Tonicity-responsive Enhancer Binding Protein Research Articles

TonEBP recognizes R-loops and initiates m6A RNA methylation for R-loop resolution.

R-loops are three-stranded, RNA–DNA hybrid, nucleic acid structures produced due to inappropriate processing of newly transcribed RNA or transcription-replication collision (TRC). Although R-loops are important for many cellular processes, their accumulation causes genomic instability and malignant diseases, so these structures are tightly regulated. It was recently reported that R-loop accumulation is resolved by methyltransferase-like 3 (METTL3)-mediated m6A RNA methylation under physiological conditions. However, it remains unclear how R-loops in the genome are recognized and induce resolution signals. Here, we demonstrate that tonicity-responsive enhancer binding protein (TonEBP) recognizes R-loops generated by DNA damaging agents such as ultraviolet (UV) or camptothecin (CPT). Single-molecule imaging and biochemical assays reveal that TonEBP preferentially binds a R-loop via both 3D collision and 1D diffusion along DNA in vitro. In addition, we find that TonEBP recruits METTL3 to R-loops through the Rel homology domain (RHD) for m6A RNA methylation. We also show that TonEBP recruits RNaseH1 to R-loops through a METTL3 interaction. Consistent with this, TonEBP or METTL3 depletion increases R-loops and reduces cell survival in the presence of UV or CPT. Collectively, our results reveal an R-loop resolution pathway by TonEBP and m6A RNA methylation by METTL3 and provide new insights into R-loop resolution processes.

TonEBP Promotes β-Cell Survival under ER Stress by Enhancing Autophagy.

The endoplasmic reticulum (ER) stress response and autophagy are important cellular responses that determine cell fate and whose dysregulation is implicated in the perturbation of homeostasis and diseases. Tonicity-responsive enhancer-binding protein (TonEBP, also called NFAT5) is a pleiotropic stress protein that mediates both protective and pathological cellular responses. Here, we examined the role of TonEBP in β-cell survival under ER stress. We found that TonEBP increases β-cell survival under ER stress by enhancing autophagy. The level of TonEBP protein increased under ER stress due to a reduction in its degradation via the ubiquitin–proteasome pathway. In response to ER stress, TonEBP increased autophagosome formations and suppressed the accumulation of protein aggregates and β-cell death. The Rel-homology domain of TonEBP interacted with FIP200, which is essential for the initiation of autophagy, and was required for autophagy and cell survival upon exposure to ER stress. Mice in which TonEBP was specifically deleted in pancreatic endocrine progenitor cells exhibited defective glucose homeostasis and a loss of islet mass. Taken together, these findings demonstrate that TonEBP protects against ER stress-induced β-cell death by enhancing autophagy.

Tonicity-responsive enhancer-binding protein promotes stemness of liver cancer and cisplatin resistance

BackgroundHigh recurrence and chemoresistance drive the high mortality in hepatocellular carcinoma (HCC). Although cancer stem cells are considered to be the source of recurrent and chemoresistant tumors, they remain poorly defined in HCC. Tonicity-responsive enhancer binding protein (TonEBP) is elevated in almost all HCC tumors and associated with recurrence and death. We aimed to identify function of TonEBP in stemness and chemoresistance of liver cancer.MethodsTumors obtained from 280 HCC patients were analyzed by immunohistochemical analyses. Stemness and chemoresistance of liver CSCs (LCSCs) were investigated using cell culture. Tumor-initiating activity was measured by implanting LCSCs into BALB/c nude mice.FindingsExpression of TonEBP is higher in LCSCs in HCC cell lines and correlated with markers of LCSCs whose expression is significantly associated with poor prognosis of HCC patients. TonEBP mediates ATM-mediated activation of NF-κB, which stimulates the promoter of a key stem cell transcription factor SOX2. As expected, TonEBP is required for the tumorigenesis and self-renewal of LSCSs. Cisplatin induces the recruitment of the ERCC1/XPF dimer to the chromatin in a TonEBP-dependent manner leading to DNA repair and cisplatin resistance. The cisplatin-induced inflammation in LSCSs is also dependent on the TonEBP-ERCC1/XPF complex, and leads to enhanced stemness via the ATM-NF-κB-SOX2 pathway. In HCC patients, tumor expression of ERCC1/XPF predicts recurrence and death in a TonEBP-dependent manner.InterpretationTonEBP promotes stemness and cisplatin resistance of HCC via ATM-NF-κB. TonEBP is a key regulator of LCSCs and a promising therapeutic target for HCC and its recurrence.

TonEBP in dendritic cells mediates pro-inflammatory maturation and Th1/Th17 responses

Dendritic cells (DCs) are potent antigen-presenting cells that link the innate and adaptive immune responses; as such they play pivotal roles in initiation and progression of rheumatoid arthritis (RA). Here, we report that the tonicity-responsive enhancer-binding protein (TonEBP or NFAT5), a Rel family protein involved in the pathogenesis of autoimmune disease and inflammation, is required for maturation and function of DCs. Myeloid cell-specific TonEBP deletion reduces disease severity in a murine model of collagen-induced arthritis; it also inhibits maturation of DCs and differentiation of pathogenic Th1 and Th17 cells in vivo. Upon stimulation by TLR4, TonEBP promotes surface expression of major histocompatibility complex class II and co-stimulatory molecules via p38 mitogen-activated protein kinase. This is followed by DC-mediated differentiation of pro-inflammatory Th1 and Th17 cells. Taken together, these findings provide mechanistic basis for the pathogenic role of TonEBP in RA and possibly other autoimmune diseases.

The evolving role of TonEBP as an immunometabolic stress protein.

Tonicity-responsive enhancer-binding protein (TonEBP), which is also known as nuclear factor of activated T cells 5 (NFAT5), was discovered 20 years ago as a transcriptional regulator of the cellular response to hypertonic (hyperosmotic salinity) stress in the renal medulla. Numerous studies since then have revealed that TonEBP is a pleiotropic stress protein that is involved in a range of immunometabolic diseases. Some of the single-nucleotide polymorphisms (SNPs) in TONEBP introns are cis-expression quantitative trait loci that affect TONEBP transcription. These SNPs are associated with increased risk of type 2 diabetes mellitus, diabetic nephropathy, inflammation, high blood pressure and abnormal plasma osmolality, indicating that variation in TONEBP expression might contribute to these phenotypes. In addition, functional studies have shown that TonEBP is involved in the pathogenesis of rheumatoid arthritis, atherosclerosis, diabetic nephropathy, acute kidney injury, hyperlipidaemia and insulin resistance, autoimmune diseases (including type 1 diabetes mellitus and multiple sclerosis), salt-sensitive hypertension and hepatocellular carcinoma. These pathological activities of TonEBP are in contrast to the protective actions of TonEBP in response to hypertonicity, bacterial infection and DNA damage induced by genotoxins. An emerging theme is that TonEBP is a stress protein that mediates the cellular response to a range of pathological insults, including excess caloric intake, inflammation and oxidative stress.

DDC expression is not regulated by NFAT5 (TonEBP) in dopaminergic neural cell lines

The nuclear factor of activated T-cells 5 (NFAT5), also known as tonicity-responsive enhancer-binding protein (TonEBP), is a transcription factor that regulates osmoadaptive response in multiple tissues and is highly expressed in the developing central nervous system. A former study reported that NFAT5 activation through hypertonic stress increases the expression of the dopa decarboxylase enzyme (DDC), also known as aromatic-l-amino-acid decarboxylase (AADC), in human renal proximal tubule cells, leading to an increase of dopamine synthesis. In a previous study, we identified NFAT5 as a candidate gene for cocaine dependence, a complex psychiatric disorder in which dopaminergic neurotransmission plays an important role. Therefore, to test the hypothesis that NFAT5 may also affect dopamine levels in the nervous system through the regulation of DDC expression, we examined this regulation using two neural dopaminergic cell lines, SH-SY5Y and PC12. The effect of NFAT5 on the expression of the neuronal isoform of DDC was evaluated by qRT-PCR. Upon hypertonic stress, NFAT5 was activated and accumulated into the nuclei and, subsequently, the expression of NFAT5 and its known targets sodium/myo-inositol cotransporter 1 (SMIT) and sodium chloride/taurine cotransporter (TAUT) increased, as expected. However, the expression of DDC decreased. When silencing the expression of NFAT5 with a specific shRNA we observed that the downregulation of DDC is independent from NFAT5 in both cell lines and is due to hypertonic stress. In conclusion, NFAT5 does not regulate the expression of the neuronal isoform of DDC in neural dopaminergic cell lines and, consequently, it does not modulate dopamine synthesis through DDC.

X-box binding protein 1 (XBP1): A key protein for renal osmotic adaptation. Its role in lipogenic program regulation

In renal cells, hyperosmolarity can induce cellular stress or differentiation. Both processes require active endoplasmic reticulum (ER)-associated protein synthesis. Lipid biosynthesis also occurs at ER surface. We showed that hyperosmolarity upregulates glycerophospholipid (GP) and triacylglycerol (GL-TG) de novo synthesis. Considering that massive synthesis of proteins and/or lipids may drive to ER stress, herein we evaluated whether hyperosmolar environment induces ER stress and the participation of inositol-requiring enzyme 1α (IRE1α)-XBP1 in hyperosmotic-induced lipid synthesis. Treatment of Madin-Darby canine kidney (MDCK) cells with hyperosmolar medium triggered ER stress-associated unfolded protein response (UPR). Hyperosmolarity significantly increased xbp1 mRNA and protein as function of time; 24 h of treatment raised the spliced form of XBP1 protein (XBP1s) and induced its translocation to nuclear compartment where it can act as a transcription factor. XBP1 silencing or IRE1α ribonuclease (RNAse) inhibition impeded the expression of lipin1, lipin2 and diacylglycerol acyl transferase-1 (DGAT1) enzymes which yielded decreased GL-TG synthesis. The lack of XBP1s also decreased sterol regulatory element binding protein (SREBP) 1 and 2. Together our data demonstrate that hyperosmolarity induces IRE1α → XBP1s activation; XBP1s drives the expression of SREBP1 and SREBP2 which in turn regulates the expression of the lipogenic enzymes lipin1 (LPIN1) and 2 (LPIN2) and DGAT1. We also demonstrated for the first time that tonicity-responsive enhancer binding protein (TonEBP), the master regulator of osmoprotective response, regulates XBP1 expression. Thus, XBP1 acts as an osmoprotective protein since it is activated by high osmolarity and upregulates lipid metabolism, membranes generation and the restoration of ER homeostasis

TonEBP-deficiency accelerates intervertebral disc degeneration underscored by matrix remodeling, cytoskeletal rearrangements, and changes in proinflammatory gene expression

The tonicity-responsive enhancer binding protein (TonEBP) plays an important role in intervertebral disc and axial skeleton embryogenesis. However, the contribution of this osmoregulatory transcription factor in postnatal intervertebral disc homeostasis is not known in vivo. Here, we show for the first time that TonEBP-deficient mice have pronounced age-related degeneration of the intervertebral disc with annular and endplate herniations. Using FTIR-imaging spectroscopy, quantitative immunohistochemistry, and tissue-specific transcriptomic analysis, we provide morphological and molecular evidence that the overall phenotype is driven by a replacement of water-binding proteoglycans with fibrocartilaginous matrix. Whereas TonEBP deficiency in the AF compartment caused tissue fibrosis associated with alterations in actin cytoskeleton and adhesion molecules, predominant changes in pro-inflammatory pathways were seen in the NP compartment of mutants, underscoring disc compartment-specific effects. Additionally, TonEBP-deficient mice presented with compromised trabecular bone properties of vertebrae. These results provide the first in vivo support to the long-held hypothesis that TonEBP is crucial for postnatal homeostasis of the spine and controls a multitude of functions in addition to cellular osmoadaptation.

Nucleus pulposus primary cilia alter their length in response to changes in extracellular osmolarity but do not control TonEBP-mediated osmoregulation

The nucleus pulposus (NP) cells adapt to their physiologically hyperosmotic microenvironment through Tonicity-responsive enhancer binding protein (TonEBP/nuclear factor of activated T-cell5 [NFAT5])-mediated osmoregulation. Primary cilia in different organs serve diverse roles including osmosensing, but its contribution to NP cell osmoadaptive response is unknown. A high percentage of cultured primary NP cells possessed primary cilia that changed length in response to osmotic stimuli. Stable silencing of Intraflagellar Transport 88 (Ift88) or Kinesin Family Member 3 A (Kif3a) to inhibit the formation of primary cilia did not affect hyperosmotic upregulation of TonEBP. While ShKif3a blocked hyperosmotic increase of TonEBP-Transactivation Domain (TAD) activity, overall the knockdown of either gene did not alter the hyperosmotic status of proximal promoter activities and transcription of key TonEBP targets. On the other hand, a small decrease in TonEBP level under hypoosmotic condition was attenuated by Ift88 or Kif3a knockdown. Noteworthy, none of the TonEBP target genes were responsive to hypoosmotic stimulus in control and Ift88 or Kif3a knockdown cells, suggesting the primary role of TonEBP in the hyperosmotic adaptation of NP cells. Similarly, in Kif3a null mouse embryonic fibroblasts (MEFs), the overall TonEBP-dependent hyperosmotic responses were preserved. Unlike NP cells, TonEBP targets were responsive to hypoosmolarity in wild-type MEFs, and these responses remained intact in Kif3a null MEFs. Together, these results suggest that primary cilia are dispensable for TonEBP-dependent osmoadaptive response.

Transcriptional Regulator TonEBP Mediates Oxidative Damages in Ischemic Kidney Injury.

TonEBP (tonicity-responsive enhancer binding protein) is a transcriptional regulator whose expression is elevated in response to various forms of stress including hyperglycemia, inflammation, and hypoxia. Here we investigated the role of TonEBP in acute kidney injury (AKI) using a line of TonEBP haplo-deficient mice subjected to bilateral renal ischemia followed by reperfusion (I/R). In the TonEBP haplo-deficient animals, induction of TonEBP, oxidative stress, inflammation, cell death, and functional injury in the kidney in response to I/R were all reduced. Analyses of renal transcriptome revealed that genes in several cellular pathways including peroxisome and mitochondrial inner membrane were suppressed in response to I/R, and the suppression was relieved in the TonEBP deficiency. Production of reactive oxygen species (ROS) and the cellular injury was reproduced in a renal epithelial cell line in response to hypoxia, ATP depletion, or hydrogen peroxide. The knockdown of TonEBP reduced ROS production and cellular injury in correlation with increased expression of the suppressed genes. The cellular injury was also blocked by inhibitors of necrosis. These results demonstrate that ischemic insult suppresses many genes involved in cellular metabolism leading to local oxidative stress by way of TonEBP induction. Thus, TonEBP is a promising target to prevent AKI.

TonEBP/NFAT5 promotes obesity and insulin resistance by epigenetic suppression of white adipose tissue beiging

Tonicity-responsive enhancer binding protein (TonEBP or NFAT5) is a regulator of cellular adaptation to hypertonicity, macrophage activation and T-cell development. Here we report that TonEBP is an epigenetic regulator of thermogenesis and obesity. In mouse subcutaneous adipocytes, TonEBP expression increases > 50-fold in response to high-fat diet (HFD) feeding. Mice with TonEBP haplo-deficiency or adipocyte-specific TonEBP deficiency are resistant to HFD-induced obesity and metabolic defects (hyperglycemia, hyperlipidemia, and hyperinsulinemia). They also display increased oxygen consumption, resistance to hypothermia, and beiging of subcutaneous fat tissues. TonEBP suppresses the promoter of β3-adrenoreceptor gene, a critical regulator of lipolysis and thermogenesis, in ex vivo and cultured adipocytes. This involves recruitment of DNMT1 DNA methylase and methylation of the promoter. In human subcutaneous adipocytes TonEBP expression displays a correlation with body mass index but an inverse correlation with β3-adrenoreceptor expression. Thus, TonEBP is an attractive therapeutic target for obesity, insulin resistance, and hyperlipidemia.

Tonicity-responsive enhancer binding protein (TonEBP) regulates TNF-α-induced hypothalamic inflammation.

Tonicity-responsive enhancer binding protein (TonEBP) is a widely expressed transcription factor and is important in the regulation of inflammatory cytokines. Here, we have identified TonEBP expression in the hypothalamus, which is particularly high in proopiomelanocortin (POMC) neurons. TonEBP overexpression stimulates POMC transcription, and TonEBP haploinsufficiency in TonEBP (+/-) mice results in a decrease in hypothalamic POMC expression. TonEBP (+/-) mice show reduced sickness responses, which include anorexia and hyperthermia, that are initially induced by tumor necrosis factor (TNF)-α. TonEBP (+/-) mice also show lower levels of TNF-α-induced hypothalamic expression of POMC and pro-inflammatory cytokines. These results suggest that TonEBP is an important molecular regulator in the development of inflammatory sickness responses through the control of POMC and pro-inflammatory cytokine expression in the hypothalamus.

TonEBP Regulates PCNA Polyubiquitination in Response to DNA Damage through Interaction with SHPRH and USP1.

SummaryPolyubiquitination of proliferating cell nuclear antigen (PCNA) regulates the error-free template-switching mechanism for the bypass of DNA lesions during DNA replication. PCNA polyubiquitination is critical for the maintenance of genomic integrity; however, the underlying mechanism is poorly understood. Here, we demonstrate that tonicity-responsive enhancer-binding protein (TonEBP) regulates PCNA polyubiquitination in response to DNA damage. TonEBP was recruited to DNA damage sites with bulky adducts and sequentially recruited E3 ubiquitin ligase SHPRH, followed by deubiquitinase USP1, to DNA damage sites, in correlation with the dynamics of PCNA polyubiquitination. Similarly, TonEBP was found to be required for replication fork protection in response to DNA damage. The Rel-homology domain of TonEBP, which encircles DNA, was essential for the interaction with SHPRH and USP1, PCNA polyubiquitination, and cell survival after DNA damage. The present findings suggest that TonEBP is an upstream regulator of PCNA polyubiquitination and of the DNA damage bypass pathway.

Abstract 3694: TonEBP promotes chemoresistance and recurrence of hepatocellular carcinoma via DNA repair of cancer stem cells

Abstract Hepatocellular carcinoma (HCC) is common cancer with a high rate of recurrence and mortality. In addition to diverse etiological agents and wide heterogeneity in individual tumors, inherent resistance to chemotherapeutic agents and a very high rate of recurrence are the major contributing factors to HCC-related death. Cancer stem cells (CSCs) are believed to play a major role in these pathological properties due to its highly efficient DNA damage responses. However, mechanistic understanding of liver CSCs is still lacking, unlike other cancers. Tonicity-responsive enhancer binding protein (TonEBP), also known as NFAT5, is a central component of the pro-inflammatory enhanceosome in which TonEBP bridges activated transcription factors to histone acetyltransferase p300 on gene promoters. Although inflammation is intimately associated with the pathogenesis of HCC, the role of TonEBP is unknown. We aimed to identify a function of TonEBP in HCC and liver CSCs. We previously reported that TonEBP promotes hepatocellular carcinogenesis and expression of TonEBP predicts recurrence, metastasis, and death of patients with HCC. Three common sites of TonEBP action in response to diverse etiological agents leading to tumorigenesis and tumor growth were found: cell injury and inflammation, induction by oxidative stress, and stimulation of the COX-2 promoter. The goal of the following study was to understand how TonEBP mediates recurrence and metastasis of HCC. In HCC cells, TonEBP was required for self-renewal and tumorigenic potential of liver CSCs. In addition, TonEBP knockdown reduced the maintenance of liver CSCs. TonEBP mediated DNA repair of the CSCs caused by cisplatin, UV, and mitomycin C - both inter- and intra-strand crosslinks. Chemoresistance by highly activated DNA repair is contributed by TonEBP. We found that TonEBP-mediated DNA repair was carried out by ERCC1/XPF dimer. TonEBP interacted with ERCC1/XPF dimer through rel-homology domain (RHD) and was required for DNA recruitment of the dimer. RHD is required for TonEBP-mediated self-renewal of liver CSCs. TonEBP-ERCC1/XPF complex activated ATM serine/threonine kinase in response to DNA damage leading to activation of NF-κB, STAT3, and expression of pro-inflammatory cytokines, which stimulate the self-renewal of CSCs. Of note, in a cohort of 296 patients with HCC, expression of ERCC1-XPF predicted recurrence, metastasis, and death with high significance in multivariate analyses in TonEBP dependent manner. We conclude that TonEBP-ERCC1/XPF plays a pivotal role in DNA repair-associated chemoresistance and recurrence via liver CSCs. As such, the TonEBP-ERCC1/XPF complex is an attractive target for the prevention of recurrence and effective chemotherapy in HCC. Citation Format: Jun Ho Lee, Jae Hee Suh, Soo Youn Choi, Hyun Je Kang, Orlando D. Schärer, Neung Hwa Park, Hyug Moo Kwon. TonEBP promotes chemoresistance and recurrence of hepatocellular carcinoma via DNA repair of cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3694.

Calcium-TonEBP1 signaling regulates the expression of matrix metalloproteinases in 29-kDA FN-f-treated chondrocytes

Purpose: Under hyperosmotic condition, tonicity-responsive enhancer-binding protein 1 (TonEBP1/nuclear factor of activated T-cells 5 (NFAT5)) modulates the expression of osmoprotective genes through Ca2+/calcineurin (CaN) signaling pathway. Fibronectin fragments (FN-fs), which are highly found in the synovial fluid of patients with osteoarthritis, induce matrix metalloproteinases (MMPs) expression via toll-like receptor-2 signaling pathway. In this study we examined whether 29-kDa FN-f has an influence on activation of Ca2+/TonEBP1 signaling pathway. Methods: Human articular chondrocytes were enzymatically isolated from articular cartilage and cultured in monolayer. The relative levels of mRNA and protein were analyzed by real-time quantitative reverse transcription-polymerase chain reaction and immuoblotting analysis, respectively. TonEBP1, CaN, and calmodulin (CaM) genes were knocked-down by transfection with small interfering RNAs (siRNAs). Results: The expressions of TonEBP1, CaM and CaN were significantly higher in human OA cartilage than normal cartilage. 29-kDa FN-f induced the expressions of TonEBP1, CaM and CaN at both mRNA and protein levels and then increased dephosphorylation of TonEBP1 and its translocation into the nucleus. Silencing of CaM, CaN, or TonEBP1 suppressed the expressions of MMP-1, -3, and -13 induced by 29-kDa FN-f, showing that CaM/CaN/TonEBP1 pathway may be implicated in 29-kDa FN-f-induced MMPs expression. In addition, inhibition of Ca2+ influx with BAPTA and thapsigargin revealed that Ca2+ is an important regulator in 29-kDa FN-f-induced MMPs expression. Furthermore, 29-kDa FN-f triggered the expression of MMPs via the phospholipase C/ phosphokinase C signaling axis. Conclusions: These results demonstrated that 29-kDa FN-f modulated the expression of MMPs through activation of Ca2+/CaM/CaN/TonEBP1 signaling pathway together with alteration of Ca2+ influx.

AT1a stimulation of tonicity‐responsive enhancer binding protein (TonEBP/NFAT5) translation through Annexin‐A2 may represent allostatic anticipation of increased tonicity

Allostasis is idea that the body may anticipate homeostatic challenges and regulate cell and organ function in preparation. Angiotensin II (ANGII) is important for regulating sodium balance and as such increases tonicity in the renal medulla. The present study identifies a novel regulatory pathway whereby ANGII increases the translation of tonicity‐responsive enhancer binding protein (TonEBP/NFAT5) by activating nucleic acid binding of Annexin‐A2, which has several binding sites on NFAT5 mRNA and increases its translation. The activation of Annexin‐A2 was shown using heparin affinity chromatography and mass spectrometry in four‐week two‐kidney, one‐clip hypertension, and verified in two‐week ANGII infusion in rats. The addition of the AT1‐receptor blocker Losartan reduced the activation. Renal gene expression was studied using micro arrays, and identified NFAT5 targets as enriched. Western blot showed that NFAT5 protein was increased, while mRNA was not significantly regulated consistent with posttranscriptional regulation by Annexin‐A2. In conclusion, ANGII/AT1/Annexin‐A2‐mediated increase of the normally tonicity‐regulated NFAT5 in the kidney may be a way to ensure a more robust response to ANGII‐induced tonicity increase, and thus better protect homeostasis.Support or Funding InformationLars Hierta Foundation, Magnus Bergvall Foundation, Swedish Society for Medical Research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Excessive salt intake increases peritoneal solute transport rate via local tonicity-responsive enhancer binding protein in subtotal nephrectomized mice.

High peritoneal transport is associated with high mortality and technical failure in peritoneal dialysis (PD). Baseline peritoneal solute transport rate (PSTR) as measured by the peritoneal equilibration test (PET) within 6 months after PD initiation varies between patients. Sodium is reported to be stored in the skin or muscle of dialysis patients. This study investigated whether excessive salt intake in uremic mice caused peritoneal alterations without exposure to PD fluid. Sham-operated (Sham) and subtotal nephrectomized (Nx) mice were randomly given tap water or 1% sodium chloride (NaCl)-containing water for 8 weeks. PET was then performed to evaluate peritoneal function. Human mesothelial cell line Met-5A was used for in vitro studies. We observed higher PSTR in Nx mice with 1% NaCl-containing drinking water (Nx + salt) compared with those with tap water (Nx + water), along with enhanced angiogenesis and inflammation in the peritoneum. Blockade of interleukin (IL)-6 signaling rescued peritoneal transport function in Nx + salt mice. In cultured Met-5A, additional NaCl in the medium upregulated IL-6 as well as vascular endothelial growth factor-A, associated with increased expression and nuclear translocation of tonicity-responsive enhancer binding protein (TonEBP). Knockdown of TonEBP lowered the induction caused by high tonicity. Peritoneal TonEBP expression was higher in Nx + salt mice, while removal of high-salt diet lowered TonEBP level and improved peritoneal transport function. Excessive dietary salt intake caused peritoneal membrane functional and structural changes under uremic status. TonEBP regulated hypertonicity-related inflammatory changes and might play a crucial role in high baseline peritoneal transport.

Role of NFAT5 in the Immune System and Pathogenesis of Autoimmune Diseases.

The nuclear factor of activated T cells (NFAT5), also known as a tonicity-responsive enhancer-binding protein, was originally identified as a key transcription factor involved in maintaining cellular homeostasis against hypertonic and hyperosmotic environments. Although NFAT5 has been expressed and studied in various types of hyperosmolar tissues, evidence has emerged that NFAT5 plays a role in the development and activation of immune cells, especially T cells and macrophages. The immune-regulatory function of NFAT5 is achieved by inducing different target genes and different signaling pathways in both tonicity-dependent and -independent manners. Particularly in response to hyperosmotic stress, NFAT5 induces the generation of pathogenic TH17 cells and pro-inflammatory macrophages, contributing to autoimmune and inflammatory diseases. Meanwhile, with tonicity-independent stimuli, including activation of the Toll-like receptors and inflammatory cytokines, NFAT5 also can be activated and promotes immune cell survival, proliferation, migration, and angiogenesis. Moreover, under isotonic conditions, NFAT5 has been implicated in the pathogenesis of a variety of inflammatory and autoimmune diseases including rheumatoid arthritis. This review describes the current knowledge of NFAT5, focusing on its immune-regulatory functions, and it highlights the importance of NFAT5 as a novel therapeutic target for chronic inflammatory diseases.

New aspects of the pathomechanism of salt-sensitive hypertension

This article shortly outlines the evolution of hypertonia from risk factors to end-organ damage. The pathogenetic role of salt intake is underlined and in the light of recent clinical and experimental observations, the importance of renal and extrarenal mechanism in the development of salt-sensitive hypertension is analysed. The generally accepted concept that the inefficient renal sodium excretion and the subsequent expansion of the extracellular space is the major factor in blood pressure elevation is challenged. Evidences have been provided that the retained sodium dissociates from the volume of extracellular space and, also from the blood pressure. It has been shown that the negatively charged macromolecules in the subcutaneous interstitium bind sodium ions in osmotically inactive form and store sodium reversibly. The local tissue hypertonicity induces monocytes/macrophages invasion and activation that causes increased expression of tonicity-responsive enhancer binding protein (TonEBP) and the secretion of vascular endothelial growth factor C that result in enhanced lymphangiogenesis. The expanded lymphatic system drains the excess sodium and volume back to the circulation. The reduction of buffer function of this system may contribute to the development or to worsening of hypertension. Similar buffer and barrier functions are attributed to the glycocalyx that covers the luminal surface of vascular endothelium. It is also recognised that the high sodium intake alone is an important pathogenetic factor in end-organ damage independent of hypertension. This may be accounted for by the induction and activation of Th17 cells as well as by the increased production of several pro-inflammatory and pro-fibrotic cytokines. Orv Hetil. 2019; 160(2): 43-49.

VEGF-C attenuates renal damage in salt-sensitive hypertension.

Salt-sensitive hypertension is a major risk factor for renal impairment leading to chronic kidney disease. High-salt diet leads to hypertonic skin interstitial volume retention enhancing the activation of the tonicity-responsive enhancer-binding protein (TonEBP) within macrophages leading to vascular endothelial growth factor C (VEGF-C) secretion and NOS3 modulation. This promotes skin lymphangiogenesis and blood pressure regulation. Whether VEGF-C administration enhances renal and skin lymphangiogenesis and attenuates renal damage in salt-sensitive hypertension remains to be elucidated. Hypertension was induced in BALB/c mice by a high-salt diet. VEGF-C was administered subcutaneously to high-salt-treated mice as well as control animals. Analyses of kidney injury, inflammation, fibrosis, and biochemical markers were performed in vivo. VEGF-C reduced plasma inflammatory markers in salt-treated mice. In addition, VEGF-C exhibited a renal anti-inflammatory effect with the induction of macrophage M2 phenotype, followed by reductions in interstitial fibrosis. Antioxidant enzymes within the kidney as well as urinary RNA/DNA damage markers were all revelatory of abolished oxidative stress under VEGF-C. Furthermore, VEGF-C decreased the urinary albumin/creatinine ratio and blood pressure as well as glomerular and tubular damages. These improvements were associated with enhanced TonEBP, NOS3, and lymphangiogenesis within the kidney and skin. Our data show that VEGF-C administration plays a major role in preserving renal histology and reducing blood pressure. VEGF-C might constitute an interesting potential therapeutic target for improving renal remodeling in salt-sensitive hypertension.