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Carcinogenesis is a multistep process that is frequently associated with p53 inactivation. The class 1 carcinogen cadmium ([Cd.sup.2+]) causes renal cancer and is known to inactivate p53. [G.sub.2]/mitosis (M) arrest contributes to stabilization of p53-deficient mutated cells, but its role and regulation in [Cd.sup.2+]-exposed p53-deficient renal cells are unknown. In p53-inactivated kidney proximal tubule (PT) cells, comet assay experiments showed that [Cd.sup.2+] (50-100 [mu]M) induced DNA damage within 1-6 h. This was associated with peak formation of reactive oxygen species (ROS) at 1-3 h, measured with dihydrorhodamine 123, and [G.sub.2]/M cell cycle arrest at 6 h, which were abolished by the antioxidant [alpha]-tocopherol (100 p,M). [Cd.sup.+]-induced G2/M arrest was enhanced approximately twofold on release from cell synchronization (double thymidine block or nocodazole) and resulted in approximately twofold increase of apoptosis, indicating that G2/M arrest mirrors DNA damage and toxicity. The Chk1/2 kinase inhibitor UCN-01 (0.3 [mu]M), which relieves [G.sub.2]/M transition block, abolished [Cd.sup.2+] or -induced [G.sub.2] arrest and increased apoptosis. This was accompanied by prevention of [Cd.sup.2+]-induced cyclin-dependent kinase cdc2 phosphorylation at tyrosine 15, as shown by immunofluorescence microscopy and immunoblotting. The data indicate that in p53-inactivated PT cells [Cd.sup.2+]-induced ROS formation and DNA damage trigger signaling of checkpoint activating kinases ataxia telangiectasia-mutated kinase (ATM) and ataxia telangiectasia and Rad3-related kinase (ATR) to cause G2/M arrest. This may promote survival of premalignant PT cells and [Cd.sup.2+] carcinogenesis. 7-hydroxystaurosporine; cyclin-dependent kinase; [p21.sup.WAF1/CIPI]; cyclin; DNA strand breaks doi: 10.1152/ajprenal.00273.2009

The positively charged fluorescent dyes ethidium ([Et.sup.+]) and propidium ([Pr.sup.2+]) are widely used as DNA and necrosis markers. [Et.sup.+] is cytotoxic and mutagenic. The polyspecific organic cation transporters OCT1 (SLC22A1), OCT2 (SLC22A2), and OCT3 (SLC22A3) mediate electrogenic facilitated diffusion of small ([less than or equal to] 500 Da) organic cations with broad specificities. In humans, OCT2 mediates basolateral uptake by kidney proximal tubules (PT), whereas in rodents OCT1/2 are involved. In mouse kidney, perfused [Et.sup.+] accumulated predominantly in the S2/S3 segments of the PT, but not [Pr.sup.2+]. In cells stably overexpressing human OCTs (hOCTs), [Et.sup.+] uptake was observed with [K.sub.m] values of 0.8 [+ or -] 0.2 [micro]M (hOCT1), 1.7 [+ or -] 0.5 [micro]M (hOCT2), and 2.0 [+ or -] 0.5 [micro]M (hOCT3), whereas [Pr.sup.2+] was not transported. Accumulation of [Et.sup.+] was inhibited by OCT substrates quinine, 3-methyl-4-phenylpyridinium ([MPP.sup.+]), cimetidine, and tetraethylammonium ([TEA.sup.+]). For hOCT1 and hOCT2, the [IC.sub.50] values for [MPP.sup.+], [TEA.sup.+], and cimetidine were higher than for inhibition of previously tested transported substrates. For hOCT2, the inhibition of [Et.sup.+] uptake by [MPP.sup.+] and cimetidine was shown to be competitive. [Et.sup.+] also inhibited transport of 0.1 [micro]M [[sup.3]H][MPP.sup.+] by all hOCT isoforms with [IC.sub.50] values between 0.4 and 1.3 [micro]M, and the inhibition of hOCT1-mediated uptake of [MPP.sup.+] by [Et.sup.+] was competitive. In [Oct1/2.sup.-/-] mice, [Et.sup.+] uptake in the PT was almost abolished. The data demonstrate that [Et.sup.+] is taken up avidly by the PT, which is mediated by OCT1 and/or OCT2. Considering the high affinity of OCTs for [Et.sup.+] and their strong expression in various organs, strict safety guidelines for [Et.sup.+] handling should be reinforced. SLC22A; cancer; epithelia; homidium; toxicity

Secretion of enzymes and fluid induced by [Ca.sup.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.sup.+] 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.sup.+] permeability (I[C.sub.50] of ~10 [micro]M). 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.sub.rev]) of -20.9 [+ or -] 0.9 mV, and a single-channel [K.sup.+] conductance (gK) of 265.8 [+ or -] 44.0 pS (n = 39). Replacement of cis 500 mM [K.sup.+] by 500 mM [Na.sup.+] shifted [E.sub.rev] to -2.4 [+ or -] 3.6 mV (n = 3), indicating [K.sup.+] selectivity. Single-channel analysis identified several [K.sup.+] channel groups with distinct channel behaviors. [K.sup.+] channels with a gK of 651.8 [+ or -] 88.0 pS, [E.sub.rev] of -22.9 [+ or -] 2.2 mV, and open probability ([P.sub.open]) of 0.43 [+ or -] 0.06 at 0 mV (n = 6) and channels with a gK of 155.0 [+ or -] 11.4 pS, [E.sub.rev] of -18.3 [+ or -] 1.8 mV, and [P.sub.open] of 0.80 [+ or -] 0.03 at 0 mV (n = 3) were inhibited by 100 [micro]M 293B or by the more selective inhibitor HMR-1556 but not by the maxi-[Ca.sup.2+]-activated [K.sup.+] 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 (I[C.sub.50] of ~10 [micro]M). Thus KCNQ1 may account for ZG [K.sup.+] conductance and contribute to pancreatic hormone-stimulated enzyme and fluid secretion. exocytosis; acinar cell; secretory granule; planar bilayer

Chronic exposure to [Cd.sup.2+] causes renal proximal tubular (PT) damage. [Cd.sup.2+] 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, [Cd.sup.2+] is thought to be released in the cytosol to activate cell death pathways. The proton-coupled divalent metal transporter DMT1 also transports [Cd.sup.2+] 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 C1.2, we tested the hypothesis that endosomal/lysosomal DMTI is involved in CdMT-1 nephrotoxicity. One out of 5 shRNAs tested (sh3) significantly reduced expression of DMTI protein detected by immunoblotting and DMT1 mRNA as determined by RT-PCR by 45.1 [+ or -] 9.6 and 36.9 [+ or -] 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 [micro]M CdMT-1 by 35.6 [+ or -] 4.2% (17 = 13). In contrast, neither fluorescently labeled metallothionein-1 (MT-1) uptake nor free [Cd.sup.2+] toxicity was altered by the effective DMT1 shRNA (sh3), indicating that cellular uptake of metal-MT-1 complexes and [Cd.sup.2+]-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. DMT1; endocytosis; heavy metals; metallothionein

A major target of cadmium ([Cd.sup.2+]) toxicity is the kidney proximal tubule (PT) cell. [Cd.sup.2+]-induced apoptosis of PT cells is mediated by sequential activation of calpains at 3-6 h and caspases-9 and -3 after 24-h exposure. Calpains also partly contribute to caspase activation, which emphasizes the importance of calpains for PT apoptosis by [Cd.sup.2+]. Upstream processes underlying [Cd.sup.2+]-induced calpain activation remain unclear. We describe for the first time that 10-50 [micro] M [Cd.sup.2+] causes a significant increase in ceramide formation by ~22% (3 h) and ~72% (24 h), as measured by diacylglycerol kinase assay. Inhibition of ceramide synthase with fumonisin [B.sub.1] (3 [micro] M) prevents ceramide formation at 3 h and abolishes calpain activation at 6 h, which is associated with significant attenuation of apoptosis at 3-6 h with Hoechst 33342 nuclear staining and/or 3(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) death assays. This indicates that [Cd.sup.2+] enhances de novo ceramide synthesis and that calpains are a downstream target of ceramides in apoptosis execution. Moreover, addition of [C.sub.6]-ceramide to PT cells increases cytosolic [Ca.sup.2+] and activates calpains. Apoptosis mediated by [C.sup.6]-ceramide at 24 h is significantly reduced by caspase-3 inhibition, which supports cross talk between calpain- and caspase-dependent apoptotic pathways. We conclude that [Cd.sup.2+]-induced apoptosis of PT cells entails endogenous ceramide elevation and subsequent [Ca.sup.2+] -dependent calpain activation, which propagates kidney damage by [Cd.sup.2+]. nephrotoxicity; cell signaling; cell biology and structure

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 [micro]M 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 [micro]M) 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 l0 [micro]M cadmium for 24 h, and accordingly apoptosis was significantly reduced by the respective inhibitors (z-LEHD-fmk, z-DEVD-fmk; 10 [micro]g/ml) at 24 h, but not at 6 h, without affecting necrosis. At 6 h, 10 [micro]M cadmium increased the activity of the calcium-activated protease calpain, but not at 24 h, and calpain inhibitors (ALLN, PD 150606; 10-30 [micro]M) blocked apoptosis by 10 [micro]M 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. heavy metals; apoptosis-inducing factor; cytochrome c; cysteine proteases

Mitochondria dominate the process of life-and-death decisions of the cell. Continuous generation of ATP is essential for cell sustenance, but, on the other hand, mitochondria play a central role in the orchestra of events that lead to apoptotic cell death. Changes of mitochondrial volume contribute to the modulation of physiological mitochondrial function, and several ion permeability pathways located in the inner mitochondrial membrane have been implicated in the mediation of physiological swelling-contraction reactions, such as the [K.sup.+] cycle. However, the channels and transporters involved in these processes have not yet been identified. Osmotic swelling is also one of the fundamental characteristics exhibited by mitochondria in pathological situations, which activates downstream cascades, culminating in apoptosis. The permeability transition pore has long been postulated to be the primary mediator for water movement in mitochondrial swelling during cell death, but its molecular identity remains obscure. Inevitably, accumulating evidence shows that mitochondrial swelling induced by apoptotic stimuli can also occur independently of permeability transition pore activation. Recently, a novel mechanism for osmotic swelling of mitochondria has been described. Aquaporin-8 and -9 channels have been identified in the inner mitochondrial membrane of various tissues, including the kidney, liver, and brain, where they may mediate water transport associated with physiological volume changes, contribute to the transport of metabolic substrates, and/or participate in osmotic swelling induced by apoptotic stimuli. Hence, the recent discovery that aquaporins are expressed in mitochondria opens up new areas of investigation in health and disease. permeability transition pore; cadmium; apoptosis; water channels doi:10.1152/ajpcell.00641.2005

[Cd.sup.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.sup.2+]-induced cytochrome c release from mitochondria in rat PT cells. Using Hoechst 33342 or MTT assay, 10 [micro]M [Cd.sup.2+] induced ~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.sup.2+] concentrations at 24 h, suggesting that elevated [Cd.sup.2+] may directly induce mitochondrial liberation of proapoptotic factors. Indeed, [Cd.sup.2+] caused swelling of energized isolated kidney cortex mitochondria (E[C.sub.50] ~9 [micro]M) 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.sup.2+] inhibited swelling and cytochrome c release induced by PTP openers (P[O.sup.3-.sub.4] or [H.sub.2][O.sub.2]+[Ca.sup.2+]). The mitochondrial [Ca.sup.2+] uniporter (MCU) played a key role in mitochondrial damage: 1) MCU inhibitors ([La.sup.3+], ruthenium red, Ru360) prevented swelling and cytochrome c release; and 2) ruthenium red attenuated [Cd.sup.2+] inhibition of P[O.sup.3-.sub.4]-induced swelling. Using the [Cd.sup.2+]-sensitive fluorescent indicator FluoZin-1, [Cd.sup.2+] was also taken up by mitoplasts. The aquaporin inhibitor AgN[O.sub.3] abolished [Cd.sup.2+]-induced swelling of mitoplasts. This could be partially mediated by activation of the mitoplast-enriched water channel aquaporin-8. Thus cytosolic [Cd.sup.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. cadmium; aquaporin; apoptosis-inducing factor; cyclosporin A

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.