Tonicity Enhancer Binding Protein Research Articles

TonEBP regulates the hyperosmotic expression of aquaporin 1 and 5 in the intervertebral disc

The central region of the intervertebral disc (IVD) is rich in proteoglycans, leading to a hyperosmotic environment, which fluctuates with daily loading. The cells of the nucleus pulposus (NP cells) have adapted to this environment via the function of tonicity enhancer binding protein (TonEBP), and NP cells have been shown to express several water channels known as aquaporins (AQP). We have previously shown that AQP1 and 5 decrease during IVD degeneration. Here, the regulation of AQP1 and 5 by hyperosmotic conditions and the role of TonEBP in this regulation was investigated. AQP1 and 5 gene expression was upregulated by hyperosmotic conditions mimicking the osmolality of the healthy IVD, which was abrogated by TonEBP knockdown. Furthermore, AQP1 and 5 immunopositivity was significantly reduced in TonEBPΔ/Δ E17.5 mice when compared with wildtype controls, indicating in vivo expression of AQP1 and 5 is controlled at least in part by TonEBP. This hyperosmotic regulation of AQP1 and 5 could help to explain the decreased AQP1 and 5 expression during degeneration, when the osmolality of the NP decreases. Together this data suggests that TonEBP-regulated osmo-adaptation may be disrupted during IVD degeneration when the expression of both AQPs is reduced.

Simulated hyperglycemia impairs insulin signaling in endothelial cells through a hyperosmolar mechanism

BackgroundHyperglycemia plays a role in promoting insulin resistance in adipocytes, hepatocytes and myocytes. Its effects on insulin signaling in endothelial cells remain, however, incompletely understood. AimTo investigate the proteomic and metabolomic profiles of human aortic endothelial cells (HAECs) exposed to insulin, normal glucose (NG), high glucose (HG) or its hyperosmolar control high mannitol (HM), and to examine whether and how HG or HM may promote insulin resistance. Methods and resultsWe exposed HAECs to HG and HM in shorter (3 h) and longer-term experiments (24 h), followed by insulin treatment for 45 min. Label-free proteomics and network analysis showed a downregulation of proteins linked to the PI3K-Akt/mTOR/eNOS signaling pathway in HAECs. Metabolomic profiling showed decreased levels of “odd-chain acylcarnitines” such as C3. At immunoblotting, HG or HM blunted insulin ability to activate the PI3K/AKT/eNOS pathway, which was reverted through a silencing of aquaporin 1 (AQP1) and Tonicity enhancer binding protein (TonEBP), while inducing p-P38 and pERK1/2. ConclusionsHG impairs the PI3K/AKT/eNOS pathway and shifts insulin signaling towards the activation of mitogenic and pro-inflammatory effectors, such as p38 and ERK1/2. These effects may explain the progression of insulin resistance as a result of endothelial glucotoxicity.

Osmolarity and calcium regulate connective tissue growth factor (CTGF/CCN2) expression in nucleus pulposus cells

ObjectiveThe objective of our study was to verify the hypothesis that the expression of connective tissue growth factor (CTGF/CCN2), a key molecule essential for the maintenance of nucleus pulposus (NP) matrix homeostasis, is regulated by osmolarity and intracellular calcium in NP cells. MethodsGene and protein expression levels of CCN2 were assessed using quantitative real-time PCR and western blot. Transfections and dual luciferase assays were performed to measure the effect of hyperosmolarity, tonicity enhancer binding protein (TonEBP) and Ca2+-calcineurin (Cn)-NFAT signaling on CCN2 promoter activity. ResultsCultured in hyperosmotic media, there was a significant decrease in the levels of CCN2 promoter activity, gene and protein expression in NP cells. The JASPAR database was used to analyze the construction of human CCN2 promoter, we found conserved TonE and NFAT binding sites. We then investigated whether TonEBP controlled CCN2 expression. Forced expression of TonEBP in NP cells showed that TonEBP negatively regulated CCN2 promoter activity, while suppression of TonEBP induced CCN2 promoter activity and expression. We then examined if Ca2+-Cn-NFAT signaling participated in the regulation of CCN2 expression. Co-expression of CCN2 reporter with individual NFAT1–4 expression plasmids and/or calcineurin A/B constructs suggested this signaling pathway played a role in the regulation of CCN2expression in NP cells. ConclusionsResults of these studies illustrated that the expression of CCN2 in NP cells was regulated by the NFAT family through a signaling pathway network involving both activator (Ca2+-Cn-NFAT signaling) and suppressor (Hyperosmolarity-TonEBP) molecules.

Genetic ablation of NFAT5/TonEBP in smooth muscle cells impairs flow- and pressure-induced arterial remodeling in mice.

The arterial wall adapts to alterations in blood flow and pressure by remodeling the cellular and extracellular architecture. Biomechanical stress of vascular smooth muscle cells (VSMCs) in the media is thought to precede this process and promote their activation and subsequent proliferation. However, molecular determinants orchestrating the transcriptional phenotype under these conditions have been insufficiently studied. We identified the transcription factor, nuclear factor of activated T cells 5 (NFAT5; or tonicity enhancer-binding protein) as a crucial regulatory element of mechanical stress responses of VSMCs. Here, the relevance of NFAT5 for arterial growth and thickening is investigated in mice upon inducible smooth muscle cell (SMC)-specific genetic ablation of Nfat5. In cultured mouse VSMCs, loss of Nfat5 inhibits the expression of gene sets involved in the control of the cell cycle and the interaction with the extracellular matrix and cytoskeletal dynamics. In vivo, SMC-specific knockout of Nfat5 did not affect the general vascular architecture and blood pressure levels under baseline conditions. However, proliferation of VSMCs and the thickening of the arterial wall were inhibited during both flow-induced collateral remodeling and hypertension-mediated arterial hypertrophy. Whereas originally described as a hypertonicity-responsive transcription factor, these findings identify NFAT5 as a novel molecular determinant of biomechanically induced phenotype changes of VSMCs and wall stress-induced arterial remodeling processes.-Arnold, C., Feldner, A., Zappe, M., Komljenovic, D., De La Torre, C., Ruzicka, P., Hecker, M., Neuhofer, W., Korff, T. Genetic ablation of NFAT5/TonEBP in smooth muscle cells impairs flow- and pressure-induced arterial remodeling in mice.

Cleavage of osmosensitive transcriptional factor NFAT5 by Coxsackieviral protease 2A promotes viral replication.

Nuclear factor of activated T cells 5 (NFAT5)/Tonicity enhancer binding protein (TonEBP) is a transcription factor induced by hypertonic stress in the kidney. However, the function of NFAT5 in other organs has rarely been studied, even though it is ubiquitously expressed. Indeed, although NFAT5 was reported to be critical for heart development and function, its role in infectious heart diseases has remained obscure. In this study, we aimed to understand the mechanism by which NFAT5 interferes with infection of Coxsackievirus B3 (CVB3), a major cause of viral myocarditis. Our initial results demonstrated that although the mRNA level of NFAT5 remained constant during CVB3 infection, NFAT5 protein level decreased because the protein was cleaved. Bioinformatic prediction and verification of the predicted site by site-directed mutagenesis experiments determined that the NFAT5 protein was cleaved by CVB3 protease 2A at Glycine 503. Such cleavage led to the inactivation of NFAT5, and the 70-kDa N-terminal cleavage product (p70-NFAT5) exerted a dominant negative effect on the full-length NFAT5 protein. We further showed that elevated expression of NFAT5 to counteract viral protease cleavage, especially overexpression of a non-cleavable mutant of NFAT5, significantly inhibited CVB3 replication. Ectopic expression of NFAT5 resulted in elevated expression of inducible nitric oxide synthase (iNOS), a factor reported to inhibit CVB3 replication. The necessity of iNOS for the anti-CVB3 effect of NFAT5 was supported by the observation that inhibition of iNOS blocked the anti-CVB3 effect of NFAT5. In a murine model of viral myocarditis, we observed that treatment with hypertonic saline or mannitol solution upregulated NFAT5 and iNOS expression, inhibited CVB3 replication and reduced tissue damage in the heart. Taken together, our data demonstrate that the anti-CVB3 activity of NFAT5 is impaired during CVB3 infection due to 2A-mediated cleavage of NFAT5. Thus induction of NFAT5 by hypertonic agents may be a promising strategy for the development of anti-CVB3 therapeutics.

The importance of the NFAT5/TonEBP-mediated osmotic stress response in B cells

Abstract Introduction Hyperosmotic stress is well investigated within renal borders, many non-renal tissues are facing fluctuations of osmolality as part of normal, physiological behavior. For example, compared to blood, osmolality of lymphoid tissues, like spleen and thymus, is substantially hypertonic. Therefore, the ability of immune cells to respond and adapt to osmotic stress is crucial for normal immune responses. The osmotic stress response is driven by nuclear factor of activated T cells (NFAT5)/tonicity enhancer binding protein (TonEBP), the only known mammalian osmosensitive transcription factor. It has been shown that mice with a heterozygous deletion of NFAT5 display impaired adaptive immune responses. To specifically analyze the functions of NFAT5 in B lymphocytes, we generated conditional B cell specific knock-out mice. Objectives Using a conditional B cell-specific NFAT5 knock-out mouse model, we want to assess the importance of NFAT5 for B cell development, B cell activation and antibody production. Results NFAT5-deficient mice fed with high salt diet (HSD; 4% NaCl in food and 0.9% NaCl in water) and immunized with TNP-KLH showed impaired IgM and IgG responses compared to wildtype controls. Accordingly, frequencies and numbers of PC and GC B cells were reduced in immunized NFAT5-deficient mice. In support of these findings, we found lower cell numbers and diminished viability of LPS-stimulated NFAT5-deficient B cells cultured in hypertonic medium (+40mM NaCl). Conclusion NFAT5 in B lymphocytes controls antibody responses by controlling B cell activation and survival. Gene expression profiling will now elucidate transcriptional programs and signaling pathways involved in NFAT5-controlled B cell responses.

High glucose-induced hyperosmolarity contributes to COX-2 expression and angiogenesis: implications for diabetic retinopathy

BackgroundWe tested the hypothesis that glucose-induced hyperosmolarity, occurring in diabetic hyperglycemia, promotes retinal angiogenesis, and that interference with osmolarity signaling ameliorates excessive angiogenesis and retinopathy in vitro and in vivo.Methods and ResultsWe incubated human aortic (HAECs) and dermal microvascular endothelial cells (HMVECs) with glucose or mannitol for 24 h and tested them for protein levels and in vitro angiogenesis. We used the Ins2 Akita mice as a model of type 1 diabetes to test the in vivo relevance of in vitro observations. Compared to incubations with normal (5 mmol/L) glucose concentrations, cells exposed to both high glucose and high mannitol (at 30.5 or 50.5 mmol/L) increased expression of the water channel aquaporin-1 (AQP1) and cyclooxygenase (COX)-2. This was preceded by increased activity of the osmolarity-sensitive transcription factor Tonicity enhancer binding protein (TonEBP), and enhanced endothelial migration and tubulization in Matrigel, reverted by treatment with AQP1 and TonEBP siRNA. Retinas of Ins2 Akita mice showed increased levels of AQP1 and COX-2, as well as angiogenesis, all reverted by AQP1 siRNA intravitreal injections.ConclusionsGlucose-related hyperosmolarity seems to be able to promote angiogenesis and retinopathy through activation of TonEBP and possibly increasing expression of AQP1 and COX-2. Osmolarity signaling may be a target for therapy.

Salt and gene expression: evidence for [Na+]i/[K+]i-mediated signaling pathways.

Our review focuses on the recent data showing that gene transcription and translation are under the control of signaling pathways triggered by modulation of the intracellular sodium/potassium ratio ([Na+]i/[K+]i). Side-by-side with sensing of osmolality elevation by tonicity enhancer-binding protein (TonEBP, NFAT5), [Na+]i/[K+]i-mediated excitation-transcription coupling may contribute to the transcriptomic changes evoked by high salt consumption. This novel mechanism includes the sensing of heightened Na+ concentration in the plasma, interstitial, and cerebrospinal fluids via augmented Na+ influx in the endothelium, immune system cells, and the subfornical organ, respectively. In these cells, [Na+]i/[K+]i ratio elevation, triggered by augmented Na+ influx, is further potentiated by increased production of endogenous Na+,K+-ATPase inhibitors documented in salt-sensitive hypertension.

Hyperosmolar stress induces TonEBP activation in human retinal pigmented epithelial cells

AbstractPurpose Macular edema, a frequently encountered complication of diabetic retinopathy (DR), results from alterations of the blood retinal barrier (BRB) and leads to modifications of the retinal pigmented epithelium (RPE) functions. Osmolar changes of the surrounding medium could be responsible for some modifications of the RPE functions leading to a disturbance of the retinal homeostasis.Methods To study the modification and stability of the key hyperosmolar response factor Tonicity Enhancer Binding Protein’s (TonEBP) expression in ARPE‐19 cells, derived from human RPE, were submitted to hyperosmolar stress of increasing concentration and for various times.Results In response to hyperosmolar stimulation of ARPE‐19 cells, a dose‐dependent increase in TonEBP mRNA and protein levels, as well as TonEBP nuclear translocation, were observed. Moreover, the mRNA levels of Aldose Reductase (AR) and Sodium‐dependent Taurine Transporter (TauT), two TonEBP target genes required for increasing the intracellular ionic force, increased in a delayed manner following hyperosmolar stimulation. Additionally, SB203580, a p38 protein kinase inhibitor, but not its inactive analogue SB202474, inhibited TonEBP nuclear translocation in ARPE‐19 cells submitted to hyperosmolar stimulation.Conclusion In ARPE‐19 cells submitted to hyperosmolar stress, TonEBP expression is induced, and its nuclear translocation stimulated through a mechanism involving p38 protein kinase activation. It is likely that TonEBP nuclear translocation mediates the increase of AR and TauT expression induced by hyperosmolar stress and plays a role in RPE cells osmoadaptation.

Macrophages in homeostatic immune function.

Macrophages are not only involved in inflammatory and anti-infective processes, but also play an important role in maintaining tissue homeostasis. In this review, we summarize recent evidence investigating the role of macrophages in controlling angiogenesis, metabolism as well as salt and water balance. Particularly, we summarize the importance of macrophage tonicity enhancer binding protein (TonEBP, also termed nuclear factor of activated T-cells 5 [NFAT5]) expression in the regulation of salt and water homeostasis. Further understanding of homeostatic macrophage function may lead to new therapeutic approaches to treat ischemia, hypertension and metabolic disorders.

NFAT5-mediated expression of S100A4 contributes to proliferation and migration of renal carcinoma cells.

The osmosensitive transcription factor nuclear factor of activated T-cells (NFAT) 5, also known as tonicity enhancer binding protein (TonEBP), has been associated with the development of a variety of tumor entities, among them breast cancer, colon carcinoma, and melanoma. The aim of the present study was to determine whether NFAT5 is also involved in the development of renal cell carcinoma (RCC). The most common type of RCC, the clear cell RCC, originates from the proximal convoluted tubule. We tested our hypothesis in the clear cell RCC cell line CaKi-1 and the non-cancerous proximal tubule cell line HK-2, as control. Basal expression of NFAT5 and NFAT5 activity in CaKi-1 cells was several times higher than in HK-2 cells. Osmotic stress induced an increased NFAT5 activity in both CaKi-1 and HK-2 cells, again with significantly higher activities in CaKi-1 cells. Analysis of NFAT5-regulating signaling pathways in CaKi-1 cells revealed that inhibition of the MAP kinases p38, c-Jun-terminal kinase (JNK) and extracellular regulated kinase (ERK) and of the focal adhesion kinase (FAK) partially blunted NFAT5 activity. FAK and ERK were both constitutively active, even under isotonic conditions, which may contribute to the high basal expression and activity of NFAT5 in CaKi-1 cells. In contrast, the MAP kinases p38 and JNK were inactive under isotonic conditions and became activated under osmotic stress conditions, indicating that p38 and JNK mediate upregulation of NFAT5 activity under these conditions. siRNA-mediated knockdown of NFAT5 in CaKi-1 cells reduced the expression of S100A4, a member of the S100 family of proteins, which promotes metastasis. Knockdown of NFAT5 was accompanied by a significant decrease in proliferation and migration activity. Taken together, our results indicate that NFAT5 induces S100A4 expression in CaKi-1 cells, thereby playing an important role in RCC proliferation and migration.

Hyperosmolarity-enhanced COX-2 expression contributes to high glucose-induced angiogenesis

Aim/hypothesis: Diabetic hyperglycemia increases plasma osmolarity, leading to adaptive cellular responses. Cycloxygenase-2 (COX-2) plays a role in angiogenesis and plaque stability. We tested the hypothesis that glucose-induced hyperosmolarity promotes angiogenesis through activation of COX-2 expression, thus orchestrating endothelial sprouting and migration. Methods: Human aortic endothelial cells (HAEC) and dermal microvascular endothelial cells (HMVEC) were incubated with 5.5 mmol/L glucose (normoglycemia), high glucose (HG, at 12.5, 25 and 45 mmol/L), or equimolar concentrations of the hyperosmolar controls mannitol (HM). Results: Both HG and HM increased the expression of the water channel aquaporin-1 (AQP1) and of COX-2. HG and HM for 1 h increased the nuclear accumulation of Tonicity enhancer binding protein (TonEBP) and its binding to Tonicity enhancer element at electrophoretic mobility shift assay. HG and HM induced endothelial migration at a fluorimetric assay, and tubulization in Matrigel. Targeting the osmosignaling pathway with small interfering RNAs to AQP1 and to TonEBP both reverted the inducing effects of HG and HM on COX-2 expression as well as angiogenic activities. Compared with age- and sex-matched C57/BL6 control mice, the aorta of D2.B6-Ins2 Akita diabetic mice (3 months old, male) showed lipid accumulation as visualized with Oil red O staining (Figure, panel A), and slight increase of AQP1 (panel B) and COX-2 (panel C) expression. ![Figure][1] Conclusion/interpretation: By activating the water channels AQP1 and TonEBP, hyperosmolarity caused by HG or HM induces COX-2 expression and angiogenesis in human endothelial cells, which may be relevant for vascular complications of diabetes. [1]: pending:yes

Abstract 180: Interleukin (IL) 16 Acts as a Regulator of Skin Electrolyte Metabolism and Blood Pressure

We showed that Na + is stored in the skin without commensurate water retention. In response to interstitial electrolyte load, mononuclear phagocyte system (MPS) cells secrete VEGF C, as regulated by tonicity enhancer binding protein (TonEBP) attaching to the VEGF C promoter. VEGF C induces hyperplasia of skin lymphcapillaries. As IL16 harbors a TonEBP binding site, we tested the hypothesis that IL16 participates in this process. We cultured bone marrow-derived macrophages in isotonic or hypertonic cell culture media and measured IL16 expression. We fed male FVB mice a low salt diet (LSD; < 0.1% NaCl + tap) or a high salt diet (HSD; 4% NaCl + 0.9% saline) for two weeks. We treated the groups with either IL16 specific antibodies or no antibody. After two weeks we measured mean arterial blood pressure (MAP), skin Na + , Cl - , and water content (rSKNa, rSKCl, rSKW), quantified lymphcapillary density, VEGF C and TonEBP expression. IL16 was specifically secreted from macrophages in response to hypertonicity in vitro . HSD increased skin electrolytes, activated TonEBP/VEGF C, and resulted in lymphcapillary hyperplasia. Anti IL16 treatment under LSD increased skin electrolyte, water content and MAP. The IL16 antibody blunted the VEGF C driven lymphcapillary response despite salt load. No differences in skin electrolytes and MAP were found between IL16 antibody treated and untreated mice after HSD. We conclude that IL16 is a regulator of electrolyte and water metabolism and MAP, even in the absence of HSD. The finding that the IL16 promoter contains a TonEBP binding site suggests that IL16 could act as an osmoprotective gene that is involved in regulation of electrolyte homeostasis and MAP control.

Tonicity enhancer binding protein (TonEBP) and hypoxia-inducible factor (HIF) coordinate heat shock protein 70 (Hsp70) expression in hypoxic nucleus pulposus cells: Role of Hsp70 in HIF-1α degradation

The objective of our study was to examine the regulation of hypoxic expression of heat shock protein 70 (Hsp70) in nucleus pulposus cells and to determine if Hsp70 promoted hypoxia-inducible factor (HIF)-1α degradation. Rat nucleus pulposus cells were maintained in culture in either 21% or 1% oxygen. To determine the regulation of Hsp70 expression by tonicity enhancer binding protein (TonEBP) and HIF-1/2, loss-of-function and gain-of-function experiments and mutational analysis of the Hsp70 promoter were performed. Hypoxia increased Hsp70 expression in nucleus pulposus cells. Noteworthy, hypoxia increased TonEBP transactivation and mutation of TonE motifs blocked hypoxic induction of the Hsp70 promoter. In contrast, mutation of hypoxia response element (HRE) motifs coupled with loss-of-function experiments suggested that HIF-1 and HIF-2 suppressed Hsp70 promoter activity and transcription. Interestingly, HIF-α interferes with TonEBP function and suppresses the inductive effect of TonEBP on the Hsp70 promoter. In terms of Hsp70 function, when treated with Hsp70 transcriptional inhibitor, KNK437, there was an increase in HIF-1α protein stability and transcriptional activity. Likewise, when Hsp70 was overexpressed, the stability of HIF-1α and its transcriptional activity decreased. Hsp70 interacted with HIF-1α under hypoxic conditions and evidenced increased binding when treated with MG132, a proteasomal inhibitor. These results suggest that Hsp70 may promote HIF-1α degradation through the proteasomal pathway in nucleus pulposus cells. In hypoxic and hyperosmolar nucleus pulposus cells, Hsp70, TonEBP, and HIFs form a regulatory loop. We propose that the positive regulation by TonEBP and negative regulation of Hsp70 by HIF-1 and HIF-2 may serve to maintain Hsp70 levels in these cells, whereas Hsp70 may function in controlling HIF-1α homeostasis.

Abstract 17216: Aquaporin-1 is Required for Vascular Differentiation of Human Induced Pluripotent Stem Cells Following Exposure to Glucose-Induced Hyperosmolarity

Background and objective: Diabetic hyperglycemia increases plasma osmolarity, leading to adaptive cell reponses. Aquaporin-1 (AQP1) is induced by hyperosmolarity and plays a role in the vascular permeability. Induced pluripotent stem (iPS) cells offer a disease-relevant cellular model for the interplay of risk factors in the vascular complications of diabetes. We tested the hypothesis that glucose-induced hyperosmolarity promotes angiogenesis in human iPS cells through activation of AQP1 and downstream osmosignaling pathway, thus orchestrating cell sprouting and migration. Methods and results: Human iPS cells were generated from skin fibroblasts by lentiviral transduction of four reprogramming factors (Oct4, SOX2, Klf4 and c-Myc). Following 3 weeks compact refractive embryonic stem cell-like colonies emerged. All the iPS cell colonies expressed OCT4, SSEA3, SSEA4 and SOX2, characteristic of successful dedifferentiation. After reprogramming, iPS cells were transfected with siRNA-AQP1 or scrambled controls, and exposed to 5.5 mmol/L glucose (normoglycemia), high glucose (HG) at 12.5, 25 and 45 mmol/L or with high mannitol (HM) at 12.5, 25 and 45 mmol/L for 24-72 hours. Exposure to either HG or HM increased expression of AQP1 and tonicity enhancer binding protein (TonEBP) while decreasing the iPS cell numbers. In iPS cells, AQP1 could be co-immunoprecipitated with β-catenin. HG and HM strongly induced the expression of β-catenin (n=3, p<0.001 vs normal glucose by ANOVA). Under these conditions, proteins co-immunoprecipitated with anti-AQP1 and β-catenin showed increased ratio of F-actin vs G-actin (n=3, p<0.01 vs normal glucose by ANOVA). iPS cells formed bundles in methylcellulose matrix and tubing networks in matrigel, especially when they were exposed to HG and HM (HG 2.8±0.2 fold; HM 3.3±0.5 fold; n=3, p<0.01 by ANOVA. SiRNA to AQP1 reverted the inducing effects of HG and HM on β-catenin expression, actin polymerization and angiogenic activities. Conclusions: The hyperosmolarity serves as a biophysical factor that promotes angiogenesis in human iPS cells. This effect may occur through an AQP1-associated cytoskeleton remodeling. Targeting the osmosignaling pathway offers a novel strategy to reduce vascular complications of diabetes.

Different effects of CsA and FK506 on aquaporin-2 abundance in rat primary cultured collecting duct cells

Calcineurin (Cn) inhibitors (CnI) such as cyclosporine A (CsA) and FK506 are nephrotoxic immunosuppressant drugs, which decrease tubular function. Here, we examined the direct effect of CnI on aquaporin-2 (AQP2) expression in rat primary cultured inner medullary collecting duct cells. CsA (0.5-5μM) but not FK 506 (0.01-1μM) decreased expression of AQP2 protein and messenger RNA (mRNA) in a concentration and time dependent manner, without affecting mRNA stability. This effect was observed despite similar inhibition of Cn activity by both CnI, thereby suggesting that the CsA-dependent decrease in AQP2 expression was Cn independent. Another inhibitor of cyclophilin A, the primary intracellular target of CsA, had no effect on AQP2 expression. In order to investigate the mechanism of decreased AQP2 transcription, we studied activation status of two suggested transcriptional regulators of AQP2, cAMP-responsive element binding protein (CREB), and tonicity enhancer binding protein (TonEBP). Localization of TonEBP, as well as TonEBP-mediated gene transcription, was not affected by CsA. Phosphorylation of CREB at an activating phosphorylation site (S133) was decreased by CsA, but not by FK506. However, both CnI did not affect cellular cAMP levels. We show that CsA decreases transcription of AQP2, a process that is in part independent of Cn or cyclophilin A and suggests dependence on decreased activity of CREB.

Sodium sensing in the interstitium and relationship to hypertension

Internal environment regulation, particularly volume and osmoregulation, has been a fundamental concept important to physiologists and clinicians for almost two centuries. Na balance, intracellular K homeostasis, the crucial role of the Na,K-ATPase pump, osmotic forces, and the overriding effect of the kidney on maintaining homeostasis are notions that have been taught by many and accepted by most for over 50 years. Nevertheless, contradictory findings, problems with simplistic balance explanations, and the notion of salt-sensitive and salt-resistant hypertension have been nagging headaches in the straightforward, two-compartment model of electrolyte balance. Na can be accumulated without commensurate water retention in the interstitium of the skin, and this skin Na storage is paralleled by increased polymerization and sulfation of glycosaminoglycans in the Na reservoir. Subcutaneous tissue macrophages express the transcription factor tonicity enhancer binding protein in response to Na-mediated interstitial osmotic stress and thereby secrete vascular endothelial growth factor C, which stimulates lymphatic formation and endothelial nitric oxide synthase expression, suggesting that the immune system is a regulator of volume and blood pressure homeostasis. Our findings do not abrogate the notion of pressure natriuresis and renal regulatory function. However, we do suggest that extracellular Na, volume and blood pressure homeostasis cannot be maintained without extrarenal regulatory mechanisms.

Induction of the renal stanniocalcin-1 gene in rodents by water deprivation

Stanniocalcin-1 (STC-1) is made by kidney collecting duct cells for targeting of nephron mitochondria to promote respiratory uncoupling and calcium uniport activity. However, the purpose of these actions and how the renal gene is regulated are poorly understood. This study has addressed the latter issue by monitoring renal STC-1 gene expression in different models of kidney function. Unilateral nephrectomy and over-hydration had no bearing on renal gene activity in adult Wistar rats. Dehydration, on the other hand, had time-dependent stimulatory effects in male and female kidney cortex, where STC-1 mRNA levels increased 8-fold by 72 h. Medullary gene activity was significantly increased as well, but muted in comparison (∼2-fold). Gene induction was accompanied by an increase in mitochondrial sequestration of STC-1 protein. Aldosterone and angiotensin II had no bearing on STC-1 gene induction, although there was evidence of a role for arginine vasopressin. Gene induction was unaltered in integrin alpha1 knockout mice, which have an impaired tonicity enhancer binding protein (TonEBP) response to dehydration. The STC-1 gene response could be cytoprotective in intent, as dehydration entails a fall in renal blood flow and a rise in medullary interstitial osmolality. Alternatively, STC-1 could have a role in salt and water balance as dehydration necessitates water conservation as well as controlled natriuresis and kaliuresis.

Gene expression profiling and immune fluorescence implicate cellular redifferentiation induced by hypertonicity in primary cultured IMCD cells

The cells of the renal inner medulla are challenged with an environment consisting of high osmolality mediated mainly by Na+ and urea. High tonicity leads to the activation of the tonicity enhancer binding protein, which induces the expression of several transporters and enzymes. This in turn leads to the cellular accumulation of organic osmolytes thereby protecting the cells from high extracellular tonicity. Although several genes that are induced by tonicity are known, a global gene expression profile and analysis of pathways is missing. We used primary cultured rat renal inner medullary collecting duct cells which were cultivated for 6 days at 300, 600 or 900 mosmol/kg. Total RNA was isolated and prepared for hybridization on Rat Gene 1.0 ST GeneChip. Differentially expressed genes were identified using the BRB Array Tools and functional molecular network were identified with Ingenuity Pathway Analysis.Comparing cells grown in 300 vs. 600 mosmol/kg, more than 1600 and vs. 900 mosmol/kg more than 3300 genes were differentially expressed. In both cases the ratio between the up and down regulated genes was approximately one. While 1300 were common in both comparisons 290 at 600 and more than 2000 genes at 900 mosmol/kg were unique. Besides known genes the expression of several other genes were identified for which regulation by tonicity had not been described before. Cellular morphology was markedly modified with increases in culture medium tonicity. Tonicity induced actin reorganization and morphological changes at the cell to cell contacts. It also induced a hypertrophic phenotype. The adaptation to a higher tonicity is not only associated with massive changes in gene expression but also with changes in cell morphology. This implicates that tonicity induces cellular redifferentiation.

Cyclosporin-A Induced Toxicity in Rat Renal Collecting Duct Cells: Interference with Enhanced Hypertonicity Induced Apoptosis

Background/ Aims: Rat renal inner medullary collecting duct (IMCD) cells are physiologically exposed to a wide range of ambient tonicity. To maintain their function upon changes in osmolality, IMCD cells induce expression of osmoprotective and antiapoptotic genes, mainly mediated by the transcription factor Tonicity Enhancer Binding Protein (TonEBP). Some drugs like Cyclosporin-A (CsA) are discussed to interfere with the activity of TonEBP and thereby mediate their nephrotoxic effects. The aim of our study was to further understand CsA toxicity during elevation of ambient osmolality. Methods: First we examined cytotoxicity of CsA in IMCD exposed to elevated tonicity. Employing microarray analysis of gene expression, real-time PCR and immunoassays, we scrutinized pathways contributing to this effect. Results: We show that in IMCD cells CsA but not FK506 increases apoptosis upon an increase in tonicity. This effect is independent of cellular TonEBP localization or activity and reactive oxygen species. Microarray studies revealed marked quantitative differences in gene expression. Functional analysis showed overrepresentation of genes associated with cell death in presence of CsA. This correlated with increased mRNA expression of genes associated with the death receptor pathway and detection of TNFΑ in culture medium of cells treated with CsA. Conclusion: Our results show that CsA cytotoxicity is induced under elevated ambient osmolality and that death receptor signaling probably contributes to CsA cytotoxicity.