A novel three-dimensional Nrf2 reporter epidermis model for skin sensitization assessment.

Skin sensitization assessment has progressed from the use of animal models towards the application of New Approach Methodologies (NAMs). Several skin sensitization NAMs are accepted for regulatory use, but a majority relies on submerged in vitro cell cultures that limit their applicability domain, posing challenges for testing hydrophobic chemicals and mixtures. A newly developed three-dimensional (3D) Nrf2 reporter epidermis model for skin sensitization assessment is reported. This NAM may help to overcome these limitations. The NAM combines the in vivo-like biology and exposure conditions of 3D epidermis models with the reliability, convenience, and cost-effectiveness of secreted reporter gene technology. The Keap1-Nrf2-ARE pathway was chosen as the reporter gene read-out, as it is induced by most skin sensitizers and already adopted in OECD Test guideline 442D. Immortalized human primary keratinocytes (Ker-CT) were stably transfected with the pIGB-Nrf2-SEAP vector to construct a Nrf2 reporter cell line. Ker-CT Nrf2 reporter cells showed negligible basal expression of the Secreted Embryonic Alkaline Phosphatase (SEAP) reporter, which was induced 13.5-fold by exposure to the skin sensitizer cinnamic aldehyde (CA). Co-exposure to CA and the Nrf2 inhibitor glucocorticoid clobetasol propionate significantly suppressed the CA-induced SEAP expression, confirming dependance of the SEAP expression on Nrf2 activation. Using air-liquid interface and animal constituent free culture conditions, the Ker-CT Nrf2 reporter cells differentiated to stratified 3D epidermis models with an in vivo-like skin architecture and functional skin barrier. Evaluation of a Ker-CT Nrf2 reporter cell-based 2D assay by testing 10 conventional reference chemicals showed a predictive accuracy for skin sensitization potential of 80% and 70% compared to LLNA and human data in two independent laboratories and a high intra- and interlaboratory reproducibility. Moreover, the 3D epidermis models predicted 3 sensitizing and 2 non-sensitizing reference chemicals correctly in a first proof-of-concept study. Further investigations foresee the testing of additional chemicals, including hydrophobic compounds and mixtures to confirm the potential of the 3D epidermis models to broaden the applicability domain for NAM-based skin sensitization assessment.

Intracellular action of the cytokine MIF to modulate AP-1 activity and the cell cycle through Jab1.

Cytokines are multifunctional mediators that classically modulate immune activity by receptor-mediated pathways. Macrophage migration inhibitory factor (MIF) is a cytokine that has a critical role in several inflammatory conditions but that also has endocrine and enzymatic functions. The molecular targets of MIF action have so far remained unclear. Here we show that MIF specifically interacts with an intracellular protein, Jab1, which is a coactivator of AP-1 transcription that also promotes degradation of the cyclin-dependent kinase inhibitor p27Kip1 (ref. 10). MIF colocalizes with Jab1 in the cytosol, and both endogenous and exogenously added MIF following endocytosis bind Jab1. MIF inhibits Jab1- and stimulus-enhanced AP-1 activity, but does not interfere with the induction of the transcription factor NFkappaB. Jab1 activates c-Jun amino-terminal kinase (JNK) activity and enhances endogenous phospho-c-Jun levels, and MIF inhibits these effects. MIF also antagonizes Jab1-dependent cell-cycle regulation by increasing p27Kip1 expression through stabilization of p27Kip1 protein. Consequently, Jab1-mediated rescue of fibroblasts from growth arrest is blocked by MIF. Amino acids 50-65 and Cys 60 of MIF are important for Jab1 binding and modulation. We conclude that MIF may act broadly to negatively regulate Jab1-controlled pathways and that the MIF-Jab1 interaction may provide a molecular basis for key activities of MIF.

Possible involvement of heat shock protein 25 in the angiotensin II-induced glomerular mesangial cell contraction via p38 MAP kinase.

In the rat kidney, mesangial cells (MCs), especially those in the extraglomerular mesangium (EGM) region of the juxtagomerular apparatus, express high amounts of heat shock protein 25 (HSP25). Because MCs are contractile in vivo and HSP25 is known to modulate polymerization/depolymerization of F-actin and to be involved in smooth muscle contraction, it is possible that HSP25 participates in the contraction process of MCs. We analyzed a permanent mouse MC line using Northern and Western blot analyses, and observed that similar to the MCs in the glomerulus, these cells also express high amounts of HSP25 constitutively. Exposure of these cells to angiotensin II (ANG II: 2 x 10(-7) M) evoked contraction and a concomitant increase in HSP25 phosphorylation, while the cytoplasmic fraction of HSP25 was transiently reduced. Because phosphorylation of HSP25 is essential for its actin-modulating function, we suppressed the activity of p38 MAP kinase, the major upstream activator of HSP25 phosphorylation, with the specific inhibitor SB 203580. This maneuver reduced HSP25 phosphorylation dramatically, abolished cell contraction, and prevented the decrease of the cytoplasmic HSP25 content. This suggests that HSP25 might be a component of the contraction machinery in MCs and that this process depends on p38 MAP kinase-mediated HSP25 phosphorylation. The decrease of cytoplasmic HSP25 content observed after ANG II exposure is probably the result of a transient redistribution of HSP25 into a buffer-insoluble fraction, because the whole cell content of HSP25 did not change, a phenomenon known to be related to the actin-modulating activity of HSP25. The fact that this function requires phosphorylation of HSP25 would explain the observation that HSP25 does not redistribute in SB 203580-pretreated cells.

Hypertonicity-induced accumulation of organic osmolytes in papillary interstitial cells.

BACKGROUND: Medullary cells of the concentrating kidney are exposed to high extracellular solute concentrations. It is well established that epithelial cells in this kidney region adapt osmotically to hypertonic stress by accumulating organic osmolytes. Little is known, however, of the adaptive mechanisms of a further medullary cell type, the papillary interstitial cell [renal papillary fibroblast (RPF)]. We therefore compared the responses of primary cultures of RPFs and papillary collecting duct (PCD) cells exposed to hypertonic medium. METHODS: In RPFs and PCD cells, organic osmolytes were determined by high-performance liquid chromatography; mRNA expression for organic osmolyte transporters [Na+/Cl(-)-dependent betaine transporter (BGT), Na(+)-dependent myo-inositol transporter (SMIT)], and the sorbitol synthetic and degrading enzymes [aldose reductase (AR) and sorbitol dehydrogenase (SDH), respectively] was determined by Northern blot analysis. RESULTS: Exposure to hypertonic medium (600 mOsm/kg by NaCl addition) caused intracellular contents of glycerophosphorylcholine, betaine, myo-inositol, and sorbitol, but not free amino acids, to increase significantly in both RPFs and PCD cells. The rise in intracellular contents of these organic osmolytes was accompanied by enhanced expression of mRNAs coding for BGT, SMIT, and AR in both RPFs and PCD cells. SDH mRNA abundance, however, was unchanged. Nonradioactive in situ hybridization studies on sections from formalin-fixed and paraffin-embedded, normally concentrating kidneys showed strong expression of BGT, SMIT, and AR mRNAs in interstitial and collecting duct cells of the papilla, whereas expression of SDH mRNA was much weaker in both cell types. CONCLUSIONS: These results suggest that both RPFs and PCD cells use similar strategies to adapt osmotically to the high interstitial NaCl concentrations characteristic for the inner medulla and papilla of the concentrating kidney.

Inhibition of NaCl-induced heat shock protein 72 expression renders MDCK cells susceptible to high urea concentrations.

Exposure of Madin-Darby canine kidney (MDCK) cells to elevated extracellular NaCl concentrations is associated with increased heat shock protein 72 (HSP72) expression and improved survival of these pretreated cells upon exposure to an additional 600 mM urea in the medium. To establish a causal relationship between HSP72 expression and cell protection against high urea concentrations, two approaches to inhibit NaCl-induced HSP72 synthesis prior to exposure to 600 mM urea were employed. First, the highly specific p38 kinase inhibitor SB203580 was added (100 microM) to the hypertonic medium (600 mosm/kg H2O by NaCl addition, 2 days of exposure), which significantly reduced HSP72 mRNA abundance and HSP72 content. Survival of these cells after a 24-h urea treatment (600 mM) was markedly curtailed compared with appropriate controls. Second, a pcDNA3-based construct, containing 322 bases of the HSP72 open reading frame in antisense orientation and the geneticine resistance gene, was transfected into MDCK cells. Clones with strong inhibition of HSP72 synthesis and others which express the protein at normal levels (comparable to nontransfected MDCK cells) after heat shock treatment or hypertonic stress were established. When these transformants were subjected to hypertonic stress for 2 days prior to exposure to an additional 600 mM urea for 24 h, cell survival was significantly reduced in those clones in which HSP72 expression was strongly inhibited. These results provide further evidence for the protective function of HSP72 against high urea concentrations in renal epithelial cells.

Expression of aldose reductase, sorbitol dehydrogenase and Na+/myo-inositol and Na+/Cl-/betaine transporter mRNAs in individual cells of the kidney during changes in the diuretic state.

The effect of changes in medullary extracellular tonicity on mRNA expression for aldose reductase (AR), sorbitol dehydrogenase (SDH), Na+/Cl-/betaine (BGT) and Na+/myo-inositol (SMIT) cotransporter in different kidney zones was studied using Northern blot analysis and non-radioactive in situ hybridization in four groups of rats: controls, acute diuresis (the loop diuretic furosemide was administered), chronic diuresis (5 days of diuresis), and antidiuresis [5 days of diuresis followed by 24 h deamino-Cys1, d-Arg8 vasopressin (dDAVP)]. Acute administration of the loop diuretic furosemide significantly reduced AR, SMIT and BGT gene expression in the inner and outer medulla compared with controls. Administration of dDAVP to chronically diuretic rats raised the expression of these three mRNAs in the inner but not the outer medulla compared with the chronically diuretic rats. None of these alterations in medullary tonicity significantly changed SDH expression. The in situ hybridization studies showed AR, BGT and SMIT mRNAs to be expressed in both epithelial and non-epithelial cells of the outer and inner medulla. The various cell types (epithelial, endothelial and interstitial cells) differed in their expression pattern and intensity of AR, SDH, BGT and SMIT mRNA, but the inner medullary cells responded uniformly to a decrease in extracellular tonicity with a reduction, and to an increase with enhancement of their AR, BGT and SMIT expression.

Cellular response to osmotic stress in the renal medulla.

Cells of the renal medulla, which are exposed under normal physiological conditions to widely fluctuating extracellular solute concentrations, respond to hypertonic stress by accumulating the organic osmolytes glycerophosphorylcholine (GPC), betaine, myo-inositol, sorbitol and free amino acids. Increased intracellular contents of these osmolytes are achieved by a combination of increased uptake (myo-inositol and betaine) and synthesis (sorbitol, possibly GPC), decreased degradation (GPC) and reduced osmolyte release. In the medulla of the concentrating kidney, accumulation of organic osmolytes, which do not perturb cell function even at high concentrations, allows the maintenance of "normal" intracellular concentrations of inorganic electrolytes. Adaptation to decreasing extracellular solute concentrations, e.g. diuresis, is achieved primarily by activation of pathways allowing the efflux of organic osmolytes, and secondarily by inactivation of production (sorbitol) and uptake (betaine, myo-inositol) and stimulation of degradation (GPC). Apart from modulation of the osmolyte content, osmolality-dependent reorganization of the cytoskeleton and expression of specific stress proteins (heat shock proteins) may be further, as yet poorly characterized, components of the regulatory systems involved in the adaptation of medullary cells to osmotic stress.

Cationic amino acid transporter mRNA expression in rat kidney and liver.

Expression of rat cationic amino acid transporter 2 (r-CAT-2) mRNA was studied in kidney and liver using Northern blot analysis and nonradioactive in situ hybridization with a probe identifying both the r-CAT-2alpha and -2beta splice variants. Expression of r-CAT-2 mRNA was higher in the liver than in the kidney. Within the kidney, r-CAT-2 mRNA was more abundant in the outer and inner medulla than in the cortex. In the liver lobule, the intensity of the hybridization signal in hepatocytes decreased between the portal area and the central vein. In the kidney, hybridization signals were detected in parietal cells of Bowman's capsule, various tubule cells of outer and inner medulla, in endothelial and interstitial cells of inner medulla, and in papillary epithelial cells.

Influence of osmotic stress on heat shock proteins 25 and 72 in mouse mesangial cells.

Previous studies have shown intense staining for heat shock protein 25 (HSP25) in the extraglomerular mesangium (EGM). Because relationships are believed to exist between osmotic stress, expression of HSP25, and protection against stress and because the EGM may be exposed to high local tonicity, we examined the expression of HSP25 and the major stress-inducible and cytoprotective HSP72 in mouse mesangial cells and embryonic lung fibroblasts (3T3) after exposure to hypertonic stress (addition of 150 mM NaCl to the medium for two to seven days). Mesangial, but not 3T3, cells expressed high levels of HSP25 already under control conditions, whereas neither cell line contained HSP72. Hypertonic treatment neither enhanced (mesangial cells) or induced (3T3 cells) HSP25 expression. HSP72, however, was induced strongly in 3T3 cells, but only minimally in mesangial cells. The high level of HSP25 in mesangial cells thus seems not to be a consequence of high tonicity in the EGM because cultured mesangial cells express HSP25 already under control conditions, and osmotic stress did not induce HSP25 in either cell line. Furthermore, high amounts of HSP25 seem to reduce the requirement for HSP72 after stress exposure, suggesting that, in mesangial cells, HSP25 might assume some functions of HSP72.

Hypertonicity affects heat shock protein 27 and F-actin localization in Madin-Darby canine kidney cells.

High concentrations of NaCl are known to perturb the cytoskeleton. In this study, expression and intracellular localization of actin, an important component of the cytoskeleton and of heat shock protein (HSP)27, which promotes the assembly of F-actin, were examined in Madin-Darby canine kidney (MDCK) cells grown chronically in hypertonic medium. HSP27 mRNA abundance was increased twofold compared with wild-type MDCK cells. Chronic hypertonic stress led to enrichment of HSP27 in the insoluble component of the cell lysate and colocalization with cortical F-actin. These results support the notion that HSP27 participates in the modulation of actin dynamics following hypertonic stress.

Effect of ischemia on localization of heat shock protein 25 in kidney.

The effects of renal ischemia on the intracellular distribution of the low-molecular weight heat shock protein (HSP)25 were examined using immunofluorescence microscopy. In all kidney zones, ischemia decreased HSP25 in the supernatant of the tissue homogenates and increased it in the pellet fraction (containing mainly nuclei and cytoskeletal components). This was associated with disappearance of HSP25 staining from the brush border of proximal convoluted tubule (PCT) cells. Because no nuclear staining of cortical tubule cells was apparent either in control or ischemic kidneys, ischemia seems to cause a closer association of HSP25 with cytoskeletal components. HSP25 probably participates in the postischemic restructuring of the cytoskeleton of PCT cells.

Expression of Na+/Cl-/betaine and Na+/myo-inositol transporters, aldose reductase and sorbitol dehydrogenase in macula densa cells of the kidney.

It has been suggested that macula densa cells may be exposed to hyperosmotic stress. Since chronic exposure to hypertonic stress causes the amount of intracellular organic osmolytes to increase, the expression of transporters and enzymes that participate in the intracellular accumulation of organic osmolytes was examined using non-radioactive in situ hybridization in the macula densa region of control rats and furosemide-treated animals. Both the sodium- and chloride-dependent betaine transporter (BGT) and sodium-dependent myo-inositol transporter (SMIT) were expressed preferentially in macula densa cells and for both mRNAs the signal intensity was visibly reduced by furosemide. The enzymes aldose reductase (which mediates the conversion of glucose to sorbitol) and sorbitol dehydrogenase (which converts sorbitol into fructose) were expressed not only in macula densa cells but also in the surrounding tubular cells, and the expression was insensitive to furosemide. Thus it remains unclear whether the expression of BGT and SMIT is related to a putative hypertonic juxtaglomerular region.

Effects of long-term changes in medullary osmolality on heat shock proteins HSp25, HSP60, HSP72 and HSP73 in the rat kidney.

The influence of diuresis and antidiuresis on the expression of heat shock proteins (HSP) 25, 60, 72 and 73 in the renal cortex and outer and inner medulla of Wistar rats was analysed. Medullary osmolality was reduced by long-term diuresis (3% sucrose in the drinking water for 3 weeks) and subsequently enhanced by transition to a concentrating state by giving normal drinking water again in combination with deamino-D-arginine vasopressin (dDAVP) for 5 days. Western blot analyses revealed that neither HSP73 nor HSP60 was influenced by any treatment. The HSP72 level in the medulla was markedly reduced (50%) when osmolality was lowered and increased when tonicity was high. RNAse protection assays showed that the effects on HSP72 are parallelled in general by changes in HSP72 mRNA. While levels of HSP25 were not influenced, isoelectric focusing revealed that the degree of phosphorylation of outer and inner medullary HSP25 increased following both treatments. It thus seems that HSP73 and HSP60 are not directly involved in the long-term adaptation to varying medullary osmolalities. The correlation between changes in osmolality and amounts of the major stress-inducible HSP72 in the medulla implies that medullary hypertonicity is stressful for kidney cells. Furthermore, adaptation to pronounced changes in the osmolality of the environment most likely involves phosphorylation of HSP25.

CFTR mRNA and its truncated splice variant (TRN-CFTR) are differentially expressed during collecting duct ontogeny.

The collecting duct epithelium originates from the embryonic ureter by branching morphogenesis. Ontogeny-dependent changes of CFTR mRNA expression were assessed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in primary monolayer cultures of rat ureteric buds (UB) and cortical collecting ducts, microdissected at different embryonic and postnatal developmental stages. The amount of wild-type CFTR-specific PCR product in UB declined to 20% of the initial value between embryonic gestational day E15 and postnatal day P1. After birth the CFTR product increased transiently between P1 and P7 by a factor of 10 and decreased towards day P14. PCR products specific for TRN-CFTR, a truncated splice variant, however, were low in early embryonic cells, increased markedly between day E17 and P2, and reached a plateau postnatally. Therefore, mRNA encoding TRN-CFTR does not appear to have a specific embryonic-morphogenetic function. By contrast, such function is suggested for wild-type CFTR mRNA as its abundance was high in early embryonic nephrogenesis, as well as during a postnatal period shortly before branching morphogenesis is completed.

Pretreatment with hypertonic NaCl protects MDCK cells against high urea concentrations.

In antidiuresis, the cells of the renal medulla are exposed to high extracellular concentrations of NaCl and urea. Since urea equilibrates with the intracellular compartment and is known to perturb intracellular macromolecules, high urea concentrations may well disturb the structure and function of cell proteins. Two types of organic substances are believed to counteract the adverse effects of high intracellular urea concentrations: specific organic osmolytes of the trimethylamine family [betaine and glycerophosphorylcholine (GPC)], which accumulate in renal medullary cells during prolonged periods of antidiuresis and cytoprotective heat shock proteins (HSPs), the tissue content of two of which (HSPs 27 and 72) is much higher in the inner medulla than in the iso-osmotic renal cortex. To evaluate the contribution of trimethylamines and HSPs to cytoprotection in the presence of high urea concentrations, the effect of HSP induction and osmolyte accumulation prior to exposure to high urea concentrations was examined in Madin-Darby canine kidney (MDCK) cells. Accumulation of organic osmolytes and synthesis of HSP27 and HSP72 was initiated by hypertonic stress (increasing the osmolality of the medium from 290 to 600 mosmol/kg H2O by NaCl addition). Control, non-conditioned cells remained in the isotonic medium for the same period. Upon subsequent exposure to an additional 600 mM urea in the medium for 24 h, 90% of the osmotically conditioned cells but only 15% of non-conditioned cells survived. The HSP72 and trimethylamine contents of the NaCl-conditioned MDCK cells, but not HSP27 content, correlated positively with cell survival. To separate the effects of organic osmolytes and HSP72, chronically NaCl-adapted MDCK cells were returned to isotonic medium for 1 or 2 days, so depleting them of trimethylamine osmolytes. HSP72, with its longer half life, remained elevated. Subsequent exposure of these cells to 600 mM urea in the medium resulted in about 80% survival. These results suggest that in MDCK cells and probably in the renal medulla, HSP72 and perhaps additional protective factors contribute substantially to the resistance against high urea concentrations.