NFAT5 Is Activated by Hypoxia: Role in Ischemia and Reperfusion in the Rat Kidney

The current hypothesis postulates that NFAT5 activation in the kidney's inner medulla is due to hypertonicity, resulting in cell protection. Additionally, the renal medulla is hypoxic (10–18 mmHg); however there is no information about the effect of hypoxia on NFAT5. Using in vivo and in vitro models, we evaluated the effect of reducing the partial pressure of oxygen (PO2) on NFAT5 activity. We found that 1) Anoxia increased NFAT5 expression and nuclear translocation in primary cultures of IMCD cells from rat kidney. 2) Anoxia increased transcriptional activity and nuclear translocation of NFAT5 in HEK293 cells. 3) The dose-response curve demonstrated that HIF-1α peaked at 2.5% and NFAT5 at 1% of O2. 4) At 2.5% of O2, the time-course curve of hypoxia demonstrated earlier induction of HIF-1α gene expression than NFAT5. 5) siRNA knockdown of NFAT5 increased the hypoxia-induced cell death. 6) siRNA knockdown of HIF-1α did not affect the NFAT5 induction by hypoxia. Additionally, HIF-1α was still induced by hypoxia even when NFAT5 was knocked down. 7) NFAT5 and HIF-1α expression were increased in kidney (cortex and medulla) from rats subjected to an experimental model of ischemia and reperfusion (I/R). 7) Experimental I/R increased the NFAT5-target gene aldose reductase (AR). 8) NFAT5 activators (ATM and PI3K) were induced in vitro (HEK293 cells) and in vivo (I/R kidneys) with the same timing of NFAT5. 8) Wortmannin, which inhibits ATM and PI3K, reduces hypoxia-induced NFAT5 transcriptional activation in HEK293 cells. These results demonstrate for the first time that NFAT5 is induced by hypoxia and could be a protective factor against ischemic damage

Clinical Characteristics and Outcomes of Drug-Induced Acute Kidney Injury Cases

Introduction Drug-induced acute kidney injury (DI-AKI) is a frequent adverse event. The identification of DI-AKI is challenged by competing etiologies, clinical heterogeneity among patients, and a lack of accurate diagnostic tools. Our research aims to describe the clinical characteristics and predictive variables of DI-AKI. Methods We analyzed data from the DIRECT study (NCT02159209), an international, multi-center, observational cohort study of enriched clinically adjudicated DI-AKI cases. Cases met the primary inclusion criteria if the patient was exposed to at least one nephrotoxic drug for a minimum of 24 hours prior to acute kidney injury (AKI) onset. Cases were clinically adjudicated and inter-rater reliability (IRR) was measured using Krippendorff's alpha. Variables associated with DI-AKI were identified using L1 regularized multivariable logistic regression. Model performance was assessed using the area under the receiver operating characteristic curve (ROC AUC). Results 314 AKI cases met the eligibility criteria for this analysis, and 271 (86%) cases were adjudicated as DI-AKI. The majority of the AKI cases were recruited from the United States (68%). The most frequent causal nephrotoxic drugs were vancomycin (48.7%), non-steroidal anti-inflammatory drugs (18.2%), and piperacillin/tazobactam (17.8%). The IRR for DI-AKI adjudication was 0.309. The multivariable model identified age, vascular capacity, hyperglycemia, infections, pyuria, serum creatinine trends, and contrast media as significant predictors of DI-AKI with good performance, ROC AUC 0.86. Conclusions The identification of DI-AKI is challenging even with comprehensive adjudication by experienced nephrologists. Our analysis identified key clinical characteristics and outcomes of DI-AKI compared to other AKI etiologies

Exploring the Expression of Pro-Inflammatory and Hypoxia-Related MicroRNA-20a, MicroRNA-30e, and MicroRNA-93 in Periodontitis and Gingival Mesenchymal Stem Cells under Hypoxia

Hypoxia associated with inflammation are common hallmarks observed in several diseases, and it plays a major role in the expression of non-coding RNAs, including microRNAs (miRNAs). In addition, the miRNA target genes for hypoxia-inducible factor-1α (HIF-1α) and nuclear factor of activated T cells-5 (NFAT5) modulate the adaptation to hypoxia. The objective of the present study was to explore hypoxia-related miRNA target genes for HIF-1α and NFAT5, as well as miRNA-20a, miRNA-30e, and miRNA-93 expression in periodontitis versus healthy gingival tissues and gingival mesenchymal stem cells (GMSCs) cultured under hypoxic conditions. Thus, a case-control study was conducted, including healthy and periodontitis subjects. Clinical data and gingival tissue biopsies were collected to analyze the expression of miRNA-20a, miRNA-30e, miRNA-93, HIF-1α, and NFAT5 by qRT-PCR. Subsequently, GMSCs were isolated and cultured under hypoxic conditions (1% O2) to explore the expression of the HIF-1α, NFAT5, and miRNAs. The results showed a significant upregulation of miRNA-20a (p = 0.028), miRNA-30e (p = 0.035), and miRNA-93 (p = 0.026) in periodontitis tissues compared to healthy gingival biopsies. NFAT5 mRNA was downregulated in periodontitis tissues (p = 0.037), but HIF-1α was not affected (p = 0.60). Interestingly, hypoxic GMSCs upregulated the expression of miRNA-20a and HIF-1α, but they downregulated miRNA-93e. In addition, NFAT5 mRNA expression was not affected in hypoxic GMSCs. In conclusion, in periodontitis patients, the expression of miRNA-20a, miRNA-30e, and miRNA-93 increased, but a decreased expression of NFAT5 mRNA was detected. In addition, GMSCs under hypoxic conditions upregulate the HIF-1α and increase miRNA-20a (p = 0.049) expression. This study explores the role of inflammatory and hypoxia-related miRNAs and their target genes in periodontitis and GMSCs. It is crucial to determine the potential therapeutic target of these miRNAs and hypoxia during the periodontal immune–inflammatory response, which should be analyzed in greater depth in future studies

NFAT5 is up-regulated by hypoxia: possible implications in preeclampsia and intrauterine growth restriction

During gestation, low oxygen environment is a major determinant of early placentation process, while persistent placental hypoxia leads to pregnancy-related complications such as preeclampsia (PE) and intrauterine growth restriction (IUGR). PE affects 5%-8% of all pregnancies worldwide and is a cause of maternal and fetal morbidity and mortality. During placental development, persistent hypoxia due to poor trophoblast invasion and reduced uteroplacental perfusion leads to maternal endothelial dysfunction and clinical manifestation of PE. Here we hypothesized that nuclear factor of activated T cells-5 (NFAT5), a well-known osmosensitive renal factor and recently characterized hypoxia-inducible protein, is also activated in vivo in placentas of PE and IUGR complications as well as in the in vitro model of trophoblast hypoxia. In JAR cells, low oxygen tension (1% O2) induced NFAT5 mRNA and increased its nuclear abundance, peaking at 16 h. This increase did not occur in parallel with the earlier HIF1A induction. Real-time PCR and Western blot analysis confirmed up-regulation of NFAT5 mRNA and NFAT5 nuclear content in human preeclamptic placentas and in rabbit placentas of an experimentally induced IUGR model, as compared with the control groups. In vitro lambda protein phosphatase (lambda PPase) treatment revealed that increased abundance of NFAT5 protein in nuclei of either JAR cells (16 h of hypoxia) or PE and IUGR placentas is at least partially due to NFAT5 phosphorylation. NFAT5 downstream targets aldose reductase (AR) and sodium-myo-inositol cotransporter (SMIT; official symbol SLC5A3) were not significantly upregulated either in JAR cells exposed to hypoxia or in placentas of PE- and IUGR-complicated pregnancies, suggesting that hypoxia-dependent activation of NFAT5 serves as a separate function to its tonicity-dependent stimulation. In conclusion, we propose that NFAT5 may serve as a novel marker of placental hypoxia and ischemia independently of HIF1A

NFAT5 and HIF-1α are independently up-regulated by hypoxia.

<p>HEK293 cells cultured at 300 mosmol were transfected with control (C), NFAT5 or HIF-1α siRNA. 48 hrs after transfection the cells were cultured in normoxia (N) or 8 hrs of hypoxia (H). <b>A.</b> Protein abundance of NFAT5 and HIF-1α were studied by Western blot in cells transfected with siRNA against NFAT5. <b>B.</b> Protein abundance of NFAT5 and HIF-1α were studied by Western blot in cells transfected with siRNA against HIF-1α. A representative picture is shown in the upper section. Bar graph represents Mean ± SEM. *, P<0.05; n = 5.</p

NFAT5 has a protective role against hypoxia.

<p>HEK293 cells cultured at 300 mosmol were transfected with control and NFAT5 siRNA. 48 hrs after transfection the cells were exposed for 8 hrs to 2.5% PO₂. <b>A.</b> Western blot of NFAT5. <b>B.</b> LDH activity was assayed in cell culture media and cell lysate by spectrometric determination of NADH. <b>C.</b> Western blot of M30. <b>D.</b> Western blot of Cleaved caspase-3. Bar graph represents Mean ± SEM. *, P<0.05; n = 5.</p