which has relevance for physiology, pathology, and nephrotoxicity due to the accumulation of proteins complexed with transition metals, e.g., cadmium or iron. Other putative renal functions of SLC22A17, such as its contribution to osmotic stress adaptation, protection against urinary tract infection, or renal carcinogenesis, are discussed.

Targeting Oncolytic Adenoviruses to Cancer Cells Using a Designed Ankyrin Repeat Protein Lipocalin-2 Fusion Protein.

Oncolytic viruses are a promising technology to attack cancer cells and to recruit immune cells to the tumor site. Since the Lipocalin-2 receptor (LCN2R) is expressed on most cancer cells, we used its ligand LCN2 to target oncolytic adenoviruses (Ads) to cancer cells. Therefore, we fused a Designed Ankyrin Repeat Protein (DARPin) adapter binding the knob of Ad type 5 (knob5) to LCN2 to retarget the virus toward LCN2R with the aim of analyzing the basic characteristics of this novel targeting approach. The adapter was tested in vitro with Chinese Hamster Ovary (CHO) cells stably expressing the LCN2R and on 20 cancer cell lines (CCLs) using an Ad5 vector encoding luciferase and green fluorescent protein. Luciferase assays with the LCN2 adapter (LA) showed 10-fold higher infection compared with blocking adapter (BA) in CHO cells expressing LCN2R and in cells not expressing the LCN2R. Most CCLs showed an increased viral uptake of LA-bound virus compared with BA-bound virus and for five CCLs viral uptake was comparable to unmodified Ad5. Flow cytometry and hexon immunostainings also revealed increased uptake of LA-bound Ads compared with BA-bound Ads in most tested CCLs. Virus spread was studied in 3D cell culture models and nine CCLs showed increased and earlier fluorescence signals for LA-bound virus compared with BA-bound virus. Mechanistically, we show that the LA increases viral uptake only in the absence of its ligand Enterobactin (Ent) and independently of iron. Altogether, we characterized a novel DARPin-based system resulting in enhanced uptake demonstrating potential for future oncolytic virotherapy.

Role of hepcidin in oxidative stress and cell death of cultured mouse renal collecting duct cells: protection against iron and sensitization to cadmium.

The liver hormone hepcidin regulates systemic iron homeostasis. Hepcidin is also expressed by the kidney, but exclusively in distal nephron segments. Several studies suggest hepcidin protects against kidney damage involving Fe2+ overload. The nephrotoxic non-essential metal ion Cd2+ can displace Fe2+ from cellular biomolecules, causing oxidative stress and cell death. The role of hepcidin in Fe2+ and Cd2+ toxicity was assessed in mouse renal cortical [mCCD(cl.1)] and inner medullary [mIMCD3] collecting duct cell lines. Cells were exposed to equipotent Cd2+ (0.5-5 µmol/l) and/or Fe2+ (50-100 µmol/l) for 4-24 h. Hepcidin (Hamp1) was transiently silenced by RNAi or overexpressed by plasmid transfection. Hepcidin or catalase expression were evaluated by RT-PCR, qPCR, immunoblotting or immunofluorescence microscopy, and cell fate by MTT, apoptosis and necrosis assays. Reactive oxygen species (ROS) were detected using CellROX™ Green and catalase activity by fluorometry. Hepcidin upregulation protected against Fe2+-induced mIMCD3 cell death by increasing catalase activity and reducing ROS, but exacerbated Cd2+-induced catalase dysfunction, increasing ROS and cell death. Opposite effects were observed with Hamp1 siRNA. Similar to Hamp1 silencing, increased intracellular Fe2+ prevented Cd2+ damage, ROS formation and catalase disruption whereas chelation of intracellular Fe2+ with desferrioxamine augmented Cd2+ damage, corresponding to hepcidin upregulation. Comparable effects were observed in mCCD(cl.1) cells, indicating equivalent functions of renal hepcidin in different collecting duct segments. In conclusion, hepcidin likely binds Fe2+, but not Cd2+. Because Fe2+ and Cd2+ compete for functional binding sites in proteins, hepcidin affects their free metal ion pools and differentially impacts downstream processes and cell fate.

A role for divalent metal transporter (DMT1) in mitochondrial uptake of iron and manganese.

Much of iron and manganese metabolism occurs in mitochondria. Uptake of redox-active iron must be tightly controlled, but little is known about how metal ions enter mitochondria. Recently, we established that the divalent metal transporter 1 (DMT1) is present in the outer mitochondrial membrane (OMM). Therefore we asked if it mediates Fe2+ and Mn2+ influx. Mitochondria were isolated from HEK293 cells permanently transfected with inducible rat DMT1 isoform 1 A/+IRE (HEK293-rDMT1). Fe2+-induced quenching of the dye PhenGreen™SK (PGSK) occurred in two phases, one of which reflected OMM DMT1 with stronger Fe2+ uptake after DMT1 overexpression. DMT1-specific quenching showed an apparent affinity of ~1.5 µM for Fe2+and was blocked by the DMT1 inhibitor CISMBI. Fe2+ influx reflected an imposed proton gradient, a response that was also observed in purified rat kidney cortex (rKC) mitochondria. Non-heme Fe accumulation assayed by ICPOES and stable 57Fe isotope incorporation by ICPMS were increased in HEK293-rDMT1 mitochondria. HEK293-rDMT1 mitochondria displayed higher 59Fe2+ and 54Mn2+ uptake relative to controls with 54Mn2+ uptake blocked by the DMT1 inhibitor XEN602. Such transport was defective in rKC mitochondria with the Belgrade (G185R) mutation. Thus, these results support a role for DMT1 in mitochondrial Fe2+ and Mn2+ acquisition.

Biochemical and Structural Characterization of the Interaction between the Siderocalin NGAL/LCN2 (Neutrophil Gelatinase-associated Lipocalin/Lipocalin 2) and the N-terminal Domain of Its Endocytic Receptor SLC22A17.

The neutrophil gelatinase-associated lipocalin (NGAL, also known as LCN2) and its cellular receptor (LCN2-R, SLC22A17) are involved in many physiological and pathological processes such as cell differentiation, apoptosis, and inflammation. These pleiotropic functions mainly rely on NGAL's siderophore-mediated iron transport properties. However, the molecular determinants underlying the interaction between NGAL and its cellular receptor remain largely unknown. Here, using solution-state biomolecular NMR in conjunction with other biophysical methods, we show that the N-terminal domain of LCN2-R is a soluble extracellular domain that is intrinsically disordered and interacts with NGAL preferentially in its apo state to form a fuzzy complex. The relatively weak affinity (≈10 µm) between human LCN2-R-NTD and apoNGAL suggests that the N terminus on its own cannot account for the internalization of NGAL by LCN2-R. However, human LCN2-R-NTD could be involved in the fine-tuning of the interaction between NGAL and its cellular receptor or in a biochemical mechanism allowing the receptor to discriminate between apo- and holo-NGAL.

Iron transport in the kidney: implications for physiology and cadmium nephrotoxicity.

The kidney has recently emerged as an organ with a significant role in systemic iron (Fe) homeostasis. Substantial amounts of Fe are filtered by the kidney, which have to be reabsorbed to prevent Fe deficiency. Accordingly Fe transporters and receptors for protein-bound Fe are expressed in the nephron that may also function as entry pathways for toxic metals, such as cadmium (Cd), by way of "ionic and molecular mimicry". Similarities, but also differences in handling of Cd by these transport routes offer rationales for the propensity of the kidney to develop Cd toxicity. This critical review provides a comprehensive update on Fe transport by the kidney and its relevance for physiology and Cd nephrotoxicity. Based on quantitative considerations, we have also estimated the in vivo relevance of the described transport pathways for physiology and toxicology. Under physiological conditions all segments of the kidney tubules are likely to utilize Fe for cellular Fe requiring processes for metabolic purposes and also to contribute to reabsorption of free and bound forms of Fe into the circulation. But Cd entering tubule cells disrupts metabolic pathways and is unable to exit. Furthermore, our quantitative analyses contest established models linking chronic Cd nephrotoxicity to proximal tubular uptake of metallothionein-bound Cd. Hence, Fe transport by the kidney may be beneficial by preventing losses from the body. But increased uptake of Fe or Cd that cannot exit tubule cells may lead to kidney injury, and Fe deficiency may facilitate renal Cd uptake.

Mitochondria represent another locale for the divalent metal transporter 1 (DMT1).

The divalent metal transporter (DMT1) is well known for its roles in duodenal iron absorption across the apical enterocyte membrane, in iron efflux from the endosome during transferrin-dependent cellular iron acquisition, as well as in uptake of non-transferrin bound iron in many cells. Recently, using multiple approaches, we have obtained evidence that the mitochondrial outer membrane is another subcellular locale of DMT1 expression. While iron is of vital importance for mitochondrial energy metabolism, its delivery is likely to be tightly controlled due to iron's damaging redox properties. Here we provide additional support for a role of DMT1 in mitochondrial iron acquisition by immunofluorescence colocalization with mitochondrial markers in cells and isolated mitochondria, as well as flow cytometric quantification of DMT1-positive mitochondria from an inducible expression system. Physiological consequences of mitochondrial DMT1 expression are discussed also in consideration of other DMT1 substrates, such as manganese, relevant to mitochondrial antioxidant defense.

Evidence for mitochondrial localization of divalent metal transporter 1 (DMT1).

In mammalian cells, mitochondria receive most incoming iron, yet no entry pathway for iron at the outer mitochondrial membrane (OMM) has been characterized. Our results show that the divalent metal transporter 1 (DMT1) occurs in the OMM. Immunoblots detected DMT1 in mitochondria from a pneumocyte cell model in their OMM. Using the split-ubiquitin yeast 2-hybrid system, we found that cytochrome c oxidase subunit II (COXII) and the translocase of OMM 6-kDa subunit (Tom6) homologue interact with DMT1. COXII coimmunoprecipitates with DMT1. There are 4 DMT1 isoforms that differ at the N and C termini. Using HEK293 cells that inducibly express all of the 4 ends of DMT1, we found all of them in the OMM, as detected by immunoblots after cell fractionation, and in isolated mitochondria, as detected by immunofluorescence. Immunoblot analysis of purified cell fractions from rat renal cortex confirmed and extended these results to the kidney, which expressed high levels of DMT1. Immunogold labeling detected DMT1 colocalization in mitochondria with the voltage-dependent anion-selective channel protein-1, which is expressed in the OMM. We suggest that DMT1 not only exports iron from endosomes, but also serves to import the metal into the mitochondria.

Substrate- and cell contact-dependent inhibitor affinity of human organic cation transporter 2: studies with two classical organic cation substrates and the novel substrate cd2+.

Polyspecific organic cation transporter Oct2 from rat (gene Slc22A2) has been previously shown to transport Cs(+). Here we report that human OCT2 (hOCT2) is able to transport Cd(2+) showing substrate saturation with a Michaelis-Menten constant (Km) of 54 ± 5.8 µM. Uptake of Cd(2+) by hOCT2 was inhibited by typical hOCT2 ligands (unlabeled substrates and inhibitors), and the rate of uptake was decreased by a point mutation in a substrate binding domain of hOCT2. Incubation of hOCT2 overexpressing human embryonic kidney 293 cells (HEK-hOCT2-C) or rat renal proximal tubule cells expressing rOct2 (NRK-52E-C) with Cd(2+) resulted in an increased level of apoptosis that was reduced by OCT2 inhibitory ligand cimetidine(+). HEK-hOCT2-C exhibited different functional properties when they were confluent or had been dissociated by removal of Ca(2+) and Mg(2+). Only confluent HEK-hOCT2-C transported Cd(2+), and confluent and dissociated cells exhibited different potencies for inhibition of uptake of 1-methyl-4-phenylpyridinium(+) (MPP(+)) by Cd(2+), MPP(+), tetraethylammonium(+), cimetidine(+), and corticosterone. In confluent HEK-hOCT2-C, largely different inhibitor potencies were obtained upon comparison of inhibition of Cd(2+) uptake, 4-[4-(dimethylamino)styryl]-N-methylpyridinium(+) (ASP(+)) uptake, and MPP(+) uptake using substrate concentrations far below the respective Km values. Employing a point mutation in the previously identified substrate binding site of rat Oct1 produced evidence that short distance allosteric effects between binding sites for substrates and inhibitors are involved in substrate-dependent inhibitor potency. Substrate-dependent inhibitor affinity is probably a common property of OCTs. To predict interactions between drugs that are transported by OCTs and inhibitory drugs, it is necessary to employ the specific transported drug rather than a model substrate for in vitro measurements.

ERK1/2-dependent bestrophin-3 expression prevents ER-stress-induced cell death in renal epithelial cells by reducing CHOP.

Upon endoplasmic reticulum (ER) stress induction, cells endeavor to survive by engaging the adaptive stress response known as the unfolded protein response or by removing aggregated proteins via autophagy. Chronic ER stress culminates in apoptotic cell death, which involves induction of pro-apoptotic CHOP. Here, we show that bestrophin-3 (Best-3), a protein previously associated with Ca(2+)-activated Cl(-) channel activity, is upregulated by the ER stressors, thapsigargin (TG), tunicamycin (TUN) and the toxic metal Cd(2+). In cultured rat kidney proximal tubule cells, ER stress, CHOP and cell death were induced after 6h by Cd(2+) (25µM), TG (3µM) and TUN (6µM), were associated with increased cytosolic Ca(2+) and downstream formation of reactive oxygen species and attenuated by the Ca(2+) chelator BAPTA-AM (10µM), the antioxidant α-tocopherol (100µM), or overexpression of catalase (CAT). Immunofluorescence staining showed Best-3 expression in the plasma membrane, nuclei and intracellular compartments, though not in the ER, in cultured cells and rat kidney cortex sections. Best-3 mRNA was augmented by ER stress and signaled through increased Ca(2+), oxidative stress and ERK1/2 phosphorylation, because it was attenuated by α-tocopherol, CAT expression, BAPTA-AM, calmodulin kinase inhibitor calmidazolium (40µM), ERK1/2 inhibitor U0126 (10µM), and ERK1/2 RNAi. Knockdown of Best-3 resulted in decreased cell number consequentially of cell death, as determined by nuclear staining and PARP-1 cleavage. Furthermore, reduced ER stress-cell death by Best-3 overexpression is attributed to diminished CHOP. Since Best-3 overexpression did not affect upstream signaling pathways, we hypothesize that Best-3 possibly interferes with CHOP transcription. From our novel observations, we conclude that ERK1/2-dependent Best-3 activation regulates cell fate decisions during ER stress by suppressing CHOP induction and death.

Lipocalin-2 (24p3/neutrophil gelatinase-associated lipocalin (NGAL)) receptor is expressed in distal nephron and mediates protein endocytosis.

In the kidney, bulk reabsorption of filtered proteins occurs in the proximal tubule via receptor-mediated endocytosis (RME) through the multiligand receptor complex megalin-cubilin. Other mechanisms and nephron sites for RME of proteins are unclear. Recently, the secreted protein 24p3 (lipocalin-2, neutrophil gelatinase-associated lipocalin (NGAL)), which is expressed in the distal nephron, has been identified as a sensitive biomarker of kidney damage. A high-affinity receptor for 24p3 (24p3R) that is involved in endocytotic iron delivery has also been cloned. We investigated the localization of 24p3R in rodent kidney and its role in RME of protein-metal complexes and albumin. Immunostaining of kidney tissue showed expression of 24p3R in apical membranes of distal tubules and collecting ducts, but not of proximal tubule. The differential expression of 24p3R in these nephron segments was confirmed in the respective cell lines. CHO cells transiently transfected with 24p3R or distal tubule cells internalized submicromolar concentrations of fluorescence-coupled proteins transferrin, albumin, or metallothionein (MT) as well as the toxic cadmium-MT (Cd2+(7)-MT) complex, which caused cell death. Uptake of MT or transferrin and Cd2+(7)-MT toxicity were prevented by picomolar concentrations of 24p3. An EC50 of 123±50 nM was determined for binding of MT to 24p3R by microscale thermophoresis. Hence, 24p3R binds proteins filtered by the kidney with high affinity and may contribute to RME of proteins, including 24p3, and to Cd2+(7)-MT toxicity in distal nephron segments.

Role of Arf1 in endosomal trafficking of protein-metal complexes and cadmium-metallothionein-1 toxicity in kidney proximal tubule cells.

Cadmium (Cd) is nephrotoxic. Circulating Cd-metallothionein complexes (CdMT) are filtered by the kidney, reabsorbed by proximal tubule cells (PTC) via receptor-mediated endocytosis, and trafficked to lysosomes which results in apoptosis. ADP-ribosylation factors (Arfs) regulate vesicular trafficking. Arf1 is traditionally associated with the secretory pathway, but has been recently found involved in endocytotic trafficking in PTC. Hence, the role of Arf1 was investigated in MT-1 and transferrin (Tf) endocytosis, and in CdMT-1-induced cell death in a PTC line by overexpressing Arf1-wildtype (WT) or dominant-negative mutant Arf1-T31N. Endogenous Arf1 distribution in PTC was punctate throughout the cytosol, but was not detected in the plasma membrane. Arf1 colocalized with markers for sorting to late endosomes (Rab7, CLC6). Arf1 weakly overlapped with the late endosomal/lysosomal marker CLC7, but not with markers for early (Rab5, CLC5) and recycling endosomes (Rab11). Arf1-T31N significantly attenuated CdMT-1 toxicity by ∼60% when compared to Arf1-WT. However, overexpression of Arf1-T31N did not prevent internalization of Alexa Fluor 546-coupled Tf or MT-1 which accumulated in an EEA1-positive early endocytotic compartment, but not in Arf1-WT overexpressing cells. We conclude that Arf1 is involved in trafficking of protein-metal complexes, including CdMT, to late endosomes/lysosomes in renal PTC.

Ferroportin 1 is expressed basolaterally in rat kidney proximal tubule cells and iron excess increases its membrane trafficking.

Ferroportin 1 (FPN1) is an iron export protein expressed in liver and duodenum, as well as in reticuloendothelial macrophages. Previously, we have shown that divalent metal transporter 1 (DMT1) is expressed in late endosomes and lysosomes of the kidney proximal tubule (PT), the nephron segment responsible for the majority of solute reabsorption. We suggested that following receptor mediated endocytosis of transferrin filtered by the glomerulus, DMT1 exports iron liberated from transferrin into the cytosol. FPN1 is also expressed in the kidney yet its role remains obscure. As a first step towards determining the role of renal FPN1, we localized FPN1 in the PT. FPN1 was found to be located in association with the basolateral PT membrane and within the cytosolic compartment. FPN1 was not expressed on the apical brush-border membrane of PT cells. These data support a role for FPN1 in vectorial export of iron out of PT cells. Furthermore, under conditions of iron loading of cultured PT cells, FPN1 was trafficked to the plasma membrane suggesting a coordinated cellular response to export excess iron and limit cellular iron concentrations.

Cadmium induces Wnt signaling to upregulate proliferation and survival genes in sub-confluent kidney proximal tubule cells.

BACKGROUND: The class 1 carcinogen cadmium (Cd2+) disrupts the E-cadherin/beta-catenin complex of epithelial adherens junctions (AJs) and causes renal cancer. Deregulation of E-cadherin adhesion and changes in Wnt/beta-catenin signaling are known to contribute to carcinogenesis. RESULTS: We investigated Wnt signaling after Cd2+-induced E-cadherin disruption in sub-confluent cultured kidney proximal tubule cells (PTC). Cd2+ (25 microM, 3-9 h) caused nuclear translocation of beta-catenin and triggered a Wnt response measured by TOPflash reporter assays. Cd2+ reduced the interaction of beta-catenin with AJ components (E-cadherin, alpha-catenin) and increased binding to the transcription factor TCF4 of the Wnt pathway, which was upregulated and translocated to the nucleus. While Wnt target genes (c-Myc, cyclin D1 and ABCB1) were up-regulated by Cd2+, electromobility shift assays showed increased TCF4 binding to cyclin D1 and ABCB1 promoter sequences with Cd2+. Overexpression of wild-type and mutant TCF4 confirmed Cd2+-induced Wnt signaling. Wnt signaling elicited by Cd2+ was not observed in confluent non-proliferating cells, which showed increased E-cadherin expression. Overexpression of E-cadherin reduced Wnt signaling, PTC proliferation and Cd2+ toxicity. Cd2+ also induced reactive oxygen species dependent expression of the pro-apoptotic ER stress marker and Wnt suppressor CHOP/GADD153 which, however, did not abolish Wnt response and cell viability. CONCLUSIONS: Cd2+ induces Wnt signaling in PTC. Hence, Cd2+ may facilitate carcinogenesis of PTC by promoting Wnt pathway-mediated proliferation and survival of pre-neoplastic cells.

Cellular distribution and subcellular localization of mCLCA1/2 in murine gastrointestinal epithelia.

mCLCA1/2 are members of the CLCA protein family that are widely expressed in secretory epithelia, but their putative physiological role still awaits elucidation. mCLCA1/2 have 95% amino acid identity, but currently no specific antibody is available. We have generated a rabbit polyclonal antibody (pAb849) against aa 424-443 of mCLCA1/2. In HEK293 cells transfected with mCLCA1; pAb849 detected two specific protein bands at approximately 125 kDa and 90 kDa, representing full-length precursor and N-terminal cleavage product, respectively. pAb849 also immunoprecipitated mCLCA1 and labeled the protein by immunostaining. But pAb849 crossreacted with mCLCA3/4/6 despite < or =80% amino acid identity of the antigenic epitope. We therefore investigated the cellular localization of mCLCA1/2 in epithelial tissues, which do not express mCLCA3/4/6 (salivary glands, pancreas, kidney) or express mCLCA3/6 with known localization (mucus cells of stomach and small intestine; villi of small intestine). mCLCA1/2 mRNA and protein expression were found in both parotid and submandibular gland, and immunohistochemistry revealed labeling in parotid acinar cells, in the luminal membrane of parotid duct cells, and in the duct cells of submandibular gland. In exocrine pancreas, mCLCA1/2 expression was restricted to acinar zymogen granule membranes, as assessed by immunoblotting, immunohistochemistry, and preembedding immunoperoxidase and immunogold electron microscopy. Moreover, mCLCA1/2 immunolabeling was present in luminal membranes of gastric parietal cells and small intestinal crypt enterocytes, whereas in the kidney, mCLCA1/2 protein was localized to proximal and distal tubules. The apical membrane localization and overall distribution pattern of mCLCA1/2 favor a transmembrane protein implicated in transepithelial ion transport and protein secretion.

Assessment of the role of renal organic anion transporters in drug-induced nephrotoxicity.

In the present review we have attempted to assess the involvement of the organic anion transporters OAT1, OAT2, OAT3, and OAT4, belonging to the SLC22 family of polyspecific carriers, in drug-induced renal damage in humans. We have focused on drugs with widely recognized nephrotoxic potential, which have previously been reported to interact with OAT family members, and whose underlying pathogenic mechanism suggests the participation of tubular transport. Thus, only compounds generally believed to cause kidney injury either by means of direct tubular toxicity or crystal nephropathy have been considered. For each drug, or class of agents, the evidence for actual transport mediated by individual OATs under in vivo conditions is discussed. We have then examined their role in the context of other carriers present in the renal proximal tubule sharing certain substrates with OATs, as these are critical determinants of the overall contribution of OAT-dependent transport to intracellular accumulation and transepithelial drug secretion, and thus the impact it may have in drug-induced nephrotoxicity.

Organic cation transporters: physiology, toxicology and special focus on ethidium as a novel substrate.

The polyspecific organic cation transporters OCT1 (SLC22A1), OCT2 (SLC22A2) and OCT3 (SLC22A3) mediate facilitated and bidirectional diffusion of small (< or = 500Da) organic cations with broad specificities for endogenous substrates such as choline, acetylcholine and monoamine neurotransmitters, as well as a variety of xenobiotics. Importantly, besides a wide range of clinically used drugs, these also include several toxins like the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) and herbicide paraquat. OCT2-OCT-3 display differential tissue distribution: OCT1 is predominantly found in liver of humans, and liver and kidney in rodents; OCT2 is most strongly expressed in both human and rodent kidney, whereas is OCT3 primarily expressed in placenta, but also more widely detected in various tissues, including brain and lung. The physiological roles of OCTs as transporters for biogenic amines or acetylcholine in these tissues are still debated, in contrast to their involvement in providing access pathways for harmful/toxic cationic substrates into the body and particular tissues. This review highlights a novel role of human and rodent OCTs as carriers of the toxic fluorescent dye ethidium, as opposed to the less harmful related phenanthridine compound propidium, which is not transported. Additional uptake and efflux pathways for ethidium in pro- and eukaryotes are discussed. OCT-mediated pathways may determine major entry routes for ethidium into the body where toxicity via specific mechanisms may develop in tissues expressing OCTs. Considering the high affinity of OCTs for ethidium (K(m) = 1-2 microM) and their strong expression in various organs, strict safety guidelines for the handling of ethidium should be reinforced.

Role of ARF6 in internalization of metal-binding proteins, metallothionein and transferrin, and cadmium-metallothionein toxicity in kidney proximal tubule cells.

Filtered metal-protein complexes, such as cadmium-metallothionein-1 (CdMT-1) or transferrin (Tf) are apically endocytosed partly via megalin/cubilin by kidney proximal tubule (PT) cells where CdMT-1 internalization causes apoptosis. Small GTPase ARF (ADP-ribosylation factor) proteins regulate endocytosis and vesicular trafficking. We investigated roles of ARF6, which has been shown to be involved in internalization of ligands and endocytic trafficking in PT cells, following MT-1/CdMT-1 and Tf uptake by PT cells. WKPT-0293 Cl.2 cells derived from rat PT S1 segment were transfected with hemagglutinin-tagged wild-type (ARF6-WT) or dominant negative (ARF6-T27N) forms of ARF6. Using immunofluorescence, endogenous ARF6 was associated with the plasma membrane (PM) as well as juxtanuclear and co-localized with Rab5a and Rab11 involved in early and recycling endosomal trafficking. Immunofluorescence staining of megalin showed reduced surface labelling in ARF6 dominant negative (ARF6-DN) cells. Intracellular Alexa Fluor 546-conjugated MT-1 uptake was reduced in ARF6-DN cells and CdMT-1 (14.8 microM for 24 h) toxicity was significantly attenuated from 27.3+/-3.9% in ARF6-WT to 11.1+/-4.0% in ARF6-DN cells (n=6, P<0.02). Moreover, reduced Alexa Fluor 546-conjugated Tf uptake was observed in ARF-DN cells (75.0+/-4.6% versus 3.9+/-3.9% of ARF6-WT cells, n=3, P<0.01) and/or remained near the PM (89.3+/-5. 6% versus 45.2+/-14.3% of ARF6-WT cells, n=3, P<0.05). In conclusion, the data support roles for ARF6 in receptor-mediated endocytosis and trafficking of MT-1/Tf to endosomes/lysosomes and CdMT-1 toxicity of PT cells.

Highly efficient neural differentiation of human somatic stem cells, isolated by minimally invasive periodontal surgery.

Neural stem cells (NSCs) are potential sources for cell therapy of neurodegenerative diseases and for drug screening. Despite their potential benefits, ethical and practical considerations limit the application of NSCs derived from human embryonic stem cells (ES) or adult brain tissue. Thus, alternative sources are required to satisfy the criteria of ready accessibility, rapid expansion in chemically defined media and reliable induction to a neuronal fate. We isolated somatic stem cells from the human periodontium that were collected during minimally invasive periodontal access flap surgery as part of guided tissue regeneration therapy. These cells could be propagated as neurospheres in serum-free medium, which underscores their cranial neural crest cell origin. Culture in the presence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) under serum-free conditions resulted in large numbers of nestin-positive/Sox-2-positive NSCs. These periodontium-derived (pd) NSCs are highly proliferative and migrate in response to chemokines that have been described as inducing NSC migration. We used immunocytochemical techniques and RT-PCR analysis to assess neural differentiation after treatment of the expanded cells with a novel induction medium. Adherence to substrate, growth factor deprivation, and retinoic acid treatment led to the acquisition of neuronal morphology and stable expression of markers of neuronal differentiation by more than 90% of the cells. Thus, our novel method might provide nearly limitless numbers of neuronal precursors from a readily accessible autologous adult human source, which could be used as a platform for further experimental studies and has potential therapeutic implications.