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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.

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. [ABSTRACT FROM AUTHOR]

The cancer multidrug resistance (MDR) phenotype encompasses a myriad of molecular, genetic and cellular alterations resulting from progressive oncogenic transformation and selection. Drug efflux transporters, in particular the MDR P-glycoprotein ABCB1, play an important role in MDR but cannot confer the complete phenotype alone indicating parallel alterations are prerequisite. Sphingolipids are essential constituents of lipid raft domains and directly participate in functionalization of transmembrane proteins, including providing an optimal lipid microenvironment for multidrug transporters, and are also perturbed in cancer. Here we postulate that increased sphingomyelin content, developing early in some cancers, recruits and functionalizes plasma membrane ABCB1 conferring a state of partial MDR, which is completed by glycosphingolipid disturbance and the appearance of intracellular vesicular ABCB1. In this review, the independent and interdependent roles of sphingolipid alterations and ABCB1 upregulation during the transformation process and resultant conferment of partial and complete MDR phenotypes are discussed. [ABSTRACT FROM AUTHOR]

R4734 --> 567.15 --> The tumour suppressor von Hippel‐Lindau (VHL) is involved in ubiquitination and subsequent proteasomal degradation of hypoxia‐inducible factors (HIFs). Under hypoxic conditions, HIFα‐subunits are stabilized, leading to dimerization with HIFβ and transactivation of genes for metabolism, survival and angiogenesis. Human renal cancer cells A498 are VHL‐deficient and harbor stabilized HIF‐2, whereas Caki‐1 cells are VHL‐intact and HIF‐1‐dominant. Previous studies demonstrated increased paired‐like homeodomain transcription factor 2 (PITX2) and its target gene, the ABCB1 drug transporter, in A498 cells. The relationship between VHL, HIF, PITX2 and ABCB1 remains unclear. Transient transfection of A498 cells with HIF‐1α or HIF‐2α siRNA (25 nM, 72 h, >85% protein knockdown) attenuated PITX2 protein by up to 75% by immunoblotting and PITX2 promoter activity by ~40% in a luciferase reporter gene assay. Immunoprecipitation studies did not propose direct HIF‐PITX2 protein‐protein interaction. HIF‐regulated PITX2 expression was proportional to ABCB1 expression, which was decreased at both mRNA (~20%) and protein (~70%) levels after HIF‐1α or HIF‐2α knockdown. Furthermore, HIF/PITX2 target gene CCND1 was reduced by >60%, but PITX2 targets LCN2 and WNT4 were increased >3‐fold following transient HIF downregulation. HIF‐2α siRNA was generally more effective than HIF‐1α siRNA in altering PITX2 and its target genes. Conversely, in Caki‐1 cells, HIF siRNA augmented PITX2 and ABCB1 mRNA and protein by ~1.5‐fold and ~2‐fold, respectively, with no significant changes in CCND1, LCN2 and WNT4. Polysome profiling show HIF‐2α siRNA negatively impacts active translation of PITX2 and ABCB1 mRNA. Finally, HIF2 knockdown sensitizes A498 cells to cell death by the chemotherapeutic drug and ABCB1 substrate, doxorubicin (1 µM, 24 h), as determined by trypan blue staining. In summary, these findings suggest PITX2 and ABCB1 are positively regulated by tumorigenic HIF2 in drug‐resistant A498 cells whereas they are suppressed by HIF‐1 in drug‐sensitive Caki‐1 cells. These data may provide better understanding of membrane transporters and possible upstream causes of multidrug resistance in different renal cancers. [ABSTRACT FROM AUTHOR]

Abstract: Upon endoplasmic reticulum (ER) stress induction, cells endeavor to survive by engaging the adaptive stress response known as the unfolded protein response or by removing aggregated proteins via autophagy. Chronic ER stress culminates in apoptotic cell death, which involves induction of pro-apoptotic CHOP. Here, we show that bestrophin-3 (Best-3), a protein previously associated with Ca2+-activated Cl− channel activity, is upregulated by the ER stressors, thapsigargin (TG), tunicamycin (TUN) and the toxic metal Cd2+. In cultured rat kidney proximal tubule cells, ER stress, CHOP and cell death were induced after 6h by Cd2+ (25μM), TG (3μM) and TUN (6μM), were associated with increased cytosolic Ca2+ and downstream formation of reactive oxygen species and attenuated by the Ca2+ chelator BAPTA-AM (10μM), the antioxidant α-tocopherol (100μM), or overexpression of catalase (CAT). Immunofluorescence staining showed Best-3 expression in the plasma membrane, nuclei and intracellular compartments, though not in the ER, in cultured cells and rat kidney cortex sections. Best-3 mRNA was augmented by ER stress and signaled through increased Ca2+, oxidative stress and ERK1/2 phosphorylation, because it was attenuated by α-tocopherol, CAT expression, BAPTA-AM, calmodulin kinase inhibitor calmidazolium (40μM), ERK1/2 inhibitor U0126 (10μM), and ERK1/2 RNAi. Knockdown of Best-3 resulted in decreased cell number consequentially of cell death, as determined by nuclear staining and PARP-1 cleavage. Furthermore, reduced ER stress-cell death by Best-3 overexpression is attributed to diminished CHOP. Since Best-3 overexpression did not affect upstream signaling pathways, we hypothesize that Best-3 possibly interferes with CHOP transcription. From our novel observations, we conclude that ERK1/2-dependent Best-3 activation regulates cell fate decisions during ER stress by suppressing CHOP induction and death. [Copyright &y& Elsevier]

Abstract: We investigated the ring-pattern characteristic of self-phase modulation in a homeotropic nematic liquid crystal cell induced by a focused Ar-ion laser beam. Laser induced self-phase retardation versus incident angle was experimentally studied, and good agreement with theory was found. Effects of applied dc electric field on the ring-pattern were studied, to our knowledge, for the first time. When the electric field was below a critical value, laser induced self-phase retardation could be varied by changing the electric field strength. Above this value, irregular shaped diffraction pattern accompanied by intense light scattering was observed, which is attributable to honeycomb-like domain formation. [Copyright &y& Elsevier]

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]

Laser heterodyne measurements have been used to study the nonradiative energy loss of isolated rhodamine 6G molecules in methanol solutions with different potassium iodide quencher concentrations. Simultaneous measurement of both the relative fluorescent intensity and photothermal phase modulation induced by argon ion laser excitation allows absolute determination of both the quantum yield and the fraction of absorbed photo-energy converted to heat. At zero quencher concentration, their values were found to be 98.2% and 12%, respectively. [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]

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.

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.)

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.)