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

The positively charged fluorescent dyes ethidium (Et+) and propidium (Pr2+) are widely used as DNA and necrosis markers. Et+ is cytotoxic and mutagenic. The polyspecific organic cation transporters OCT1 (SLC22AI), OCT2 (SLC22A2), and OCT3 (SLC22A3) mediate electrogenic facilitated diffusion of small (⩽500 Da) organic cations with broad specificities. In humans, OCT2 mediates basolateral uptake by kidney proximal tubules (PT), whereas in rodents OCTI/2 are involved. In mouse kidney, perfused Et+ accumulated predominantly in the S2/S3 segments of the PT, but not Pr2+. In cells stably overexpressing human OCTs (hOCTs), Et+ uptake was observed with Km values of 0.8 ± 0.2 μM (hOCT1), 1.7 ± 0.5 μM (hOCT2), and 2.0 ± 0.5 μM (hOCT3), whereas Pr2+ was not transported. Accumulation of Et+ was inhibited by OCT substrates quinine, 3-methyl-4-phenylpyridinium (MPP+), cimetidine, and tetraethylammonium (TEA+). For hOCT1 and hOCT2, the IC50 values for MPP+, TEA+, and cimetidine were higher than for inhibition of previously tested transported substrates. For hOCT2, the inhibition of Et+ uptake by MPP+ and cimetidine was shown to be competitive. Et also inhibited transport of 0.1 μM [3H]MPP+ by all hOCT isoforms with IC50 values between 0.4 and 1.3 μM, and the inhibition of hOCT1-mediated uptake of MPP+ by Et+ was competitive. In Oct1/2-/- mice, Et+ uptake in the PT was almost abolished. The data demonstrate that Et+ is taken up avidly by the PT, which is mediated by OCT1 and/or OCT2. Considering the high affinity of OCTs for Et and their strong expression in various organs, strict safety guidelines for Et+ handling should be reinforced. [ABSTRACT FROM AUTHOR]

Oncogenic multidrug resistance (MDR) is a multifactorial phenotype intimately linked to deregulated expression of detoxification transporters. Drug efflux transporters, particularly the MDR P-glycoprotein ABCB1, represent a central mechanism by which not only chemotherapeutic drugs are extruded or sequestered to prevent drug delivery to their intracellular targets, but also for inhibiting apoptotic cell death cues, such as removal of proapoptotic signals. Several cell populations exhibiting the MDR phenotype co-exist within a tumor, such as cells forming the bulk tumor cell mass, cancer stem cells, and cancer persister cells. The key to regulation of ABCB1 expression is the cellular transcriptional machinery. Developmental signaling pathways (e.g, Hedgehog, Notch, Wnt/β-catenin, TGFβ, PITX2) are pivotal in governing cell proliferation, survival, differentiation and guiding cell migration during embryogenesis, and their reactivation during carcinogenesis, which is of particular significance for tumor initiation, progression, and metastasis, also leads to the upregulation of ABCB1. These pathways also drive and maintain cancer cell stemness, for which ABCB1 is used as a marker. In this review, the contribution of canonical and non-canonical developmental signaling pathways in transcriptional regulation of ABCB1 to confer MDR in cancer is delineated., Competing Interests: The authors declare no conflicts of interest., (© The Author(s) 2021.)

Secretion of enzymes and fluid induced by Ca(2+) in pancreatic acini is not completely understood and may involve activation of ion conductive pathways in zymogen granule (ZG) membranes. We hypothesized that a chromanol 293B-sensitive K(+) conductance carried by a KCNQ1 protein is expressed in ZG membranes (ZGM). In suspensions of rat pancreatic ZG, ion flux was determined by ionophore-induced osmotic lysis of ZG suspended in isotonic salts. The KCNQ1 blocker 293B selectively blocked K(+) permeability (IC(50) of approximately 10 microM). After incorporation of ZGM into planar bilayer membranes, cation channels were detected in 645/150 mM potassium gluconate cis/trans solutions. Channels had linear current-voltage relationships, a reversal potential (E(rev)) of -20.9 +/- 0.9 mV, and a single-channel K(+) conductance (g(K)) of 265.8 +/- 44.0 pS (n = 39). Replacement of cis 500 mM K(+) by 500 mM Na(+) shifted E(rev) to -2.4 +/- 3.6 mV (n = 3), indicating K(+) selectivity. Single-channel analysis identified several K(+) channel groups with distinct channel behaviors. K(+) channels with a g(K) of 651.8 +/- 88.0 pS, E(rev) of -22.9 +/- 2.2 mV, and open probability (P(open)) of 0.43 +/- 0.06 at 0 mV (n = 6) and channels with a g(K) of 155.0 +/- 11.4 pS, E(rev) of -18.3 +/- 1.8 mV, and P(open) of 0.80 +/- 0.03 at 0 mV (n = 3) were inhibited by 100 microM 293B or by the more selective inhibitor HMR-1556 but not by the maxi-Ca(2+)-activated K(+) channel (BK channel) inhibitor charybdotoxin (5 nM). KCNQ1 protein was demonstrated by immunoperoxidase labeling of pancreatic tissue, immunogold labeling of ZG, and immunoblotting of ZGM. 293B also inhibited cholecystokinin-induced amylase secretion of permeabilized acini (IC(50) of approximately 10 microM). Thus KCNQ1 may account for ZG K(+) conductance and contribute to pancreatic hormone-stimulated enzyme and fluid secretion.

Chronic exposure to Cd2+ causes renal proximal tubular (PT) damage. Cd2+ reaches the PT mainly as cadmium-metallothionein 1 (CdMT-1) complexes that are filtered at the glomerulus and then internalized in part via endocytosis mediated by megalin and cubulin. Subsequently, Cd2+ is thought to be released in the cytosol to activate cell death pathways. The proton-coupled divalent metal transporter DMT1 also transports Cd2+ and is expressed exclusively in endosomes/lysosomes in rat PT cells. Using vector-based RNA interference with short-hairpin small-interfering RNAs (shRNAs) to downregulate DMT1 in the rat renal PT cell line WKPT-0293 Cl.2, we tested the hypothesis that endosomal/lysosomal DMT1 is involved in CdMT-1 nephrotoxicity. One out of 5 shRNAs tested (sh3) significantly reduced expression of DMT1 protein detected by immunoblotting and DMT1 mRNA as determined by RT-PCR by 45.1 +/- 9.6 and 36.9 +/- 14.4% (n = 5-6), respectively. Similarly, sh3 reduced perinuclear DMT1 immunostaining in transfected cells. Consistent with the assumed role of DMT1 in CdMT-1 cytotoxicity, sh3, but not the empty vector or sh5, significantly attenuated cell death induced by a 24-h exposure to 14.3 microM CdMT-1 by 35.6 +/- 4.2% (n = 13). In contrast, neither fluorescently labeled metallothionein-1 (MT-1) uptake nor free Cd2+ toxicity was altered by the effective DMT1 shRNA (sh3), indicating that cellular uptake of metal-MT-1 complexes and Cd2+-induced cell death signaling are not affected by DMT1 knockdown. Thus we conclude that endosomal/lysosomal DMT1 plays a role in renal PT CdMT-1 toxicity.

The nephrotoxic metal cadmium at micromolar concentrations induces apoptosis of rat kidney proximal tubule (PT) cells within 3-6 h of exposure. The underlying cell death pathways remain poorly defined. Using Hoechst 33342/ethidium bromide nuclear staining and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell death assays, 10-50 microM cadmium induced apoptosis of immortalized rat kidney cells derived from the S1-segment of PT at 6 and 24 h, but necrosis at 24 h only. Cadmium (10-50 microM) also caused mitochondrial cytochrome c (cyt. c)- and apoptosis-inducing factor release at 24 h, but not at 6 h, as measured by immunofluorescence imaging and immunoblotting. Caspases-9 and -3 were activated only by 10 microM cadmium for 24 h, and accordingly apoptosis was significantly reduced by the respective inhibitors (z-LEHD-fmk, z-DEVD-fmk; 10 microg/ml) at 24 h, but not at 6 h, without affecting necrosis. At 6 h, 10 microM cadmium increased the activity of the calcium-activated protease calpain, but not at 24 h, and calpain inhibitors (ALLN, PD 150606; 10-30 microM) blocked apoptosis by 10 microM cadmium at 3-6 h. However, PD-150606 also attenuated caspase-3 activity and apoptosis at 24 h, suggesting calpain-dependent caspase activation. Thus cadmium-induced apoptosis of PT cells involves a complex and sensitive interplay of signaling cascades involving mitochondrial proapoptotic factors, calpains and caspases, whose activation is also determined by cadmium concentration and the duration of cadmium exposure.

An orientational grating with reduced scattering noise is formed in fullerene-doped and undoped homeotropic nematic liquid crystal cells by simultaneous application of both spatially modulated light and ac electric field. The first-order diffraction efficiency obtained in the fullerene-doped cell reaches ~30 %, approaching the maximum value predicted by theory. Effective nonlinear index coefficient is ~0.9 cm2/W and ~1000 times larger than previous observations in C(60)-doped nematic liquid crystals using a dc field. Grating formation time can be as short as 1 s, which is ~100 times shorter than those found by us using a dc field and previous studies.

The nephrotoxic metal Cd(2+) causes mitochondrial damage and apoptosis of kidney proximal tubule cells. A K(+) cycle involving a K(+) uniporter and a K(+)/H(+) exchanger in the inner mitochondrial membrane (IMM) is thought to contribute to the maintenance of the structural and functional integrity of mitochondria. In the present study, we have investigated the effect of Cd(2+) on K(+) cycling in rat kidney cortex mitochondria. Cd(2+) (EC(50) approximately 19 microM) induced swelling of nonenergized mitochondria suspended in isotonic salt solutions according to the sequence KCl = NaCl > LiCl >> choline chloride. Cd(2+)-induced swelling of energized mitochondria had a similar EC(50) value and showed the same cation dependence but was followed by a spontaneous contraction. Mitochondrial Ca(2+) uniporter (MCU) blockers, but not permeability transition pore inhibitors, abolished swelling, suggesting the need for Cd(2+) influx through the MCU for swelling to occur. Complete loss of mitochondrial membrane potential (DeltaPsi(m)) induced by K(+) influx did not prevent contraction, but addition of the K(+)/H(+) exchanger blocker, quinine (1 mM), or the electroneutral protonophore nigericin (0.4 microM), abolished contraction, suggesting the mitochondrial pH gradient (DeltapH(m)) driving contraction. Accordingly, a quinine-sensitive partial dissipation of DeltapH(m) was coincident with the swelling-contraction phase. The data indicate that Cd(2+) enters the matrix through the MCU to activate a K(+) cycle. Initial K(+) load via a Cd(2+)-activated K(+) uniporter in the IMM causes osmotic swelling and breakdown of DeltaPsi(m) and triggers quinine-sensitive K(+)/H(+) exchange and contraction. Thus Cd(2+)-induced activation of a K(+) cycle contributes to the dissipation of the mitochondrial protonmotive force.

Cd(2+) induces apoptosis of kidney proximal tubule (PT) cells. Mitochondria play a pivotal role in toxic compound-induced apoptosis by releasing cytochrome c. Our objective was to investigate the mechanisms underlying Cd(2+)-induced cytochrome c release from mitochondria in rat PT cells. Using Hoechst 33342 or MTT assay, 10 muM Cd(2+) induced approximately 5-10% apoptosis in PT cells at 6 and 24 h, which was associated with cytochrome c and apoptosis-inducing factor release at 24 h only. This correlated with previously described maximal intracellular Cd(2+) concentrations at 24 h, suggesting that elevated Cd(2+) may directly induce mitochondrial liberation of proapoptotic factors. Indeed, Cd(2+) caused swelling of energized isolated kidney cortex mitochondria (EC(50) approximately 9 muM) and cytochrome c release, which were independent of permeability transition pore (PTP) opening since PTP inhibitors cyclosporin A or bongkrekic acid had no effect. On the contrary, Cd(2+) inhibited swelling and cytochrome c release induced by PTP openers (PO(4)(3-) or H(2)O(2)+Ca(2+)). The mitochondrial Ca(2+) uniporter (MCU) played a key role in mitochondrial damage: 1) MCU inhibitors (La(3+), ruthenium red, Ru360) prevented swelling and cytochrome c release; and 2) ruthenium red attenuated Cd(2+) inhibition of PO(4)(3-)-induced swelling. Using the Cd(2+)-sensitive fluorescent indicator FluoZin-1, Cd(2+) was also taken up by mitoplasts. The aquaporin inhibitor AgNO(3) abolished Cd(2+)-induced swelling of mitoplasts. This could be partially mediated by activation of the mitoplast-enriched water channel aquaporin-8. Thus cytosolic Cd(2+) concentrations exceeding a certain threshold may directly cause mitochondrial damage and apoptotic development by interacting with MCU and water channels in the inner mitochondrial membrane.