Cadmium transport by mammalian ATP-binding cassette transporters.

Cellular responses to toxic metals depend on metal accessibility to intracellular targets, reaching interaction sites, and the intracellular metal concentration, which is mainly determined by uptake pathways, binding/sequestration and efflux pathways. ATP-binding cassette (ABC) transporters are ubiquitous in the human body-usually in epithelia-and are responsible for the transfer of indispensable physiological substrates (e.g. lipids and heme), protection against potentially toxic substances, maintenance of fluid composition, and excretion of metabolic waste products. Derailed regulation and gene variants of ABC transporters culminate in a wide array of pathophysiological disease states, such as oncogenic multidrug resistance or cystic fibrosis. Cadmium (Cd) has no known physiological role in mammalians and poses a health risk due to its release into the environment as a result of industrial activities, and eventually passes into the food chain. Epithelial cells, especially within the liver, lungs, gastrointestinal tract and kidneys, are particularly susceptible to the multifaceted effects of Cd because of the plethora of uptake pathways available. Pertinent to their broad substrate spectra, ABC transporters represent a major cellular efflux pathway for Cd and Cd complexes. In this review, we summarize current knowledge concerning transport of Cd and its complexes (mainly Cd bound to glutathione) by the ABC transporters ABCB1 (P-glycoprotein, MDR1), ABCB6, ABCC1 (multidrug resistance related protein 1, MRP1), ABCC7 (cystic fibrosis transmembrane regulator, CFTR), and ABCG2 (breast cancer related protein, BCRP). Potential detoxification strategies underlying ABC transporter-mediated efflux of Cd and Cd complexes are discussed.

Distinct concentration-dependent oxidative stress profiles by cadmium in a rat kidney proximal tubule cell line.

Levels and chemical species of reactive oxygen/nitrogen species (ROS/RNS) determine oxidative eustress and distress. Abundance of uptake pathways and high oxygen consumption for ATP-dependent transport makes the renal proximal tubule particularly susceptible to cadmium (Cd2+)-induced oxidative stress by targeting ROS/RNS generation or antioxidant defence mechanisms, such as superoxide dismutase (SOD) or H2O2-metabolizing catalase (CAT). Though ROS/RNS are well-evidenced, the role of distinct ROS profiles in Cd2+ concentration-dependent toxicity is not clear. In renal cells, Cd2+ (10-50 µM) oxidized dihydrorhodamine 123, reaching a maximum at 2-3 h. Increases (up to fourfold) in lipid peroxidation by TBARS assay and H2O2 by Amplex Red were evident within 30 min. ROS and loss in cell viability by MTT assay with 50 µM Cd2+ could not be fully reversed by SOD mimetics Tempol and MnTBAP nor by SOD1 overexpression, whereas CAT expression and α-tocopherol were effective. SOD and CAT activities were attenuated below controls only with >6 h 50 µM Cd2+, yet augmented by up to 1.5- and 1.2-fold, respectively, by 10 µM Cd2+. Moreover, 10 µM, but not 25-50 µM Cd2+, caused 1.7-fold increase in superoxide anion (O2•-), detected by dihydroethidium, paralled by loss in cell viability, that was abolished by Tempol, MnTBAP, α-tocopherol and SOD1 or CAT overexpression. H2O2-generating NADPH oxidase 4 (NOX4) was attenuated by ~50% with 10 µM Cd2+ at 3 h compared to upregulation by 50 µM Cd2+ (~1.4-fold, 30 min), which was sustained for 24 h. In summary, O2•- predominates with low-moderate Cd2+, driving an adaptive response, whereas oxidative stress by elevated H2O2 at high Cd2+ triggers cell death signaling pathways.Highlights Different levels of reactive oxygen species are generated, depending on cadmium concentration. Superoxide anion predominates and H2O2 is suppressed with low cadmium representing oxidative eustress. High cadmium fosters H2O2 by inhibiting catalase and increasing NOX4 leading to oxidative distress. Superoxide dismutase mimetics and overexpression were less effective with high versus low cadmium. Oxidative stress profile could dictate downstream signalling pathways.

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.

Renal hypoxia-HIF-PHD-EPO signaling in transition metal nephrotoxicity: friend or foe?

The kidney is the main organ that senses changes in systemic oxygen tension, but it is also the key detoxification, transit and excretion site of transition metals (TMs). Pivotal to oxygen sensing are prolyl-hydroxylases (PHDs), which hydroxylate specific residues in hypoxia-inducible factors (HIFs), key transcription factors that orchestrate responses to hypoxia, such as induction of erythropoietin (EPO). The essential TM ion Fe is a key component and regulator of the hypoxia-PHD-HIF-EPO (HPHE) signaling axis, which governs erythropoiesis, angiogenesis, anaerobic metabolism, adaptation, survival and proliferation, and hence cell and body homeostasis. However, inadequate concentrations of essential TMs or entry of non-essential TMs in organisms cause toxicity and disrupt health. Non-essential TMs are toxic because they enter cells and displace essential TMs by ionic and molecular mimicry, e. g. in metalloproteins. Here, we review the molecular mechanisms of HPHE interactions with TMs (Fe, Co, Ni, Cd, Cr, and Pt) as well as their implications in renal physiology, pathophysiology and toxicology. Some TMs, such as Fe and Co, may activate renal HPHE signaling, which may be beneficial under some circumstances, for example, by mitigating renal injuries from other causes, but may also promote pathologies, such as renal cancer development and metastasis. Yet some other TMs appear to disrupt renal HPHE signaling, contributing to the complex picture of TM (nephro-)toxicity. Strikingly, despite a wealth of literature on the topic, current knowledge lacks a deeper molecular understanding of TM interaction with HPHE signaling, in particular in the kidney. This precludes rationale preventive and therapeutic approaches to TM nephrotoxicity, although recently activators of HPHE signaling have become available for therapy.

Investigating the Molecular Mechanisms of Renal Hepcidin Induction and Protection upon Hemoglobin-Induced Acute Kidney Injury.

Hemolysis is known to cause acute kidney injury (AKI). The iron regulatory hormone hepcidin, produced by renal distal tubules, is suggested to exert a renoprotective role during this pathology. We aimed to elucidate the molecular mechanisms of renal hepcidin synthesis and its protection against hemoglobin-induced AKI. In contrast to known hepatic hepcidin induction, incubation of mouse cortical collecting duct (mCCDcl1) cells with IL-6 or LPS did not induce Hamp1 mRNA expression, whereas iron (FeS) and hemin significantly induced hepcidin synthesis (p < 0.05). Moreover, iron/heme-mediated hepcidin induction in mCCDcl1 cells was caused by the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, as indicated by increased nuclear Nrf2 translocation and induced expression of Nrf2 downstream targets GCLM (p < 0.001), NQO1 (p < 0.001), and TXNRD1 (p < 0.005), which could be prevented by the known Nrf2 inhibitor trigonelline. Newly created inducible kidney-specific hepcidin KO mice demonstrated a significant reduction in renal Hamp1 mRNA expression. Phenylhydrazine (PHZ)-induced hemolysis caused renal iron loading and oxidative stress in both wildtype (Wt) and KO mice. PHZ treatment in Wt induced inflammatory markers (IL-6, TNFα) but not Hamp1. However, since PHZ treatment also significantly reduced systemic hepcidin levels in both Wt and KO mice (both p < 0.001), a dissection between the roles of systemic and renal hepcidin could not be made. Combined, the results of our study indicate that there are kidney-specific mechanisms in hepcidin regulation, as indicated by the dominant role of iron and not inflammation as an inducer of renal hepcidin, but also emphasize the complex interplay of various iron regulatory mechanisms during AKI on a local and systemic level.

Kidney tubule iron loading in experimental focal segmental glomerulosclerosis.

Kidney iron deposition may play a role in the progression of tubulointerstitial injury during chronic kidney disease. Here, we studied the molecular mechanisms of kidney iron loading in experimental focal segmental glomerulosclerosis (FSGS) and investigated the effect of iron-reducing interventions on disease progression. Thy-1.1 mice were injected with anti-Thy-1.1 monoclonal antibody (mAb) to induce proteinuria. Urine, blood and tissue were collected at day (D)1, D5, D8, D15 and D22 after mAb injection. Thy-1.1 mice were subjected to captopril (CA), iron-deficient (ID) diet or iron chelation (deferoxamine; DFO). MAb injection resulted in significant albuminuria at all time points (p < 0.01). Kidney iron loading, predominantly in distal tubules, increased in time, along with urinary kidney injury molecule-1 and 24p3 concentration, as well as kidney mRNA expression of Interleukin-6 (Il-6) and Heme oxygenase-1 (Ho-1). Treatment with CA, ID diet or DFO significantly reduced kidney iron deposition at D8 and D22 (p < 0.001) and fibrosis at D22 (p < 0.05), but not kidney Il-6. ID treatment increased kidney Ho-1 (p < 0.001). In conclusion, kidney iron accumulation coincides with progression of tubulointerstitial injury in this model of FSGS. Reduction of iron loading halts disease progression. However, targeted approaches to prevent excessive kidney iron loading are warranted to maintain the delicate systemic and cellular iron balance.

Dependence of ABCB1 transporter expression and function on distinct sphingolipids generated by ceramide synthases-2 and -6 in chemoresistant renal cancer.

Oncogenic multidrug resistance is commonly intrinsic to renal cancer based on the physiological expression of detoxification transporters, particularly ABCB1, thus hampering chemotherapy. ABCB1 activity is directly dependent on its lipid microenvironment, localizing to cholesterol- and sphingomyelin (SM)-rich domains. As ceramides are the sole source for SMs, we hypothesized that ceramide synthase (CerS)-derived ceramides regulate ABCB1 activity. Using data from RNA-Seq databases, we found that patient kidney tumors exhibited increased CerS2 mRNA, which was inversely correlated with CerS6 mRNA in ABCB1+ clear cell carcinomas. Endogenous elevated CerS2 and lower CerS5/6 mRNA and protein resulted in disproportionately higher CerS2 to CerS5/6 activities (approximately twofold) in chemoresistant ABCB1high (A498, Caki-1) compared with chemosensitive ABCB1low (ACHN, normal human proximal convoluted tubule cell) cells. In addition, lipidomics analyses by HPLC-MS/MS showed bias toward CerS2-associated C20:0/C20:1-ceramides compared with CerS5/6-associated C14:0/C16:0-ceramides (2:1). SMs were similarly altered. We demonstrated that chemoresistance to doxorubicin in ABCB1high cells was partially reversed by inhibitors of de novo ceramide synthesis (l-cycloserine) and CerS (fumonisin B1) in cell viability assays. Downregulation of CerS2/6, but not CerS5, attenuated ABCB1 mRNA, protein, plasma membrane localization, rhodamine 123+ efflux transport activity, and doxorubicin resistance. Similar findings were observed with catalytically inactive CerS6-H212A. Furthermore, CerS6-targeting siRNA shifted ceramide and SM composition to ultra long-chain species (C22-C26). Inhibitors of endoplasmic reticulum-associated degradation (eeyarestatin I) and the proteasome (MG132, bortezomib) prevented ABCB1 loss induced by CerS2/6 downregulation. We conclude that a critical balance in ceramide/SM species is prerequisite to ABCB1 expression and functionalization, which could be targeted to reverse multidrug resistance in renal cancers.

Increased Endocytosis of Cadmium-Metallothionein through the 24p3 Receptor in an In Vivo Model with Reduced Proximal Tubular Activity.

BACKGROUND: The proximal tubule (PT) is the major target of cadmium (Cd2+) nephrotoxicity. Current dogma postulates that Cd2+ complexed to metallothionein (MT) (CdMT) is taken up through receptor-mediated endocytosis (RME) via the PT receptor megalin:cubilin, which is the predominant pathway for reuptake of filtered proteins in the kidney. Nevertheless, there is evidence that the distal parts of the nephron are also sensitive to damage induced by Cd2+. In rodent kidneys, another receptor for protein endocytosis, the 24p3 receptor (24p3R), is exclusively expressed in the apical membranes of distal tubules (DT) and collecting ducts (CD). Cell culture studies have demonstrated that RME and toxicity of CdMT and other (metal ion)-protein complexes in DT and CD cells is mediated by 24p3R. In this study, we evaluated the uptake of labeled CdMT complex through 24p3R after acute kidney injury (AKI) induced by gentamicin (GM) administration that disrupts PT function. Subcutaneous administration of GM at 10 mg/kg/day for seven days did not alter the structural and functional integrity of the kidney's filtration barrier. However, because of PT injury, the concentration of the renal biomarker Kim-1 increased. When CdMT complex coupled to FITC was administered intravenously, both uptake of the CdMT complex and 24p3R expression in DT increased and also colocalized after PT injury induced by GM. Although megalin decreased in PT after GM administration, urinary protein excretion was not changed, which suggests that the increased levels of 24p3R in the distal nephron could be acting as a compensatory mechanism for protein uptake. Altogether, these results suggest that PT damage increases the uptake of the CdMT complex through 24p3R in DT (and possibly CD) and compensate for protein losses associated with AKI.

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.

Corrigendum: Iron and Cadmium Entry Into Renal Mitochondria: Physiological and Toxicological Implications.

[This corrects the article DOI: 10.3389/fcell.2020.00848.].

Iron and Cadmium Entry Into Renal Mitochondria: Physiological and Toxicological Implications.

Regulation of body fluid homeostasis is a major renal function, occurring largely through epithelial solute transport in various nephron segments driven by Na+/K+-ATPase activity. Energy demands are greatest in the proximal tubule and thick ascending limb where mitochondrial ATP production occurs through oxidative phosphorylation. Mitochondria contain 20-80% of the cell's iron, copper, and manganese that are imported for their redox properties, primarily for electron transport. Redox reactions, however, also lead to reactive, toxic compounds, hence careful control of redox-active metal import into mitochondria is necessary. Current dogma claims the outer mitochondrial membrane (OMM) is freely permeable to metal ions, while the inner mitochondrial membrane (IMM) is selectively permeable. Yet we recently showed iron and manganese import at the OMM involves divalent metal transporter 1 (DMT1), an H+-coupled metal ion transporter. Thus, iron import is not only regulated by IMM mitoferrins, but also depends on the OMM to intermembrane space H+ gradient. We discuss how these mitochondrial transport processes contribute to renal injury in systemic (e.g., hemochromatosis) and local (e.g., hemoglobinuria) iron overload. Furthermore, the environmental toxicant cadmium selectively damages kidney mitochondria by "ionic mimicry" utilizing iron and calcium transporters, such as OMM DMT1 or IMM calcium uniporter, and by disrupting the electron transport chain. Consequently, unraveling mitochondrial metal ion transport may help develop new strategies to prevent kidney injury induced by metals.

Cell organelles as targets of mammalian cadmium toxicity.

Ever increasing environmental presence of cadmium as a consequence of industrial activities is considered a health hazard and is closely linked to deteriorating global health status. General animal and human cadmium exposure ranges from ingestion of foodstuffs sourced from heavily polluted hotspots and cigarette smoke to widespread contamination of air and water, including cadmium-containing microplastics found in household water. Cadmium is promiscuous in its effects and exerts numerous cellular perturbations based on direct interactions with macromolecules and its capacity to mimic or displace essential physiological ions, such as iron and zinc. Cell organelles use lipid membranes to form complex tightly-regulated, compartmentalized networks with specialized functions, which are fundamental to life. Interorganellar communication is crucial for orchestrating correct cell behavior, such as adaptive stress responses, and can be mediated by the release of signaling molecules, exchange of organelle contents, mechanical force generated through organelle shape changes or direct membrane contact sites. In this review, cadmium effects on organellar structure and function will be critically discussed with particular consideration to disruption of organelle physiology in vertebrates.

Inverse Regulation of Lipocalin-2/24p3 Receptor/SLC22A17 and Lipocalin-2 Expression by Tonicity, NFAT5/TonEBP and Arginine Vasopressin in Mouse Cortical Collecting Duct Cells mCCD(cl.1): Implications for Osmotolerance.

The rodent collecting duct (CD) expresses a 24p3/NGAL/lipocalin-2 (LCN2) receptor (SLC22A17) apically, possibly to mediate high-affinity reabsorption of filtered proteins by endocytosis, although its functions remain uncertain. Recently, we showed that hyperosmolarity/-tonicity upregulates SLC22A17 in cultured mouse inner-medullary CD cells, whereas activation of toll-like receptor 4 (TLR4), via bacterial lipopolysaccharides (LPS), downregulates SLC22A17. This is similar to the upregulation of Aqp2 by hyperosmolarity/-tonicity and arginine vasopressin (AVP), and downregulation by TLR4 signaling, which occur via the transcription factors NFAT5 (TonEBP or OREBP), cAMP-responsive element binding protein (CREB), and nuclear factor-kappa B, respectively. The aim of the study was to determine the effects of osmolarity/tonicity and AVP, and their associated signaling pathways, on the expression of SLC22A17 and its ligand, LCN2, in the mouse (m) cortical collecting duct cell line mCCD(cl.1). Normosmolarity/-tonicity corresponded to 300 mosmol/L, whereas the addition of 50-100 mmol/L NaCl for up to 72 h induced hyperosmolarity/-tonicity (400-500 mosmol/L). RT-PCR, qPCR, immunoblotting and immunofluorescence microscopy detected Slc22a17/SLC22A17 and Lcn2/LCN2 expression. RNAi silenced Nfat5, and the pharmacological agent 666-15 blocked CREB. Activation of TLR4 was induced with LPS. Similar to Aqp2, hyperosmotic/-tonic media and AVP upregulated Slc22a17/SLC22A17, via activation of NFAT5 and CREB, respectively, and LPS/TLR4 signaling downregulated Slc22a17/SLC22A17. Conversely, though NFAT5 mediated the hyperosmolarity/-tonicity induced downregulation of Lcn2/LCN2 expression, AVP reduced Lcn2/LCN2 expression and predominantly apical LCN2 secretion, evoked by LPS, through a posttranslational mode of action that was independent of CREB signaling. In conclusion, the hyperosmotic/-tonic upregulation of SLC22A17 in mCCD(cl.1) cells, via NFAT5, and by AVP, via CREB, suggests that SLC22A17 contributes to adaptive osmotolerance, whereas LCN2 downregulation could counteract increased proliferation and permanent damage of osmotically stressed cells.

Cadmium Complexed with ß2-Microglubulin, Albumin and Lipocalin-2 rather than Metallothionein Cause Megalin:Cubilin Dependent Toxicity of the Renal Proximal Tubule.

Cadmium (Cd2+) in the environment is a significant health hazard. Chronic low Cd2+ exposure mainly results from food and tobacco smoking and causes kidney damage, predominantly in the proximal tubule. Blood Cd2+ binds to thiol-containing high (e.g., albumin, transferrin) and low molecular weight proteins (e.g., the high-affinity metal-binding protein metallothionein, ß2-microglobulin, α1-microglobulin and lipocalin-2). These plasma proteins reach the glomerular filtrate and are endocytosed at the proximal tubule via the multiligand receptor complex megalin:cubilin. The current dogma of chronic Cd2+ nephrotoxicity claims that Cd2+-metallothionein endocytosed via megalin:cubilin causes renal damage. However, a thorough study of the literature strongly argues for revision of this model for various reasons, mainly: (i) It relied on studies with unusually high Cd2+-metallothionein concentrations; (ii) the KD of megalin for metallothionein is ~105-times higher than (Cd2+)-metallothionein plasma concentrations. Here we investigated the uptake and toxicity of ultrafiltrated Cd2+-binding protein ligands that are endocytosed via megalin:cubilin in the proximal tubule. Metallothionein, ß2-microglobulin, α1-microglobulin, lipocalin-2, albumin and transferrin were investigated, both as apo- and Cd2+-protein complexes, in a rat proximal tubule cell line (WKPT-0293 Cl.2) expressing megalin:cubilin at low passage, but is lost at high passage. Uptake was determined by fluorescence microscopy and toxicity by MTT cell viability assay. Apo-proteins in low and high passage cells as well as Cd2+-protein complexes in megalin:cubilin deficient high passage cells did not affect cell viability. The data prove Cd2+-metallothionein is not toxic, even at >100-fold physiological metallothionein concentrations in the primary filtrate. Rather, Cd2+-ß2-microglobulin, Cd2+-albumin and Cd2+-lipocalin-2 at concentrations present in the primary filtrate are taken up by low passage proximal tubule cells and cause toxicity. They are therefore likely candidates of Cd2+-protein complexes damaging the proximal tubule via megalin:cubilin at concentrations found in the ultrafiltrate.

Proximal tubule transferrin uptake is modulated by cellular iron and mediated by apical membrane megalin-cubilin complex and transferrin receptor 1.

Receptor-mediated endocytosis is responsible for reabsorption of transferrin (Tf) in renal proximal tubules (PTs). Although the role of the megalin-cubilin receptor complex (MCRC) in this process is unequivocal, modalities independent of this complex are evident but as yet undefined. Here, using immunostaining and Tf-flux assays, FACS analysis, and fluorescence imaging, we report localization of Tf receptor 1 (TfR1), the cognate Tf receptor mediating cellular holo-Tf (hTf) acquisition, to the apical brush border of the PT, with expression gradually declining along the PT in mouse and rat kidneys. In functional studies, hTf uptake across the apical membrane of cultured PT epithelial cell (PTEC) monolayers increased in response to decreased cellular iron after desferrioxamine (DFO) treatment. We also found that apical hTf uptake under basal conditions is receptor-associated protein (RAP)-sensitive and therefore mediated by the MCRC but becomes RAP-insensitive under DFO treatment, with concomitantly decreased megalin and cubilin expression levels and increased TfR1 expression. Thus, as well as the MCRC, TfR1 mediates hTf uptake across the PT apical brush border, but in conditions of decreased cellular iron, hTf uptake is predominated by augmented apical TfR1. In conclusion, both the MCRC and TfR1 mediate hTf uptake across apical brush border membranes of PTECs and reciprocally respond to decreased cellular iron. Our findings have implications for renal health, whole-body iron homeostasis, and pathologies arising from disrupted iron balance.

Channels, transporters and receptors for cadmium and cadmium complexes in eukaryotic cells: myths and facts.

Cadmium (Cd2+) is a toxic and non-essential divalent metal ion in eukaryotic cells. Cells can only be targeted by Cd2+ if it hijacks physiological high-affinity entry pathways, which transport essential divalent metal ions in a process termed "ionic and molecular mimicry". Hence, "free" Cd2+ ions and Cd2+ complexed with small organic molecules are transported across cellular membranes via ion channels, carriers and ATP hydrolyzing pumps, whereas receptor-mediated endocytosis (RME) internalizes Cd2+-protein complexes. Only Cd2+ transport pathways validated by stringent methodology, namely electrophysiology, 109Cd2+ tracer studies, inductively coupled plasma mass spectrometry, atomic absorption spectroscopy, Cd2+-sensitive fluorescent dyes, or specific ligand binding and internalization assays for RME are reviewed whereas indirect correlative studies are excluded. At toxicologically relevant concentrations in the submicromolar range, Cd2+ permeates voltage-dependent Ca2+ channels ("T-type" CaV3.1, CatSper), transient receptor potential (TRP) channels (TRPA1, TRPV5/6, TRPML1), solute carriers (SLCs) (DMT1/SLC11A2, ZIP8/SLC39A8, ZIP14/SLC39A14), amino acid/cystine transporters (SLC7A9/SLC3A1, SLC7A9/SLC7A13), and Cd2+-protein complexes are endocytosed by the lipocalin-2/NGAL receptor SLC22A17. Cd2+ transport via the mitochondrial Ca2+ uniporter, ATPases ABCC1/2/5 and transferrin receptor 1 is likely but requires further evidence. Cd2+ flux occurs through the influx carrier OCT2/SLC22A2, efflux MATE proteins SLC47A1/A2, the efflux ATPase ABCB1, and RME of Cd2+-metallothionein by the receptor megalin (low density lipoprotein receptor-related protein 2, LRP2):cubilin albeit at high concentrations thus questioning their relevance in Cd2+ loading. Which Cd2+-protein complexes are internalized by megalin:cubilin in vivo still needs to be determined. A stringent conservative and reductionist approach is mandatory to verify relevance of transport pathways for Cd2+ toxicity and to overcome dissemination of unsubstantiated conjectures.

Oncogenic PITX2 facilitates tumor cell drug resistance by inverse regulation of hOCT3/SLC22A3 and ABC drug transporters in colon and kidney cancers.

Oncogenic pituitary homeobox 2 (PITX2), a de facto master regulator of developmental organ asymmetry, previously upregulated multidrug resistance (MDR) P-glycoprotein ABCB1 in A498 renal cell carcinoma (RCC) cells. The role of PITX2 isoforms in MDR cancers was investigated. Data mining correlated elevated PITX2 in >30% of cancers analyzed, maximally in colon (4.4-fold), confirmed in co-immunostaining of colon and renal cancer microarrays wherein ABCB1 concomitantly increased in RCC. Drug-resistant colorectal adenocarcinoma Colo320DM cells exhibited increased nuclear PITX2 (40-fold), PITX2 promoter activity (27-fold) and ABCB1 (8000-fold) compared to drug-sensitive Colo205. ABCB1 inhibitor PSC833/valspodar or PITX2 siRNA reversed doxorubicin resistance. Nuclei from Colo320DM and A498 cells harbored PITX2A/B1 and PITX2A/B1/B2/Cα/Cß, respectively. ChIP-qPCR evidenced PITX2 promoter binding in drug exporters ABCB1, ABCC1, ABCG2 and importer hOCT3/SLC22A3. In A498, 786-O, Caki-1, Colo320DM, and Caco2 cells, PITX2 siRNA diminished exporters, increased hOCT3/SLC22A3 expression and activity, and reverted vincristine resistance. Heterologous PITX2 expression induced ABCB1, repressed hOCT3/SLC22A3, enhanced vincristine resistance and diminished proliferation inhibition wherein PITX2A and PITX2C were most effective. Furthermore, PITX2 activity and MDR depended on phosphorylation by GSK3 in A498 cells. Conclusively, oncogenic PITX2 limits sensitizing drug uptake and potentiates cytoprotective drug efflux, contributing to MDR phenotype.

Intrauterine Exposure to Cadmium Reduces HIF-1 DNA-Binding Ability in Rat Fetal Kidneys.

During embryonic development, some hypoxia occurs due to incipient vascularization. Under hypoxic conditions, gene expression is mainly controlled by hypoxia-inducible factor 1 (HIF-1). The activity of this transcription factor can be altered by the exposure to a variety of compounds; among them is cadmium (Cd), a nephrotoxic heavy metal capable of crossing the placenta and reaching fetal kidneys. The goal of the study was to determine Cd effects on HIF-1 on embryonic kidneys. Pregnant Wistar rats were exposed to a mist of isotonic saline solution or CdCl2 (DDel = 1.48 mg Cd/kg/day), from gestational day (GD) 8 to 20. Embryonic kidneys were obtained on GD 21 for RNA and protein extraction. Results show that Cd exposure had no effect on HIF-1α and prolyl hydroxylase 2 protein levels, but it reduced HIF-1 DNA-binding ability, which was confirmed by a decrease in vascular endothelial growth factor (VEGF) mRNA levels. In contrast, the protein levels of VEGF were not changed, which suggests the activation of additional regulatory mechanisms of VEGF protein expression to ensure proper kidney development. In conclusion, Cd exposure decreases HIF-1-binding activity, posing a risk on renal fetal development.