A novel tonicity-responsive microRNA miR-23a-5p modulates renal cell survival under osmotic stress through targeting heat shock protein 70 HSPA1B.

There is growing evidence that microRNAs (miRNAs) are implicated in cellular adaptation to osmotic stress, but the underlying osmosignaling pathways are still not completely understood. In this study, we found that a passenger strand miRNA, miR-23a-5p, was significantly downregulated in response to high NaCl treatment in mouse inner medullary collecting duct cells (mIMCD3) through an miRNA profiling assay. The decrease of miR-23a-5p is hypertonicity-dependent and osmotolerant cell type-specific. Knockdown of miR-23a-5p increased cellular survival and proliferation in mIMCD3. In contrast, miR-23a-5p overexpression repressed cell viability and proliferation under hypertonic stress. RNA deep-sequencing revealed that a heat shock protein 70 (HSP70) isoform, HSP70 member 1B (HSPA1B), was significantly increased under hypertonic treatment. Based on the prediction analysis by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and TargetScan, and a further validation via a dual-luciferase assay, HSPA1B was identified as a potential target of miR-23a-5p. Overexpressed miR-23a-5p suppressed HSPA1B, whereas downregulated miR-23a-5p promoted HSPA1B expression in mIMCD3. In addition, an in vivo study demonstrated that there is a reverse correlation between the levels of miR-23a-5p and HSPA1B in mouse renal inner medulla (papilla) that is exposed to extremely high osmolality. In summary, this study elucidates that passenger strand miR-23a-5p is a novel tonicity-responsive miRNA. The downregulation of miR-23a-5p facilitates cellular adaptation to hypertonic stress in mammalian renal cells through modulating HSPA1B.

The Role of Nuclear Factor of Activated T Cells 5 in Hyperosmotic Stress-Exposed Human Lens Epithelial Cells.

We investigated the role of nuclear factor of activated T cells 5 (NFAT5) under hyperosmotic conditions in human lens epithelial cells (HLECs). Hyperosmotic stress decreased the viability of human lens epithelial B-3 cells and significantly increased NFAT5 expression. Hyperosmotic stress-induced cell death occurred to a greater extent in NFAT5-knockout (KO) cells than in NFAT5 wild-type (NFAT5 WT) cells. Bcl-2 and Bcl-xl expression was down-regulated in NFAT5 WT cells and NFAT5 KO cells under hyperosmotic stress. Pre-treatment with a necroptosis inhibitor (necrostatin-1) significantly blocked hyperosmotic stress-induced death of NFAT5 KO cells, but not of NFAT5 WT cells. The phosphorylation levels of receptor-interacting protein kinase 1 (RIP1) and RIP3, which indicate the occurrence of necroptosis, were up-regulated in NFAT5 KO cells, suggesting that death of these cells is predominantly related to the necroptosis pathway. This finding is the first to report that necroptosis occurs when lens epithelial cells are exposed to hyperosmolar conditions, and that NFAT5 is involved in this process.

Distinct cellular pathways for resistance to urea stress and hypertonic stress.

During antidiuresis with elevated vasopressin, urea accumulates in the renal medulla to very high concentrations, imposing considerable cellular stress. How local cells cope with urea stress is relevant to the whole kidney because the renal medulla is the major site of residence for the renal stem cells. Previous studies showed that renal cells were incapable of preconditioning in moderate urea concentrations to enhance resistance to urea stress. Instead, preconditioning in moderately high salinity (moderate hypertonicity) has been shown to promote resistance to urea stress due to the induction of the molecular chaperone heat shock protein 70 (Hsp70), which is mediated by the transcription factor tonicity-responsive enhancer binding protein (TonEBP). Here we report that cell lines derived from the kidney and fibroblasts display enhanced resistance to urea stress after pretreatment in moderate, nonstressful concentrations of urea. Using TonEBP knockdown and immunoblot analyses, we demonstrate that TonEBP and Hsp70 are dispensable for the increased resistance to urea stress. These data suggest that cells in the renal medulla are capable of overcoming urea stress by activating distinct cellular pathways.

Cryobiological properties of immature zebrafish oocytes assessed by their ability to be fertilized and develop into hatching embryos.

As a step to develop a cryopreservation method for zebrafish oocytes, we investigated the cryobiological properties of immature oocytes at stage III by examining their ability to mature and to develop into hatching embryos after fertilization. When oocytes were chilled at -5°C for 30min, the maturation rate decreased, but the rates of fertilization and hatching were not significantly different from those of controls. When oocytes were exposed to hypotonic solutions for 60min at 25°C, the rates of maturation, fertilization, and hatching decreased in a solution with 0.16Osm/kg or below. When oocytes were exposed to hypertonic solutions (containing sucrose) at 25°C for 30min, the maturation rate decreased in solution with 0.51Osm/kg, whereas the hatching rate decreased with lower osmolality (0.40Osm/kg). In an experiment on the toxicity of cryoprotectants (∼10%, at 25°C), it was found that glycerol and ethylene glycol were toxic both by the assessment of maturation and hatching. Propylene glycol, DMSO and methanol were less toxic by the assessment of maturation, but were found to be toxic by the assessment of hatching. Methanol was the least toxic, but it was less effective to make a solution vitrify than propylene glycol. Therefore, a portion of methanol was replaced with propylene glycol. The replacement increased the toxicity, but could be effective to reduce chilling injury at -5°C. These results clarified the sensitivity of immature oocytes to various cryobiological properties accurately, which will be useful for realizing cryopreservation of zebrafish oocytes.

Hyperosmolar stress induces global mRNA responses in placental trophoblast stem cells that emulate early post-implantation differentiation.

Hyperosmolar stress acts in two ways on the implanting embryo and its major constituent, placental trophoblast stem cells (TSC). Stress causes homeostasis that slows development with lesser cell accumulation, increased cell cycle arrest, and apoptosis. Stress may also cause placental differentiation at implantation. To test for the homeostatic and differentiation-inducing consequences of stress, TSC were exposed to hyperosmolar stress for 24 h and tested using whole mouse genome arrays and Real-time quantitative (Q)PCR. At 0.5 h, all 31 highly changing mRNA (>1.5-fold compared with unstressed TSC) decreased, but by 24 h 158/288 genes were upregulated. Many genes upregulated at 24 h were near baseline levels in unstressed TSC, suggesting new transcription. Thus few genes change during the early stress response, but by 24 h TSC have adapted to start new transcription with large gene sets. Types of genes upregulated at 24 h included homeostatic genes regulating growth and DNA damage induced (GADD45beta/gamma), activator protein (AP)-1 (junB/junC/ATF3/4), heat shock proteins (HSP22/68), and cyclin-dependent kinase inhibitor [CDKI; p15, p21]. But, stress also induced transcription factors that mediate TSC differentiation to trophoblast giant cells (TGC) (Stra13, HES1, GATA-binding2), placental hormones [proliferin, placental lactogen (PL)1, prolactin-like protein (PLP)M], and extracellular matrix genes (CCN1/2). Transcription factors for later placental cell lineages, spongiotrophoblast (MASH2, TPBPalpha) and syncytiotrophoblast (GCM1, TEF5) and placental hormones (PLPA, PLII) were not induced by 24 h stress. Thus stress induced the temporal and spatial placental differentiation normal after implantation. Although differentiation was induced, markers of TSC stemness such as inhibitor of differentiation (ID)2 remained at 100% of levels of unstressed TSC, suggesting that retained mRNA might mediate dedifferentiation were stress to subside.

Selective tonicity-induced expression of the neutral amino-acid transporter SNAT2 in oligodendrocytes in rat brain following systemic hypertonicity.

Sodium-coupled neutral amino-acid transporter member 2 (SNAT2) belongs to the family of neutral amino-acid transporters. SNAT2 is encoded by the gene Slc38a2, whose expression was reported to increase in vitro in fibroblasts, endothelial and renal cells exposed to a hypertonic medium. SNAT2 tonicity-induced expression brings about cellular accumulation of amino-acid, which contributes to osmoadaptation to hypertonicity. Since brain osmoadaptation is observed in relationship to neurological disorders resulting from pathological osmotic imbalances in blood plasma, we have investigated, through immunocytochemistry, SNAT2 expression in brain of rats subjected to systemic hypertonicity. Following prolonged systemic hypertonicity (24 h), small, strongly immunolabeled elements were observed that were not present in sham-treated animals. They were evenly distributed in the gray matter, with a lower density in the forebrain and a higher density in the brain stem. However the highest density by far was observed in white matter, where they were frequently aligned in chain-like rows. These observations suggested an oligodendrocyte location that was further established by double immunofluorescent labeling, using the oligodendrocyte phenotypic markers 2'-3'-cyclic nucleotide 3'phosphodiesterase and carbonic anhydrase II. SNAT2-positive elements were found associated with oligodendrocyte cell bodies, while oligodendrocyte processes were devoid of labeling. A quantitative analysis performed in the cerebral cortex indicated that virtually all SNAT2-positive elements were associated with oligodendrocyte cell bodies and conversely that the overwhelming majority of oligodendrocytes showed SNAT2 immunolabeling. The tonicity-induced expression of SNAT2 was not observed following acute systemic hypertonicity (6 h). Our results suggest that the osmoadaptation of brain oligodendrocytes to hypertonicity relies upon amino-acid accumulation through the tonicity-induced expression of SNAT2. The possible significance of these findings in relationship to the selective loss of oligodendrocytes observed in osmotic demyelination syndrome is discussed.

Inherent toxicity of organ preservation solutions to cultured hepatocytes.

Organ preservation solutions have been designed to protect grafts against the injury inflicted by cold ischemia. However, toxicity of University of Wisconsin (UW) solution during rewarming has been reported. Therefore, we here assessed the toxicity of UW, histidine-tryptophan-ketoglutarate (HTK), Euro-Collins, histidine-lactobionate (HL), sodium-lactobionate-sucrose and Celsior solutions in cultured hepatocytes under hypothermic (4 degrees C), intermediate (21 degrees C) and physiological (37 degrees C) conditions. Marked toxicity of UW, HTK, HL and Euro-Collins solutions was observed at both 37 and 21 degrees C. With the exception of UW solution, these solutions also increased cell injury during cold incubation (LDH release after 18 h at 4 degrees C: HTK 76+/-2%, Euro-Collins 78+/-17%, HL 81+/-15%; control: Krebs-Henseleit buffer 20+/-6%). Testing of individual components using modified Krebs-Henseleit buffers suggested that histidine and phosphate are responsible for (part of) this toxicity. These potential toxicities should be taken into account in the development of future preservation solutions.

The effect of iodine-based contrast agents on the levels of radiation-induced chromosomal aberrations.

The effects of iodine-based contrast agents on the repair of radiation-induced chromosomal damage were investigated employing peripheral blood from a healthy male donor. The blood samples were irradiated with 0.5-4.0 Gy 137Cs gamma rays. Contrast agents and NaCl solutions of various concentrations were added to the blood within the first 15 min or at 60 min after irradiation, and the samples were subsequently cultured for 45 h at 37 degrees C. Significantly elevated frequencies of chromosomal abnormalities caused by postirradiation treatment with hypertonic contrast agents appeared to increase with increasing hypertonicity. Elevated aberration frequencies were found to be greatest in the samples treated within 15 min of irradiation. The contrast agents had little effect if they were added at 60 min after irradiation, probably because the process of chromosome rejoining had been completed. Isotonic iodine-based contrast agents did not enhance the frequencies of chromosomal aberrations to a significant degree.

Functional endothelial damage by high-potassium cardioplegic solutions to saphenous vein bypass grafts.

High-potassium cardioplegic solutions (CSs) may induce endothelial cell damage in vascular grafts, promoting graft thrombosis after coronary bypass operations. We studied prostacyclin (PGI2) production by saphenous veins as a marker of endothelial cell function in a model mimicking actual operative conditions. Fresh saphenous vein segments from patients who had undergone coronary bypass were cut in half; each part was perfused and incubated sequentially with CS (with 20, 40, or 80 mEq potassium/L) or a control buffer (5 mEq potassium/L) at 4 degrees C for 30 minutes (perfusion I), buffer at 37 degrees C for 15 minutes (perfusion II), and buffer plus 25 microM sodium arachidonate at 37 degrees C for 15 minutes (perfusion III). This permitted evaluation of changes in PGI2 production during or after exposure to CS, in basal and stimulated conditions. CS with 20 mEq potassium/L did not alter PGI2 production as compared with control buffer. CS with 40 mEq potassium/L decreased PGI2 production during perfusions I and II. CS with 80 mEq potassium/L also decreased sodium arachidonate-stimulated PGI2 production. Endothelial coverage (immunoperoxidase staining for factor VIII antigen) was intact at all potassium concentrations tested. Thus potassium in CSs can depress endothelial PGI2 production without causing immediate endothelial detachment. This effect may favor thrombosis in bypass grafts.

Flow cytometric analysis of organ preservation-induced endothelial cell membrane damage.

Organ preservation for transplantation is associated with endothelial cell damage. This vascular injury results in increased capillary permeability, graft edema, and early graft dysfunction. This damage may be the limiting factor in preservation of these organs. This study uses flow cytometric assessment of membrane integrity to examine the effects of various organ preservation solutions on human umbilical vein endothelial cell cultures. Confluent plates of human umbilical vein endothelial cells were incubated at 4 degrees C for 24, 48, and 72 hours in commonly used preservation solutions. After cold incubation, the cells were harvested and stained with propridium iodide and fluorescein diacetate. Cells were examined using a flow cytometer for membrane integrity and cytosolic activity. When examined after 24, 48, and 72 hours, cells stored at 4 degrees C in a 5% polyethylene glycol salt solution were significantly less damaged than those stored in any other solution (p less than 0.05). After 48 and 72 hours at 4 degrees C, cells stored in ViaSpan were significantly more intact than cells stored in EuroCollins and 0.9% saline solution (p less than 0.05). This study demonstrates that endothelial cell damage occurs during cold storage and that a polyethylene glycol-based solution showed superior cellular preservation.

Endothelial cell toxicity of solid-organ preservation solutions.

Endothelial cell damage caused by myocardial cardioplegic solutions (Bretschneider HTK and St. Thomas' Hospital No. 2) or renal and hepatic cold storage solutions (modified Collins and University of Wisconsin solution) was assessed in monolayer cultures of adult human venous endothelial cells at 4 degrees to 10 degrees C with phase-contrast microscopy. St. Thomas' Hospital solution caused the cells to contract, resulting in disruption of monolayer integrity and opening of intercellular gaps, and resulted in a 24-hour postexposure survival of 51.0% +/- 2.4%. Bretschneider HTK solution altered cellular morphology less and produced the best postexposure survival (80.2% +/- 2.6%; p less than 0.001). Although morphology was altered the least with University of Wisconsin solution, postexposure survival with this solution, which was similar to that with modified Collins solution, was superior to that with St. Thomas' (p less than 0.01) but inferior to that with Bretschneider HTK (p less than 0.05). The superior protection provided by Bretschneider HTK was due to its additives histidine, tryptophan, and KH-2-oxygluterate (p less than 0.005), and to its low chloride content (p less than 0.005). Furthermore, modifying St. Thomas' solution by decreasing its chloride content improved cell survival to 71.2% +/- 2.3% (p less than 0.001). Normothermic (37 degrees C) exposure to Bretschneider HTK, modified Collins, and University of Wisconsin solution was cytotoxic, whereas normothermic exposure to St. Thomas' cardioplegia was not. In conclusion, the preservation solution that is the least harmful to endothelial cells at hypothermia is Bretschneider HTK cardioplegic solution.

Studies on the increased potassium concentration of plasma produced by a hypertonic solution of low potent substances intraperitoneally administered to rats.

Intracranial hemorrhage was induced in rats by low potent substances, glucose, NaCl and Na2SO4, intraperitoneally injected in enormous amounts, that is, 2800 mOsmol/L (J. Toxicol. Sci. 5, 290, 1980). The present study was undertaken to examine a mechanism of the above mentioned phenomenon in detail from the aspects of the disturbance of water-electrolyte balances and the change in blood osmolality. After administration of hypertonic solutions, blood and abdominal fluid were obtained at intervals of 5 to 15 min and at death. Hypertonic solutions injected intraperitoneally induced rapid exchange in water and solute across the peritoneum, thus causing an increase in abdominal fluid volume and plasma osmolality. Most interesting was the fact that a marked potassemia was produced and that the value of plasma potassium reached 10 mEq/L at death in all of groups of intraperitoneally injected rats. Thus, it is clear that the intracranial hemorrhage is accompanied by an increase in the plasma concentration of potassium which does not always run parallel with an increase in sodium concentration and osmotic pressure in rat plasma.

Effects of hypertonic dextrose injections in the rabbit carpal tunnel.

OBJECTIVE: This study investigated the effects of different doses of hypertonic dextrose injection on the carpal tunnel subsynovial connective tissue (SSCT) and median nerve in a rabbit model. METHODS: Thirty-eight New Zealand white rabbits weighing 4.0-4.5 kg were used. One forepaw carpal tunnel was randomly injected with one of five different treatments: saline-single injection; saline-two injections 1 week apart; 10% dextrose-single injection; 20% dextrose-single injection; or 10% dextrose-two injections 1 week apart. Animals were sacrificed at 12 weeks after the initial injection and were evaluated by electrophysiology (EP), SSCT mechanical testing and histology. RESULTS: There were significant increases in the energy absorption of the SSCT in the 10% dextrose-double injection group compared to the saline injection groups. SSCT stiffness was also significantly increased in the 10% dextrose-double injection group compared to the other groups. There was a significant increase in the thickness of the SSCT in the 10% dextrose-double injection group compared to the saline-single injection group and a significant decrease in the nerve short-long diameter ratio in the 10% dextrose-double injection group compared to the saline-single injection group. There were no changes in EP among the groups. CONCLUSIONS: SSCT fibrosis is present for up to 12 weeks after dextrose injection; multiple injections have bigger effects, including what appears to be a secondary change in nerve flattening. This model may be useful to study the effects of external fibrosis on nerve morphology and physiology, such as occurs clinically in carpal tunnel syndrome.

The effects of various chemical and physical factors on encystment and excystment of Zygocotyle lunata.

The effects of various chemical and physical treatments on the cercariae and metacercariae of Zygocotyle lunata were studied. Cercariae that actively emerged from Helisoma trivolvis snails were examined in 4 ml of test solution in a 3-cm diameter Stender dish. Observations were usually made on encystment and excystment from 2 to 24 h after cercariae were placed in the Stender dishes. Preferences for certain substrates for encystment were noted, such as smooth glass rather than rough glass, the macrophyte Eleocharis acicularis, and plastic Petri dishes. Many of the solutions tested allowed for encystment, i.e., artificial spring water (ASW), 10% artificial ocean water (AOW), and saline (0.85% NaCl). Certain hypertonic solutions allowed for both in vitro encystment and subsequent excystment, i.e., Locke's solution, 25% AOW, and Locke's 1:1. Serial dilutions of ammonium hydroxide, neutral buffered formalin, potassium permanganate, hydrochloric acid, and acetic acid killed cercariae at all concentrations. Ethanol was effective in killing cercariae at all concentrations except at 3.1%, which allowed for encystment. Cercariae were able to encyst at a low temperature of 16 degrees C and a high temperature of 41 degrees C. Morphometric analysis was performed on both live and fixed cercariae of Z. lunata as well as metacercarial cysts formed in Locke's, Locke's 1:1, and ASW. Live cercariae had larger body dimensions than cercariae fixed in hot 5% neutral buffered formalin and mounted in glycerin jelly. Cysts formed in ASW had very similar length and width measurements. Cyst dimensions became less similar to one another as the tonicity of the medium increased. The phenomenon of spontaneous excystation in Z. lunata after in vitro encystment in a hypertonic medium appears to be reported for the first time in digeneans.

Effects of osmoprotectants on hyperosmolar stress in cultured human corneal epithelial cells.

PURPOSE: Increased tear osmolarity in dry eye disease has been found to stimulate production of inflammatory cytokines and matrix metalloproteinases by ocular surface epithelial cells. Prokaryotic and mammalian organ system cells maintain normal function under hypertonic conditions by the synthesis or accumulation of osmoprotectant compounds. This study assessed the effect of osmoprotectant compounds on the activation state of mitogen-activated protein (MAP) kinases in human corneal epithelial cells incubated in hyperosmolar conditions. METHODS: Human corneal epithelial cells were incubated in media of isotonic, physiological osmolarity (300 mOsm) and in hyperosmolar media (400 mOsm), in the presence and absence of osmoprotectants, including several amino acids (L-carnitine and betaine), glycerol, and the polyol erythritol. The phosphorylation (activation) states of c-Jun N-terminal kinases (JNK) and p38 MAP kinases were monitored by Western blot and bead-based immunoassays. RESULTS: Hyperosmolar conditions achieved by addition of sodium chloride or sucrose increased ratios of phosphorylated JNK and p38 to total JNK and p38. Compared with controls, 10 mM L-carnitine or 40 mM erythritol significantly lowered levels of activated MAP kinases in response to hyperosmolar stress. They also lowered ratios of phosphorylated to total kinases to barely detectable levels in cells cultured in isotonic media. CONCLUSIONS: The osmoprotectants L-carnitine and erythritol, alone or in combination, were found to protect against stress activation of corneal epithelial cells cultured in hyperosmolar media.

Acute pulmonary edema after over-infusion of crystalloids versus plasma: histological observations in the rat.

An experimental model of acute pulmonary edema was obtained in the rat, with over-infusion of normal saline and of plasma: histology demonstrated essential differences between edema from crystalloids, developed in the interstitial space, and that from colloids, affecting the alveoli.

Response of the small intestine to the application of a hypertonic solution.

The morphologic and functional alterations caused by a commonly used hypertonic radiographic dye (Hypaque(R)-50%) were compared with changes observed during the absorption of 150 mM saline in closed segments of the ileum of 2- to 3-kg rabbits. Hypertonic dye caused a rapid decrease in height and width of the villi, a decrease in height of the epithelial cells and closure of the intercellular space. Concomitantly, the tissue fluid content of the bowel wall and the volume of venous outflow from the segment of ileum decreased, presumably in response to the osmotic gradient between ileal lumen and blood. The fluid added to the luminal contents was hypotonic and contained sodium, potassium, chloride and bicarbonate. In contrast, the ileum exposed to 150 mM saline had prominent intercellular spaces between adjacent epithelial cells and absorbed the solution at isotonic conditions. These studies indicate that production of diarrheal fluid by this hypertonic solution is different from that reported for enteric pathogens.

Endothelial cell toxicity of preservation solutions: comparison of endothelial cells of different origin and dependence on growth state.

Previously, we have shown that cultured liver endothelial cells are affected by an energy-dependent injury when incubated in cold University of Wisconsin (UW) or histidine-tryptophan-ketoglutarate solution. Here, we studied the susceptibility of other endothelial cells to this type of injury. Aortic endothelial cells in early-confluent, i.e., still proliferating, monolayer cultures were damaged more quickly during cold incubation in UW solution than during cold incubation in Krebs-Henseleit buffer. At this stage the addition of KCN did not alter the loss of viability in UW solution, but when the culture period was prolonged, cells were protected by the addition of cyanide. A paradoxical, protective effect of KCN could also be observed in late-confluent, i.e., nonproliferating, cultures of coronary endothelial cells incubated in UW solution. Similarly, liver endothelial cells in subconfluent, growing cultures were damaged by the addition of cyanide (loss of viability after 48 h, 3 +/- 1% in UW, 65 +/- 19% in UW + KCN), whereas in late-confluent cultures the addition of cyanide to UW solution was protective (loss of viability after 48 h, 100 +/- 0% in UW, 31 +/- 15% in UW + KCN). Variations of culture period and seeding density and the use of inhibitors of cell proliferation demonstrated that liver endothelial cells acquire their susceptibility to energy-dependent injury along with confluence. Subcultured cells retained this susceptibility for some hours. These results suggest that the energy-dependent injury described previously is not confined to liver endothelial cells and that the occurrence of energy-dependent injury requires a capacity of the cells that develops only after cultures have grown to confluence.