Osmoprotective proteome adjustments in mouse kidney papilla.

The papilla of the mammalian kidney must tolerate greatly varying degrees of hyperosmotic stress during urine concentration and depending on whole organism hydration state. To identify proteome adaptations supporting cell function and survival in such a harsh environment we compared the proteome of a) the hyperosmotic renal papilla with that of adjacent iso-osmotic cortex tissue and b) the renal papilla of diuretic versus that of anti-diuretic mice. Though functionally distinct the papilla is in close physical proximity to the renal cortex, an iso-osmotic region. Proteomic differences between the papilla and cortex of C57BL6 mice were identified using two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrometry. We found 37 different proteins characteristic of the cortex and 16 proteins over-represented in the papilla. Regional specificity was confirmed by Western blot and further substantiated by immunohistochemistry for selected proteins. Proteins that are characteristic of the renal papilla include αB crystallin, Hsp beta-1, Hsp90, 14-3-3 protein, glutathione S-transferase, aldose reductase, actin and tropomyosin. Gene ontology analysis confirmed a significant increase in molecular functions associated with protein chaperoning and cell stabilization. Proteins over-represented in the cortex were largely related to routine metabolism. During antidiuresis 15 different proteins changed significantly while 18 different proteins changed significantly during diuresis relative to normally hydrated controls. Changes were confirmed by Western blot for selected proteins. Proteins that are significantly altered by diuretic state are associated with cell structure (actin, tubulin), signaling (Rho GDP dissociation inhibitor, abhydrolase domain-containing protein 14B), chaperone functioning (Hsp beta-1, αB crystallin, T complex protein-1) and anti-oxidant functions (α-enolase, GAPDH and LDH). Taken together our study reveals that specific proteins involved in protein folding, cytoskeletal stabilization, antioxidant responses, and stress signaling contribute greatly to the unique hyperosmotic stress resistant phenotype of the kidney papilla.

Proteomic and physiological responses of leopard sharks (Triakis semifasciata) to salinity change.

Partially euryhaline elasmobranchs may tolerate physiologically challenging, variable salinity conditions in estuaries as a trade-off to reduce predation risk or to gain access to abundant food resources. To further understand these trade-offs and to evaluate the underlying mechanisms, we examined the responses of juvenile leopard sharks to salinity changes using a suite of measurements at multiple organizational levels: gill and rectal gland proteomes (using 2-D gel electrophoresis and tandem mass spectrometry), tissue biochemistry (Na(+)/K(+)-ATPase, caspase 3/7 and chymotrypsin-like proteasome activities), organismal physiology (hematology, plasma composition, muscle moisture) and individual behavior. Our proteomics results reveal coordinated molecular responses to low salinity - several of which are common to both rectal gland and gill - including changes in amino acid and inositol (i.e. osmolyte) metabolism, energy metabolism and proteins related to transcription, translation and protein degradation. Overall, leopard sharks employ a strategy of maintaining plasma urea, ion concentrations and Na(+)/K(+)-ATPase activities in the short-term, possibly because they rarely spend extended periods in low salinity conditions in the wild, but the sharks osmoconform to the surrounding conditions by 3 weeks. We found no evidence of apoptosis at the time points tested, while both tissues exhibited proteomic changes related to the cytoskeleton, suggesting that leopard sharks remodel existing osmoregulatory epithelial cells and activate physiological acclimatory responses to solve the problems posed by low salinity exposure. The behavioral measurements reveal increased activity in the lowest salinity in the short-term, while activity decreased in the lowest salinity in the long-term. Our data suggest that physiological/behavioral trade-offs are involved in using estuarine habitats, and pathway modeling implicates tumor necrosis factor alpha (TNFalpha) as a key node of the elasmobranch hyposmotic response network.

Mitogen-activated protein kinases are in vivo transducers of osmosensory signals in fish gill cells.

The abundance and activity of three subgroups of mitogen-activated protein (MAP) kinases, the extracellular signal regulated kinase 1 (ERK1), stress-activated protein kinase 1/ Jun N-terminal kinase (SAPK1), and stress-activated protein kinase 2/ p38 (SAPK2), were measured in gill epithelium of the euryhaline teleost Fundulus heteroclitus exposed for 1 h to 4 weeks to hyper- and hyposmotic stress. The abundance of ERK1, SAPK1 and SAPK2 was analyzed by standard Western immunodetection. MAP kinase activity is a function of phosphorylation and was measured using phospho-specific and MAP kinase subgroup-specific antibodies. The abundance of the 63 kDa fish isoform of SAPK2 increases significantly during hyper- but not hyposmotic stress while ERK1 and SAPK1 protein levels remain unchanged during both types of osmotic stress. In contrast to this small effect of osmotic stress on MAP kinase abundance, the activity of all MAP kinases decreases significantly in response to hyperosmotic stress and increases significantly during hyposmotic stress. These results demonstrate for the first time that the activity of all major MAP kinases is osmoregulated in gill epithelium of euryhaline fish. Based on these results we conclude that MAP kinases are important components of salinity adaptation and participate in osmosensory signaling pathways in gill epithelium of euryhaline fishes.

Cellular osmoregulation: beyond ion transport and cell volume.

All cells are characterized by the expression of osmoregulatory mechanisms, although the degree of this expression is highly variable in different cell types even within a single organism. Cellular osmoregulatory mechanisms constitute a conserved set of adaptations that offset antagonistic effects of altered extracellular osmolality/environmental salinity on cell integrity and function. Cellular osmoregulation includes the regulation of cell volume and ion transport but it does not stop there. We know that organic osmolyte concentration, protein structure, cell turnover, and other cellular parameters are osmoregulated as well. In this brief review two important aspects of cellular osmoregulation are emphasized: 1) maintenance of genomic integrity, and 2) the central role of protein phosphorylation. Novel insight into these two aspects of cellular osmoregulation is illustrated based on two cell models, mammalian kidney inner medullary cells and teleost gill epithelial cells. Both cell types are highly hypertonicity stress-resistant and, therefore, well suited for the investigation of osmoregulatory mechanisms. Damage to the genome is discussed as a newly discovered aspect of hypertonic threat to cells and recent insights on how mammalian kidney cells deal with such threat are presented. Furthermore, the importance of protein phosphorylation as a core mechanism of osmosensory signal transduction is emphasized. In this regard, the potential roles of the 14-3-3 family of phospho-protein adaptor molecules for cellular osmoregulation are highlighted primarily based on work with fish gill epithelial cells. These examples were chosen for the reader to appreciate the numerous and highly specific interactions between stressor-specific and non-specific pathways that form an extensive cellular signaling network giving rise to adaptive compensation of hypertonicity. Furthermore, the example of 14-3-3 proteins illustrates that a single protein may participate in several pathways that are non-specific with regard to the type of stress and, at the same time, in stress-specific pathways to promote cell integrity and function during hypertonicity.

Prevalence of alphas1-casein genotypes in American dairy goats.

Widespread genotyping of US dairy goat breeds for casein variants has not been reported, even though the genetic data could be of use in selective breeding programs. For instance, variability in the content of protein and solids in goat milk is attributed to allelic differences in the goat alpha(s1)-casein gene. Concentrations of alpha(s1)-casein in goat milk are positively correlated with milk components and coagulation properties. The alleles A and B are designated as strong alleles, resulting in the greatest amount of alpha(s1)-casein in goat milk, whereas the E allele produces intermediate amounts and the weak allele F produces the least concentrations of alpha(s1)-casein in goat milk. Here we report on one of the first surveys of the distribution of alpha(s1)-casein genotypes in US dairy goats. The population surveyed, consisting of a total of 257 American dairy goats representing 7 main dairy breeds, contained a greater predominance of the weaker alleles, E and F, than the strong alleles, A and B. Allele distribution was related to breed, with Toggenburg, Alpine, Saanen, and Oberhasli containing the most E and F alleles and LaMancha, Nubian, and Nigerian Dwarf the fewest. Quantification of alpha(s1)-casein production by 2-dimensional gel electrophoresis demonstrated that F/F animals had the least amount of alpha(s1)-casein protein in their milk compared with all other genotypes. The results indicate that genetic improvement of dairy goats in the United States could be achieved if an alpha(s1)-casein breeding scheme were adopted.

Plasticity and stressor specificity of osmotic and heat shock responses of Gillichthys mirabilis gill cells.

Short-term effects of osmotic and heat shock on proteins of Gillichthys mirabilis gill cells were analyzed. The protein synthesis rate (PSR) of gill cells was influenced by hyperosmotic shock (335-->635 mosmol/kgH2O) and heat shock (25-->37 degrees C), but not by hyposmotic shock (335-->190 mosmol/kgH2O). Between 4 and 6 h after hyperosmotic shock, gill cell protein synthesis was inhibited relative to controls in serum-free medium but increased threefold over control values in medium supplemented with 10% serum. Serum-dependent stimulation of PSR was also observed after heat shock. By use of two-dimensional electrophoresis, 21 proteins, induced after hyperosmotic shock, 14 after hyposmotic shock, and 16 after heat shock were found. The osmotic shock response of gill cells was highly stressor specific because only five or three proteins that were induced after hyperosmotic or hyposmotic shock, respectively, were identical to proteins induced in response to heat shock. Heat shock protein 70 isoforms were only induced after heat shock, but not in response to osmotic shock. In gill and kidney epithelium, the transcription factor c-Jun was modified within 30 min after transfer of whole fish from 1,086 mosmol/kgH2O to 5 or 2,172 mosmol/kgH2O, but osmotic shock in vitro had no effect on c-Jun in isolated gill cells. Ion-substitution experiments revealed that the increase of PSR after hyperosmotic shock in serum-supplemented medium significantly depended on an elevation of extracellular Na+ concentration. These data provide evidence for the plasticity and stressor specificity of osmotic and heat shock responses of fish gill cells.

Phylogenetic and functional classification of mitogen- and stress-activated protein kinases.

All currently sequenced stress-activated protein kinases (SAPKs), extracellular signal-regulated kinases (ERKs), and other mitogen-activated protein kinases (MAPKs) were analyzed by sequence alignment, phylogenetic tree construction, and three-dimensional structure modeling in order to classify members of the MAPK family. Based on this analysis the MAPK family was divided into three subgroups (SAPKs, ERKs, and MAPK3) that consist of at least nine subfamilies. Members of a given subfamily were exclusively from animals, plants, or yeast/fungi. A single signature sequence, [LIVM][TS]XX[LIVM]XT[RK][WY]YRXPX[LIVM] [LIVM], was identified that is characteristic for all MAPKs and sufficient to distinguish MAPKs from other members of the protein kinase superfamily. This signature sequence contains the phosphorylation site and is located on loop 12 of the three-dimensional structure of MAPKs. I also identified signature sequences that are characteristic for each of the nine subfamilies of MAPKs. By modeling the three-dimensional structure of three proteins for each MAPK subfamily based on the resolved atomic structures of rat ERK2 and murine p38, it is demonstrated that amino acids conserved in all MAPKs are located primarily in the center of the protein around the catalytic cleft. I conclude that these residues are important for maintaining proper folding into the gross structure common to all MAPKs. On the other hand, amino acids conserved in a given subfamily are located mainly in the periphery of MAPKs, indicating their possible importance for defining interactions with substrates, activators, and inhibitors. Within these subfamily-specific regions, amino acids were identified that represent unique residues occurring in only a single subfamily and their location was mapped in three-dimensional structure models. These unique residues are likely to be crucial for subfamily-specific interactions of MAPKs with substrates, inhibitors, or activators and, therefore, represent excellent targets for site-directed mutagenesis experiments.

Evolution of osmotic stress signaling via MAP kinase cascades.

Cells respond to changes in osmotic pressure with compensatory molecular adaptations that allow them to re-establish homeostasis of osmotically disturbed aspects of cell structure and function. In addition, some cell types respond to osmotic stress by changing their phenotype or, if their tolerance threshold is exceeded, by initiating programmed cell death. To understand how cells achieve these different types of adaptive response to osmotic stress, it is necessary to identify the key elements of osmosensory signal transduction and to analyze the complex networks that process osmotic stimuli imposed upon cells by their environment. This review highlights mitogen-activated protein kinase (MAPK) cascades as important intracellular signal-transduction pathways activated in response to changes in osmolality. A unifying theme of osmotic stress signaling via MAPKs seems to be regulation of the cell cycle as part of the cellular stress response. This very important physiological capacity may have been conserved throughout evolution as a major function of MAPKs from many different subfamilies. The evidence for this conjecture is discussed, and our current knowledge about osmotic stress signaling pathways in yeast, animals and plants is briefly reviewed.

Hyperosmolality in the form of elevated NaCl but not urea causes DNA damage in murine kidney cells.

This study demonstrates, by using neutral comet assay and pulsed field gel electrophoresis, that hyperosmotic stress causes DNA damage in the form of double strand breaks (dsb). Different solutes increase the rate of DNA dsb to different degrees at identical strengths of hyperosmolality. Hyperosmolality in the form of elevated NaCl (HNa) is most potent in this regard, whereas hyperosmolality in the form of elevated urea (HU) does not cause DNA dsb. The amount of DNA dsb increases significantly as early as 15 min after the onset of HNa. By using neutral comet and DNA ladder assays, we show that this rapid induction of DNA damage is not attributable to apoptosis. We demonstrate that renal inner medullary cells are able to efficiently repair hyperosmotic DNA damage within 48 h after exposure to hyperosmolality. DNA repair correlates with cell survival and is repressed by 25 microM LY294002, an inhibitor of DNA-activated protein kinases. These results strongly suggest that the hyperosmotic stress resistance of renal inner medullary cells is based not only on adaptations that protect cellular proteins from osmotic damage but, in addition, on adaptations that compensate DNA damage and maintain genomic integrity.

Maintenance of genomic integrity in mammalian kidney cells exposed to hyperosmotic stress.

Changes in environmental salinity/osmolality impose an osmotic stress upon cells because, if left uncompensated, such changes will alter the conserved intracellular ionic milieu and macromolecular density, for which cell metabolism in most extant cells has been optimized. Cell responses to osmotic stress include rapid posttranslational and slower transcriptional events for the compensatory regulation of cell volume, intracellular electrolyte concentrations, and protein stability/activity. Changes in external osmolality are perceived by osmosensors that control the activation of signal transduction pathways giving rise to the above responses. We have recently shown that the targets of such pathways include cell cycle-regulatory and DNA damage-inducible genes (reviewed in Kültz, D., 2000. Environmental stressors and gene responses, Elsevier, Amsterdam. pp 157-179). Moreover, recent evidence suggests that hyperosmotic stress causes chromosomal aberrations and DNA double-strand breaks in mammalian cells. We propose that the modulation of cell cycle checkpoints and the preservation of genomic integrity are important aspects of cellular osmoprotection and as essential for cellular osmotic stress resistance as the capacity for cell volume regulation and maintaining inorganic ion homeostasis and protein stability/activity.

Differences in protein patterns of gill epithelial cells of the fish Gillichthys mirabilis after osmotic and thermal acclimation.

Different protein patterns in gill epithelium of a euryhaline and eurythermal teleost fish (Gillichthys mirabilis, Family Gobiidae) in response to long-term (2 months) osmotic and thermal acclimation were found for the first time. Gill epithelial cells were isolated to remove extracellular proteins and quantify specialized cell types. Chloride cells were identified on the basis of size (> 10 microns) and bright appearance after [2-(p-dimethylaminostyryl)-1-methyl-pyridinium-iodine] staining. Small mitochondria-rich cells were < 5 microns in diameter and showed intermediate fluorescence. Abundance of chloride cells and small mitochondria-rich cells was significantly influenced by osmotic but not thermal acclimation (dilute seawater/25 degrees C: 1.4 +/- 0.2% chloride cells, 11.9 +/- 4.6% small mitochondria-rich cells; seawater/25 degrees C: 2.4 +/- 0.6% chloride cells, 2.2 +/- 1.3% small mitochondria-rich cells; seawater/10 degrees C: 2.9 +/- 0.3% chloride cells, 1.2 +/- 0.7% small mitochondria-rich cells). Pavement cells, identified by low fluorescence and intermediate size (5-10 microns), largely predominated under all conditions (> 85% of cells). Thus, they represented the major protein source in gill epithelium. Differences in protein patterns were detectable using two-dimensional but not one-dimensional electrophoresis. Of 602 proteins identified by charge and molecular weight properties, only two were induced by high temperature (25 degrees C) and three in response to cold acclimation (10 degrees C). Nine proteins were induced in diluted seawater-acclimated fish, whereas no seawater-induced proteins were found. We hypothesize that proteins induced under dilute seawater conditions are important for the function of pavement cells in gills of hyper-osmoregulating G. mirabilis.

Ion transport in gills of the euryhaline fish Gillichthys mirabilis is facilitated by a phosphocreatine circuit.

The function of creatine kinase (CK) isozymes in energy metabolism and the short-term regulation of active ion transport in gills of the euryhaline teleost Gillichthys mirabilis was investigated. After a transfer of fish from regular seawater [36 parts/thousand (ppt)] to hypersaline water (60 ppt), the plasma osmolality increased significantly from 361.0 +/- 5.2 to 434.2 +/- 20.6 mosmol/kgH2O within 2 h and was regulated down to 391.8 +/- 11.3 mosmol/kgH2O within 12 h. Although the ATP concentration in the gill tissue remained unchanged, the creatine concentration increased significantly from 17.3 +/- 3.2 to 37.6 +/- 5.9 nmol/mg protein within 2 h after the salinity change. CK and Na(+)-K(+)-adenosinetriphosphatase-(Na(+)-K(+)-ATPase) activities were unchanged 48 h after transfer. Independent of salinity, the activities of CK were three to seven times those of the Na(+)-K(+)-ATPase, and the creatine concentration in the gill was at least one order of magnitude higher than the ATP concentration. The occurrence of muscle-type CK (CK-M), brain-type CK, and mitochondrial CK was demonstrated. CK-M was predominant in gills (59 +/- 7.1% of total CK activity). Evidence for a direct functional coupling between CK and Na(+)-K(+)-ATPase was obtained with permeabilized gill cells, by using the CK inhibitor iodoacetamide, which abolishes the competitive channeling of ADP from the external pyruvate kinase reaction to the endogeneous CK reaction in a coupled in situ Na(+)-K(+)-ATPase assay. Our results show the significance and the central regulatory role for energy metabolism and adaptive ionoregulation of a phosphocreatine-CK circuit in situations of high and fluctuating energy demands for euryhaline fishes.

A novel 14-3-3 gene is osmoregulated in gill epithelium of the euryhaline teleost Fundulus heteroclitus.

We have cloned and analyzed the full-length coding sequence and 3' untranslated region (UTR) of a unique 14-3-3 gene of the euryhaline teleost Fundulus heteroclitus, which we named 14-3-3.a. Phylogenetic analysis of the deduced amino acid sequence revealed that the 14-3-3.a gene product is most similar to vertebrate 14-3-3 zeta and beta, yet it displays considerable divergence to known classes of vertebrate 14-3-3 isoforms. The N and C termini of 14-3-3.a are the most unique regions, whereas the amino acid residues forming the amphipathic ligand-binding groove are highly conserved. F. heteroclitus 14-3-3.a mRNA expression is high in gill epithelium, moderate in intestine and brain, and low in gonads, white muscle and heart. Because 14-3-3 proteins are important molecular scaffolds and cofactors for phosphoproteins and signaling complexes, the high level of 14-3-3.a expression in gill epithelium of the euryhaline teleost F. heteroclitus suggests that it is crucial for signal transduction in gill epithelial cells. We provide evidence that 14-3-3.a is involved in osmosensory signal transduction by showing that its mRNA and protein levels in gill epithelium, but not in any other tissue analyzed, increase two- to fourfold within 24h of salinity transfer of fish from sea water to fresh water. These data are clear evidence for an important role of 14-3-3.a in the remodeling of gill epithelium during transition of euryhaline fish between plasma-hyperosmotic and plasma-hypoosmotic environments.

Regulation of gene expression by hypertonicity.

Adaptation of cells to hypertonicity often involves changes in gene expression. Since the concentration of salt in the interstitial fluid surrounding renal inner medullary cells varies with operation of the renal concentrating mechanism and generally is very high, the adaptive mechanisms of these cells are of special interest. Renal medullary cells compensate for hypertonicity by accumulating variable amounts of compatible organic osmolytes, including sorbitol, myo-inositol, glycine betaine, and taurine. In this review we consider how these solutes help relieve the stress of hypertonicity and the nature of transporters and enzymes responsible for their variable accumulation. We emphasize recent developments concerning the molecular basis for osmotic regulation of these genes, including identification and characterization of osmotic response elements. Although osmotic stresses are much smaller in other parts of the body than in the renal medulla, similar mechanisms operate throughout, yielding important physiological and pathophysiological consequences.

Osmotic regulation of gene expression.

Cells react to increased osmolality with numerous changes in gene expression. The specific genes affected differ between species, but the known osmoprotective effects of the gene products are remarkably similar, particularly with regard to cellular accumulation of compatible organic osmolytes. Here we concentrate on the molecular basis for osmotic regulation of gene expression, emphasizing certain genes expressed in bacteria, yeast, and the mammalian renal medulla because their expression is best understood. Thus, we emphasize 1) bacterial and yeast two-component histidine kinase systems, each consisting of a membrane osmolality sensor and a separate cytoplasmic response regulator that, when phosphorylated, alters transcription, 2) volume regulatory increases in cellular K+ salts that can prompt increased gene transcription in bacteria through direct effects on DNA and that in mammalian renal cells increase transcription, seemingly via trans-activating proteins, 3) a yeast kinase cascade that transmits an osmotic signal to the gene regulating the level of glycerol, and 4) in mammalian cells, several homologous cascades that are activated by hypertonicity, but whose osmoregulatory targets are not yet known.

Hyperosmolality causes growth arrest of murine kidney cells. Induction of GADD45 and GADD153 by osmosensing via stress-activated protein kinase 2.

Murine kidney cells of the inner medullary collecting duct (mIMCD) were exposed to either isosmotic (300 mosmol/kg) or hyperosmotic medium (isosmotic medium + 150 mM NaCl) after seeding. We determined cell numbers, total nucleic acid, DNA, and RNA contents in both groups every day for a total period of 7 days. Based on all 4 parameters it was evident that growth of mIMCD3 cells is arrested for approximately 18 h following onset of hyperosmolality. However, none of the parameters measured indicated cell death because of hyperosmolality. Growth curves of hyperosmotic samples were shifted compared with isosmotic samples showing a gap of 18 h but had the same shape otherwise. We demonstrated that at 24 and 48 h after onset of hyperosmolality, but not in isosmotic controls, growth arrest and DNA damage-inducible (GADD) proteins GADD45 and GADD153 are strongly induced. This result is consistent with growth arrest observed in hyperosmotic medium. We tested if mitogen- and stress-activated protein kinase (SAPK) cascades are involved in osmosignaling that leads to GADD45 and GADD153 induction. Using phosphospecific antibodies we showed that extracellular signal-regulated kinases 1 and 2 (ERK), SAPK1 (JNK), and SAPK2 (p38) are hyperosmotically activated in mIMCD cells. Hyperosmotic GADD45 induction was significantly decreased by 37.5% following inhibition of the SAPK2 pathway, whereas it was significantly increased (65.2%) after inhibition of the ERK pathway. We observed similar, although less pronounced effects of SAPK2 and ERK inhibition on hyperosmotic GADD153 induction. In conclusion, we demonstrate that mIMCD cells arrest growth following hyperosmotic shock, that this causes strong induction of GADD45 and GADD153, that GADD induction is partially dependent on osmosignaling via SAPK2 and ERK, and that SAPK2 and ERK pathways have opposite effects on GADD expression.

Distinct regulation of osmoprotective genes in yeast and mammals. Aldose reductase osmotic response element is induced independent of p38 and stress-activated protein kinase/Jun N-terminal kinase in rabbit kidney cells.

In yeast glycerol-3-phosphate dehydrogenase 1 is essential for synthesis of the osmoprotectant glycerol and is osmotically regulated via the high osmolarity glycerol (HOG1) kinase pathway. Homologous protein kinases, p38, and stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) are hyperosmotically activated in some mammalian cell lines and complement HOG1 in yeast. In the present study we asked whether p38 or SAPK/JNK signal synthesis of the osmoprotectant sorbitol in rabbit renal medullary cells (PAP-HT25), analogous to the glycerol system in yeast. Sorbitol synthesis is catalyzed by aldose reductase (AR). Hyperosmolality increases AR transcription through an osmotic response element (ORE) in the 5'-flanking region of the AR gene, resulting in elevated sorbitol. We tested if AR-ORE is targeted by p38 or SAPK/JNK pathways in PAP-HT25 cells. Hyperosmolality (adding 150 mM NaCl) strongly induces phosphorylation of p38 and of c-Jun, a specific target of SAPK/JNK. Transient lipofection of a dominant negative mutant of SAPK kinase, SEK1-AL, into PAP-HT25 cells specifically inhibits hyperosmotically induced c-Jun phosphorylation. Transient lipofection of a dominant negative p38 kinase mutant, MKK3-AL, into PAP-HT25 cells specifically suppresses hyperosmotic induction of p38 phosphorylation. We cotransfected either one of these mutants or their empty vector with an AR-ORE luciferase reporter construct and compared the hyperosmotically induced increase in luciferase activity with that in cells lipofected with only the AR-ORE luciferase construct. Hyperosmolality increased luciferase activity equally (5-7-fold) under all conditions. We conclude that hyperosmolality induces p38 and SAPK/JNK cascades in mammalian renal cells, analogous to inducing the HOG1 cascade in yeast. However, activation of p38 or SAPK/JNK pathways is not necessary for transcriptional regulation of AR through the ORE. This finding stands in contrast to the requirement for the HOG1 pathway for hyperosmotically induced activation of yeast GPD1.

Protection of renal inner medullary epithelial cells from apoptosis by hypertonic stress-induced p53 activation.

Acute hypertonicity causes cell cycle delay and apoptosis in mouse renal inner medullary collecting duct cells (mIMCD3) and increases GADD45 expression. Because the tumor suppressor protein p53 may be involved in these effects, we have investigated the role of p53 in mIMCD3 response to hyperosmotic stress. Acute elevation of osmolality with NaCl addition from the control level of 320 mosmol/kg to 500-600 mosmol/kg greatly increased the levels of total and Ser(15)-phosphorylated p53 within 15 min. However, similar elevation of osmolality with urea did not increase p53 levels. Our studies indicate that induced p53 is transcriptionally active because NaCl addition to 500-600 mosmol/kg stimulated transcription of a luciferase reporter containing a p53 consensus element and appropriately altered mRNA levels of known transcriptional targets of p53, i.e. increased MDM-2 and decreased BCL-2 levels. Elevating NaCl further to 700-800 mosmol/kg rapidly killed most of the cells by apoptosis. At these higher NaCl concentrations, p53 levels were further increased although Ser(15) phosphorylation and transcriptional activity were significantly lower than levels at 500-600 mosmol/kg. At NaCl-induced 500 mosmol/kg, apoptosis was rare in the presence of control, nonspecific oligonucleotide but highly prevalent upon addition of p53 antisense oligonucleotide that substantially reduced p53 levels. We conclude that induction of active p53 in mIMCD3 cells by hypertonic stress contributes to cell survival.