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Ca2+-induced enzyme secretion in the exocrine pancreas is not completely understood. We have proposed that Ca2+-induced enzyme secretion in the exocrine pancreas involves activation of ion conductances in the membrane of zymogen granules (ZG). Here we have identified a Ca2+-activated anion conductance in rat pancreatic ZG membranes (ZGM). Ca2+ (2.5–50 μM) increased the conductance for I−, NO3−, Br−, or HCO3−, but not for Cl−, as determined by the rate of valinomycin-induced osmotic lysis of ZG suspended in isotonic K+-salts. 4,4′-Diisothiocyanatodihydrostilbene-2,2′-disulfonate (100 μM) or 25 μM dithiothreitol strongly inhibited Ca2+-dependent lysis. The permeability sequence, Ca2+ dependence, and inhibitor sensitivity of ZG anion conductance are reminiscent of a family of epithelial Ca2+-activated anion channels (CLCA). CLCA expression was confirmed by RT-PCR with rat pancreatic mRNA and mouse CLCA1 primers. A PCR product (580 bp) exhibited 81%, 77%, and 57% amino acid similarity to the three mouse isoforms mCLCA-1, -2, and -3 (mgob-5), respectively. Antibodies against bovine tracheal CLCA1 showed CLCA expression in ZGM by immunoblotting, immunoperoxidase light microscopy, and immunogold labeling. These findings suggest that a CLCA-related protein could account for the Ca2+-activated HCO3− conductance of rat pancreatic ZGM and contribute to hormone-stimulated enzyme secretion. [Copyright &y& Elsevier]

Cd2+ induces apoptosis of PT cells. Cd2+ also induces cell cycle arrest, but the underlying mechanisms remain unclear. We hypothesized that Cd2+-+induced stress causes G2 phase arrest by activating the ATM/ATR pathway. This results in activation of two downstream kinases, chk1 and chk2, which turn off the phosphatase cdc25c, thus preventing activation of the cdc2/cyclin B complex that enables cells to enter mitosis. The effects of Cd2+ were investigated in an SV-40 immortalized (p53-inactivated) rat kidney PT cell line. FACS analysis was used for cell cycle analysis, MTT test for cell death quantification and immunoblotting for detection of cdc25c and cdc2. 50µM Cd2+ for 6 h significantly increased PT cells in the G2/M phase by ∼20%. This was increased ∼two-fold in nocodazole synchronized cells. Cd2+ also induced ∼20% cell death, which was further enhanced in synchronized cells. Inhibition of chk1 and chk2 with 300nM UCN-01 significantly increased Cd2+ toxicity by 30%. Immunoblots with antibodies to cdc25c and phospho-cdc2 supported Cd2+ induced activation of the ATM/ATR pathway in the G2 phase. We speculate that the increase in cell death induced by Cd2+ after inhibition of chk1/chk2 with UCN-01 results from damaged PT cells, which progress from the G2 phase to mitosis and then undergo cell death by mitotic catastrophe. [ABSTRACT FROM AUTHOR]

Highlights: [•] PC and MT are transcytosed via the human NGAL receptor in colonic Caco-2 BBE cells. [•] Transcytosis efficiency of PC>MT, toxicity of Cd complexes is CdMT>CdPC. [•] MT, but not PC, co-localizes with lysosomes, which is prevented by nocodazole. [•] Nocodazole increases MT transcytosis efficiency and reduces CdMT toxicity. [•] Differential enterocyte trafficking of Cd complexes may affect toxicity in vivo. [Copyright &y& Elsevier]

Nickel and nickel compounds are carcinogens that target the lungs and kidneys causing cell death or cell survival adaptation. The multidrug resistance P-glycoprotein ABCB1 protects cells against toxic metabolites and xenobiotics and is upregulated in many cancer cell types. Here, we investigated the role of ABCB1 in nickel-induced stress signaling mediated by reactive oxygen species (ROS) and ceramides. In renal proximal tubule cells, nickel chloride (0.1-0.25 mM) increased both ROS formation, detected by 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, and cellular ceramides, which were determined by lipid dot blot and surface immunostaining, culminating in decreased cell viability, increased DNA fragmentation, augmented PARP-1 cleavage, and increased ABCB1 mRNA and protein. Inhibitors of the de novo ceramide synthesis pathway (fumonisin B, l-cycloserine) and an antioxidant (α-tocopherol) attenuated nickel-induced toxicity as well as induction of ABCB1. ABCB1 protects against nickel toxicity as PSC833, an ABCB1 blocker, augmented the decrease in cell viability by nickel. Moreover, nickel toxicity was attenuated in renal MDCK cells stably overexpressing ABCB1. In agreement with previous data that demonstrated extrusion of (glucosyl)ceramides by ABCB1 (Lee et al. in Toxicol Sci 121:343, 2011), PSC833 increased total cellular ceramides by >2-fold after nickel treatment. Further, glucosylceramide synthase (GCS) mRNA is upregulated by nickel at 3 h by ~1.5-fold but declined with prolonged exposures (6-24 h). Inhibition of GCS with CDGJ or knockdown of GCS with siRNA significantly attenuated nickel toxicity. In conclusion, nickel induces a ROS-ceramide pathway to cause apoptotic cell death as well as activate adaptive survival responses, including upregulation of ABCB1, which improves cell survival by extruding proapoptotic (glucosyl)ceramides. [ABSTRACT FROM AUTHOR]

[Display omitted] • Dent disease type 1 (DD1) is a rare inherited disorder induced by Clcn5 mutations. • We generated a clinically relevant DD1 transgenic mouse model. • These mice show altered proximal tubule functions and abnormal accumulation/loss of metabolites. • DD1 progression is accompanied by increased expression/secretion of the kidney injury biomarker NGAL and its receptor 24P3R. • This new DD1 mouse model is an interesting tool for the development of therapeutic approaches. Dent disease (DD) is a hereditary renal disorder characterized by low molecular weight (LMW) proteinuria and progressive renal failure. Inactivating mutations of the CLCN5 gene encoding the 2Cl-/H+exchanger ClC-5 have been identified in patients with DD type 1. ClC-5 is essentially expressed in proximal tubules (PT) where it is thought to play a role in maintaining an efficient endocytosis of LMW proteins. However, the exact pathological roles of ClC-5 in progressive dysfunctions observed in DD type 1 are still unclear. To address this issue, we designed a mouse model carrying the most representative type of ClC-5 missense mutations found in DD patients. These mice showed a characteristic DD type 1 phenotype accompanied by altered endo-lysosomal system and autophagy functions. With ageing, KI mice showed increased renal fibrosis, apoptosis and major changes in cell metabolic functions as already suggested in previous DD models. Furthermore, we made the interesting new discovery that the Lipocalin-2-24p3R pathway might be involved in the progression of the disease. These results suggest a crosstalk between the proximal and distal nephron in the pathogenesis mechanisms involved in DD with an initial PT impairment followed by the Lipocalin-2 internalisation and 24p3R overexpression in more distal segments of the nephron. This first animal model of DD carrying a pathogenic mutation of Clcn5 and our findings pave the way aimed at exploring therapeutic strategies to limit the consequences of ClC-5 disruption in patients with DD type 1 developing chronic kidney disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) induces oxidative stress that ultimately defines cell fate and pathology. Mitochondria are the main energy-producing organelles in mammalian cells, but they also have a central role in formation of reactive oxygen species, cell injury, and death signaling. As the kidney is the major target in Cd toxicity, the roles of oxidative signature and mitochondrial function and biogenesis in Cd-related stress outcomes were investigated in vitro in cultured rat kidney proximal tubule cells (PTCs) (WKPT-0293 Cl.2) for acute Cd toxicity (1-30 µM, 24 h) and in vivo in Fischer 344 rats for sub-chronic Cd toxicity (1 mg/kg CdCl subcutaneously, 13 days). Whereas 30 µM Cd caused ~50 % decrease in cell viability, apoptosis peaked at 10 µM Cd in PTCs. A steep, dose-dependent decline in reduced glutathione (GSH) content occurred after acute exposure and an increase of the oxidized glutathione (GSSG)/GSH ratio. Quantitative PCR analyses evidenced increased antioxidative enzymes (Sod1, Gclc, Gclm), proapoptotic Bax, metallothioneins 1A/2A, and decreased antiapoptotic proteins (Bcl-xL, Bcl-w). The positive regulator of mitochondrial biogenesis Pparγ and mitochondrial DNA was increased, and cellular ATP was unaffected with Cd (1-10 µM). In vivo, active caspase-3, and hence apoptosis, was detected by FLIVO injection in the kidney cortex of Cd-treated rats together with an increase in Bax mRNA. However, antiapoptotic genes (Bcl-2, Bcl-xL, Bcl-w) were also upregulated. Both GSSG and GSH increased with chronic Cd exposure with no change in GSSG/GSH ratio and augmented expression of antioxidative enzymes (Gpx4, Prdx2). Mitochondrial DNA, mitofusin 2, and Pparα were increased indicating enhanced mitochondrial biogenesis and fusion. Hence, these results demonstrate a clear involvement of higher mitochondria copy numbers or mass and mitochondrial function in acute defense against oxidative stress induced by Cd in renal PTCs as well as in adaptive processes associated with chronic renal Cd toxicity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cadmium (Cd2+) damages the kidney proximal tubule (PT) by ceramide-dependent apoptosis and is also a class I carcinogen. Multidrug resistance P-glycoprotein (MDR1, ABCB1) confers resistance to Cd2+ apoptosis, and it has been hypothesized that ABCB1 can directly transport Cd2+ as a mode of cellular protection. Our aim was to investigate the role of ABCB1 in Cd2+ transport and ceramide apoptosis. In rat PT or Madin-Darby canine kidney (MDCK) cells overexpressing ABCB1, ABCB1-dependent efflux of rhodamine 123+ (Rh123+) or 109Cd2+ were determined, and cell death was assayed with MTT, H-33342 nuclear staining, and monolayer integrity by impedance sensing (Electric cell-substrate impedance sensing [ECIS]). ABCB1 inhibitors (PSC833, UIC-2 antibody) did not affect 109Cd2+ efflux in PT cells though Rh123+ transport was blocked. Furthermore, increased ABCB1 expression did not augment 109Cd2+ efflux but attenuated apoptosis by 10–50μM Cd2+ or 5–25μM C6-ceramide, which was abolished by PSC833 (1μM). ECIS measurements of ABCB1-MDCK monolayers exhibited similar effects. Moreover, in ABCB1-MDCK cells, Cd2+-induced ceramide formation, determined by a diacylglycerol kinase assay, was abolished and increased extrusion of nitro-2-1,3-benzoxadiazol-4-yl (NBD)-C6-ceramide, and NBD-C6-glucosylceramide was observed compared with MDCK cells. Whereas pharmacological block of sphingomyelin synthase (0.1mM D609) or sphingosine kinase (1μM dimethylsphingosine), which increase the levels of ceramide and its metabolites, augmented Cd2+-induced apoptosis, Cd2+ apoptosis was significantly decreased not only by prevention of de novo ceramide synthesis (0.1μM fumonisin B1) but also by inhibition of glucosylceramide synthase (2μM C9DGJ). We therefore conclude that Cd2+ efflux is not the mechanism behind ABCB1-mediated protection from Cd2+ apoptosis. Rather, the sphingolipid glucosylceramide may be the proapoptotic substrate extruded by ABCB1. [ABSTRACT FROM AUTHOR]

Abstract: The transition metal cadmium (Cd) is an environmental pollutant which damages the kidneys. Chronic Cd exposure may induce renal fibrosis and/or cancer, but the signaling pathways involved are not understood. The Wnt pathway is a key signaling cascade responsible for renal development, fibrosis and cancer. Hence the effect of chronic in vivo Cd exposure (100mg/l drinking water for 12 weeks) on transcriptional activation of the Wnt pathway and markers of epithelial-to-mesenchymal transition (EMT) was investigated in mouse kidneys. Cd exposure increased kidney Cd content from 0.023±0.001μg/g to 61±7μg/g wet weight (means±S.D. of 6–7 animals). This was accompanied by increased expression of Wnt ligands (Wnt3a/6/7a/7b/9a/9b/10a/11), as determined by RT-PCR. The Wnt receptors Frizzled (Fz1/2/4,5,7–10) were also upregulated, as were the co-receptors low-density lipoprotein receptor-related proteins 5/6. Immunoblots with Wnt10a and Fz7 antibodies also revealed increased protein expression induced by Cd exposure. In contrast, Wnt antagonists were largely unaffected. Upregulation of Wnt signaling components induced by Cd was corroborated by increased expression of Wnt target genes, i.e. cell proliferation and survival genes c-Myc, cyclin D1 and the multidrug transporter P-glycoprotein Abcb1b, which promote malignancy. Lastly the EMT markers Twist, fibronectin and collagen I, but not α-smooth muscle actin, were also upregulated, suggesting that Cd-induced changes of renal epithelial tissue characteristics towards fibrosis and cancer may be mediated by Wnt signaling. [ABSTRACT FROM AUTHOR]

Abstract: 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 μM 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. [Copyright &y& Elsevier]

The expression of K+–Cl− cotransporters (KCC) was examined in pancreatic islet cells. mRNA for KCC1, KCC3a, KCC3b and KCC4 were identified by RT-PCR in islets isolated from rat pancreas. In immunocytochemical studies, an antibody specific for KCC1 and KCC4 revealed the expression of KCC protein in α-cells, but not pancreatic β-cells nor δ-cells. A second antibody which does not discriminate among KCC isoforms identified KCC expression in both α-cell and β-cells. Exposure of isolated α-cells to hypotonic solutions caused cell swelling was followed by a regulatory volume decrease (RVD). The RVD was blocked by 10 μM [dihydroindenyl-oxy] alkanoic acid (DIOA; a KCC inhibitor). DIOA was without effect on the RVD in β-cells. NEM (0.2 mM), a KCC activator, caused a significant decrease of α-cell volume, which was completely inhibited by DIOA. By contrast, NEM had no effects on β-cell volume. In conclusion, KCCs are expressed in pancreatic α-cells and β-cells. However, they make a significant contribution to volume homeostasis only in α-cells. [Copyright &y& Elsevier]

Expression of multidrug resistance P-glycoprotein in kidney allografts from cyclosporine A-treated patients. Background. The multidrug resistance (MDR) gene product P-glycoprotein (P-gp) is a transmembrane efflux pump for hydrophobic, potentially toxic compounds, including the immunosuppressant cyclosporine A (CsA). We have previously shown that CsA increases P-gp expression in proximal tubule and endothelial cells in vitro. The aim of the present study was to investigate the in vivo relevance of these observations in renal allograft biopsies from CsA-treated patients. Methods. P-gp expression was determined by immunohistochemistry of paraffin sections using two different monoclonal antibodies (UIC2 and MRK16). Biopsies were taken from CsA-treated renal transplant patients with different histopathological diagnoses (N = 79) and were compared with biopsies from normal human kidneys (N = 13) or with allograft biopsies from patients under a CsA-free immunosuppression (N = 15). Moreover, biopsies from 10 donor kidneys before implantation and during rejection episodes (“zero biopsies”) were investigated. Results. P-gp expression in biopsies with acute tubular necrosis (ATN; N = 10) after CsA treatment was significantly higher in arterial endothelia, proximal tubules, and epithelial cells of Bowman's capsule (BC), whereas P-gp was sparsely induced in CsA nephrotoxicity (N = 19) compared with controls. Acute cellular (N = 30) and vascular rejection (N = 10) or chronic allograft nephropathy (N = 10) after CsA was associated with strong P-gp expression in infiltrating leukocytes and increased P-gp expression in arterial endothelia, proximal tubules, and BC. In contrast, biopsies of patients treated with a CsA-free immunosuppression regimen did not show increases in P-gp expression compared with controls. Zero biopsies showed a weak, homogeneous, nonpolarized expression of P-gp in tubules and an increased expression of P-gp after CsA therapy in the brush border, arterial endothelia, and BC. Conclusions. CsA treatment was associated with increased P-gp expression in parenchymal cells of kidney transplants with ATN, acute or chronic transplant rejection, but P-gp was not increased in patients with CsA nephrotoxicity. This indicates that CsA induces its own detoxification by P-gp and that inadequate up-regulation of P-gp in renal parenchymal cells contributes to CsA nephrotoxicity. Increased expression of P-gp in infiltrating leukocytes correlated with the severity of allograft rejection, suggesting that P-gp may decrease the immunosuppressive efficacy of CsA. Thus, individual differences in the P-gp induction response of CsA-exposed renal parenchymal cells and/or infiltrating leukocytes may predispose to either CsA nephrotoxicity or rejection, respectively. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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, β2-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, β2-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+-β2-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. [ABSTRACT FROM AUTHOR]

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., (© 2024. The Author(s).)

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 (Cd 2+ )-induced oxidative stress by targeting ROS/RNS generation or antioxidant defence mechanisms, such as superoxide dismutase (SOD) or H 2 O 2 -metabolizing catalase (CAT). Though ROS/RNS are well-evidenced, the role of distinct ROS profiles in Cd 2+ concentration-dependent toxicity is not clear. In renal cells, Cd 2+ (10-50 µM) oxidized dihydrorhodamine 123, reaching a maximum at 2-3 h. Increases (up to fourfold) in lipid peroxidation by TBARS assay and H 2 O 2 by Amplex Red were evident within 30 min. ROS and loss in cell viability by MTT assay with 50 µM Cd 2+ 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 Cd 2+ , yet augmented by up to 1.5- and 1.2-fold, respectively, by 10 µM Cd 2+ . Moreover, 10 µM, but not 25-50 µM Cd 2+ , caused 1.7-fold increase in superoxide anion (O 2 •- ), detected by dihydroethidium, paralled by loss in cell viability, that was abolished by Tempol, MnTBAP, α-tocopherol and SOD1 or CAT overexpression. H 2 O 2 -generating NADPH oxidase 4 (NOX4) was attenuated by ~50% with 10 µM Cd 2+ at 3 h compared to upregulation by 50 µM Cd 2+ (~1.4-fold, 30 min), which was sustained for 24 h. In summary, O 2 •- predominates with low-moderate Cd 2+ , driving an adaptive response, whereas oxidative stress by elevated H 2 O 2 at high Cd 2+ triggers cell death signaling pathways.Highlights Different levels of reactive oxygen species are generated, depending on cadmium concentration. Superoxide anion predominates and H 2 O 2 is suppressed with low cadmium representing oxidative eustress. High cadmium fosters H 2 O 2 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., (© 2024. The Author(s).)

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.

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.

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., (© 2022. The Author(s).)

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 ABCB1 high (A498, Caki-1) compared with chemosensitive ABCB1 low (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 ABCB1 high cells was partially reversed by inhibitors of de novo ceramide synthesis (l-cycloserine) and CerS (fumonisin B 1 ) 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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2020 Thévenod, Lee and Garrick.)

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.

Cadmium (Cd 2+ ) is a toxic and non-essential divalent metal ion in eukaryotic cells. Cells can only be targeted by Cd 2+ if it hijacks physiological high-affinity entry pathways, which transport essential divalent metal ions in a process termed "ionic and molecular mimicry". Hence, "free" Cd 2+ ions and Cd 2+ complexed with small organic molecules are transported across cellular membranes via ion channels, carriers and ATP hydrolyzing pumps, whereas receptor-mediated endocytosis (RME) internalizes Cd 2+ -protein complexes. Only Cd 2+ transport pathways validated by stringent methodology, namely electrophysiology, 109 Cd 2+ tracer studies, inductively coupled plasma mass spectrometry, atomic absorption spectroscopy, Cd 2+ -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, Cd 2+ permeates voltage-dependent Ca 2+ channels ("T-type" Ca V 3.1, 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 Cd 2+ -protein complexes are endocytosed by the lipocalin-2/NGAL receptor SLC22A17. Cd 2+ transport via the mitochondrial Ca 2+ uniporter, ATPases ABCC1/2/5 and transferrin receptor 1 is likely but requires further evidence. Cd 2+ flux occurs through the influx carrier OCT2/SLC22A2, efflux MATE proteins SLC47A1/A2, the efflux ATPase ABCB1, and RME of Cd 2+ -metallothionein by the receptor megalin (low density lipoprotein receptor-related protein 2, LRP2):cubilin albeit at high concentrations thus questioning their relevance in Cd 2+ loading. Which Cd 2+ -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 Cd 2+ toxicity and to overcome dissemination of unsubstantiated conjectures.

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/Cβ, 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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (© 2019 Smith et al.)

Hemoglobinuria is associated with kidney injury in various hemolytic pathologies. Currently, there is no treatment available and its pathophysiology is not completely understood. Here we studied the potential detrimental effects of hemoglobin (Hb) exposure to the distal nephron (DN). Involvement of the DN in Hb kidney injury was suggested by the induction of renal hepcidin synthesis (p < 0.001) in mice repeatedly injected with intravenous Hb. Moreover, the hepcidin induction was associated with a decline in urinary kidney injury markers 24p3/NGAL and KIM1, suggesting a role for hepcidin in protection against Hb kidney injury. We demonstrated that uptake of Hb in the mouse cortical collecting duct cells (mCCD cl1 ) is mediated by multi-protein ligand receptor 24p3R, as indicated by a significant 90% reduction in Hb uptake (p < 0.001) after 24p3R silencing. Moreover, incubation of mCCD cl1 cells with Hb or hemin for 4 or 24 h resulted in hepcidin synthesis and increased mRNA expression of markers for oxidative, inflammatory and ER stress, but no cell death as indicated by apoptosis staining. A protective role for cellular hepcidin against Hb-induced injury was demonstrated by aggravation of oxidative, inflammatory and ER stress after 4 h Hb or hemin incubation in hepcidin silenced mCCD cl1 cells. Hepcidin silencing potentiated hemin-mediated cell death that could be diminished by co-incubation of Nec-1, suggesting that endogenous hepcidin prevents necroptosis. Combined, these results demonstrate that renal hepcidin synthesis protects the DN against hemin and hemoglobin-mediated injury.

Iron (Fe) is an essential metal, vital for biological functions, including electron transport, DNA synthesis, detoxification, and erythropoiesis that all contribute to metabolism, cell growth, and proliferation. Interactions between Fe and O2 can result in the generation of reactive oxygen species (ROS), which is based on the ability of Fe to redox cycle. Excess Fe may cause oxidative damage with ensuing cell death, but DNA damage may also lead to permanent mutations. Hence Fe is carcinogenic and may initiate tumor formation and growth, and also nurture the tumor microenvironment and metastasis. However, Fe can also contribute to cancer defense. Fe may induce toxic ROS and/or initiate specific forms of cell death, including ferroptosis that will benefit cancer treatment. Furthermore, Fe-binding and Fe-regulatory proteins, such as hepcidin, lipocalin-2/NGAL, heme oxygenase-1, ferritin, and iron-sulfur clusters can display antitumor properties under specific conditions and in particular cancer types. In addition, the milk protein lactoferrin may synergize with other established anticancer agents in the prevention and therapy of cancer. Consequently, drugs that target Fe metabolism in tumors are promising candidates for the prevention and therapy of cancer, but consideration of context specificity (e.g., tumor type; systemic versus tumor microenvironment Fe homeostasis) is mandatory.

Cellular stress elicited by the toxic metal Cd(2+) does not coerce the cell into committing to die from the onset. Rather, detoxification and adaptive processes are triggered concurrently, allowing survival until normal function is restored. With high Cd(2+), death pathways predominate. However, if sublethal stress levels affect cells for prolonged periods, as in chronic low Cd(2+) exposure, adaptive and survival mechanisms may deregulate, such that tumorigenesis ensues. Hence, death and malignancy are the two ends of a continuum of cellular responses to Cd(2+), determined by magnitude and duration of Cd(2+) stress. Signaling cascades are the key factors affecting cellular reactions to Cd(2+). This review critically surveys recent literature to outline major features of death and survival signaling pathways as well as their activation, interactions and cross talk in cells exposed to Cd(2+). Under physiological conditions, receptor activation generates 2nd messengers, which are short-lived and act specifically on effectors through their spatial and temporal dynamics to transiently alter effector activity. Cd(2+) recruits physiological 2nd messenger systems, in particular Ca(2+) and reactive oxygen species (ROS), which control key Ca(2+)- and redox-sensitive molecular switches dictating cell function and fate. Severe ROS/Ca(2+) signals activate cell death effectors (ceramides, ASK1-JNK/p38, calpains, caspases) and/or cause irreversible damage to vital organelles, such as mitochondria and endoplasmic reticulum (ER), whereas low localized ROS/Ca(2+) levels act as 2nd messengers promoting cellular adaptation and survival through signal transduction (ERK1/2, PI3K/Akt-PKB) and transcriptional regulators (Ref1-Nrf2, NF-κB, Wnt, AP-1, bestrophin-3). Other cellular proteins and processes targeted by ROS/Ca(2+) (metallothioneins, Bcl-2 proteins, ubiquitin-proteasome system, ER stress-associated unfolded protein response, autophagy, cell cycle) can evoke death or survival. Hence, temporary or permanent disruptions of ROS/Ca(2+) induced by Cd(2+) play a crucial role in eliciting, modulating and linking downstream cell death and adaptive and survival signaling cascades.