Levels of Small Extracellular Vesicles Containing hERG-1 and Hsp47 as Potential Biomarkers for Cardiovascular Diseases.

The diagnosis of cardiovascular disease (CVD) is still limited. Therefore, this study demonstrates the presence of human ether-a-go-go-related gene 1 (hERG1) and heat shock protein 47 (Hsp47) on the surface of small extracellular vesicles (sEVs) in human peripheral blood and their association with CVD. In this research, 20 individuals with heart failure and 26 participants subjected to cardiac stress tests were enrolled. The associations between hERG1 and/or Hsp47 in sEVs and CVD were established using Western blot, flow cytometry, electron microscopy, ELISA, and nanoparticle tracking analysis. The results show that hERG1 and Hsp47 were present in sEV membranes, extravesicularly exposing the sequences 430AFLLKETEEGPPATE445 for hERG1 and 169ALQSINEWAAQTT- DGKLPEVTKDVERTD196 for Hsp47. In addition, upon exposure to hypoxia, rat primary cardiomyocytes released sEVs into the media, and human cardiomyocytes in culture also released sEVs containing hERG1 (EV-hERG1) and/or Hsp47 (EV-Hsp47). Moreover, the levels of sEVs increased in the blood when cardiac ischemia was induced during the stress test, as well as the concentrations of EV-hERG1 and EV-Hsp47. Additionally, the plasma levels of EV-hERG1 and EV-Hsp47 decreased in patients with decompensated heart failure (DHF). Our data provide the first evidence that hERG1 and Hsp47 are present in the membranes of sEVs derived from the human cardiomyocyte cell line, and also in those isolated from human peripheral blood. Total sEVs, EV-hERG1, and EV-Hsp47 may be explored as biomarkers for heart diseases such as heart failure and cardiac ischemia.

The Ischemia and Reperfusion Injury Involves the Toll-Like Receptor-4 Participation Mainly in the Kidney Cortex.

BACKGROUND/AIMS: The renal inflammatory response and kidney regeneration in ischemia-reperfusion injury (IRI) are associated with Toll-like receptor 4 (TLR4). Here we study the role of TLR4 during IRI in the renal cortex and medulla separately, using wild-type (TLR4-WT) and Knockout (TLR4-KO) TLR4 mice. METHODS: We used 30 minutes of bilateral renal ischemia, followed by 48 hours of reperfusion in C57BL/6 mice. We measured the expression of elements associated with kidney injury, inflammation, macrophage polarization, mesenchymal transition, and proteostasis in the renal cortex and medulla by qRT-PCR and Western blot. In addition, we studied kidney morphology by H/E and PAS. RESULTS: Renal ischemia (30min) and reperfusion (48hrs) induced the mRNA and protein of TLR4 in the renal cortex. In addition, Serum Creatinine (SCr), blood urea nitrogen (BUN), Neutrophil gelatinase-associated lipocalin (NGAL), and acute tubular necrosis (ATN) were increased in TLR4-WT by IRI. Interestingly, the SCr and BUN had normal levels in TLR-KO during IRI. However, ATN and high levels of NGAL were present in the kidneys of TLR4-KO mice. The pro-inflammatory (IL-6 and TNF-α) and anti-inflammatory (Foxp3 and IL-10) markers increased by IRI only in the cortex of TLR4-WT but not in TLR4-KO mice. Furthermore, the M1 (CD38 and Frp2) and M2 (Arg-I, Erg-2, and c-Myc) macrophage markers increased by IRI only in the cortex of TLR4-WT. The TLR4-KO blunted the IRI-upregulation of M1 but not the M2 macrophage polarization. Vimentin increased in the renal cortex and medulla of TLR4-WT animals but not in the cortex of TLR4-KO mice. In addition, iNOS and clusterin were increased by IRI only in the cortex of TLR4-WT, and the absence of TLR4 inhibited only clusterin upregulation. Finally, Hsp27 and Hsp70 protein levels increased by IRI in the cortex and medulla of TLR4-WT and TRL4-KO lost the IRI-upregulation of Hsp70. In summary, TLR4 participates in renal ischemia and reperfusion through pro-inflammatory and anti-inflammatory responses inducing impaired kidney function (SCr and BUN). However, the IRI-upregulation of M2 macrophage markers (cortex), iNOS (cortex), IL-6 (medulla), vimentin (medulla), and Hsp27 (cortex and medulla) were independent of TLR4. CONCLUSION: The TLR4 inactivation during IRI prevented the loss of renal function due to the inactivation of inflammation response, avoiding M1 and preserving the M2 macrophage polarization in the renal cortex.

1400W Prevents Renal Injury in the Renal Cortex But Not in the Medulla in a Murine Model of Ischemia and Reperfusion Injury.

BACKGROUND/AIMS: Acute kidney injury (AKI) carries high morbidity and mortality, and the inducible nitric oxide synthase (iNOS) is a potential molecular target to prevent kidney dysfunction. In previous work, we reported that the pharmacological inhibitions of iNOS before ischemia/reperfusion (I/R) attenuate the I/R-induced AKI in mice. Here, we study the iNOS inhibitor 1400W [N-(3-(Aminomethyl)benzyl] acetamide, which has been described to be much more specific to iNOS inhibition than other compounds. METHODS: We used 30 minutes of bilateral renal ischemia, followed by 24 hours of reperfusion in Balb/c mice. 1400w (10 mg/kg i.p) was applied before I/R injury. We measured the expression of elements associated with kidney injury, inflammation, macrophage polarization, mesenchymal transition, and nephrogenic genes by qRT-PCR in the renal cortex and medulla. The Periodic Acid-Schiff (PAS) was used to study the kidney morphology. RESULTS: Remarkably, we found that 1400W affects the renal cortex and medulla in different ways. Thus, in the renal cortex, 1400W prevented the I/R-upregulation of 1. NGAL, Clusterin, and signs of morphological damage; 2. IL-6 and TNF-α; 3. TGF-ß; 4. M2(Arg1, Erg2, cMyc) and M1(CD38, Fpr2) macrophage polarization makers; and 5. Vimentin and FGF2 levels but not in the renal medulla. CONCLUSION: 1400W conferred protection in the kidney cortex compared to the kidney medulla. The present investigation provides relevant information to understand the opportunity to use 1400W as a therapeutic approach in AKI treatment.

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.

Glutathione S-Transferase and Clusterin, New Players in the Ischemic Preconditioning Renal Protection in a Murine Model of Ischemia and Reperfusion.

BACKGROUND/AIMS: Renal ischemia and reperfusion injury (IRI) involves oxidative stress, disruption of microvasculature due to endothelial cell damage, loss of epithelial cell polarity secondary to cytoskeletal alterations, inflammation, and the subsequent transition into a mesenchymal phenotype. Ischemic preconditioning (IPC) has been proposed as a therapeutic strategy to avoid/ameliorate the IRI. Since previous results showed that IPC could have differential effects in kidney cortex vs. kidney medulla, in the present study we analyzed the effectiveness and molecular mechanisms implicated in IPC in both kidney regions. METHODS: We evaluated 3 experimental groups of BALB/c male mice: control (sham surgery); renal ischemia (30 min) by bilateral occlusion of the renal pedicle and reperfusion (48 hours) (I/R); and renal IPC (two cycles of 5 min of ischemia and 5 min of reperfusion) applied just before I/R. Acute kidney injury was evaluated by glomerular filtration rate (GFR), Neutrophil Gelatinase-Associated Lipocalin (NGAL) blood level, and histologic analysis. Oxidative stress was studied measurement the Glutathione S-Transferase (GST) activity, GSH/GSSG ratio, and lipoperoxidation levels. Inflammatory mediators (IL-1ß, IL-6, Foxp3, and IL-10) were quantified by qRT-PCR. The endothelial (PECAM-1), epithelial (AQP-1), mesenchymal (Vimentin, Fascin, and Hsp47), iNOS, clusterin, and Hsp27 expression were evaluated (qRT-PCR and/or Western blot). RESULTS: The IPC protocol prevented the decrease of GFR, reduced the plasma NGAL, and ameliorated morphological damage in the kidney cortex after I/R. The IPC also prevented the downregulation of GST activity, lipoperoxidation and ameliorated the oxidized glutathione. In addition, IPC prevented the upregulation of vimentin, fascin, and Hsp47, which was associated with the prevention of the downregulation of AQP1 after I/R. The protective effect of IPC was associated with the upregulation of Hsp27, Foxp3, and IL-10 expression in the renal cortex. However, the upregulation of iNOS, IL-1ß, IL-6, and clusterin by I/R were not modified by IPC. CONCLUSION: IPC conferred better protection in the kidney cortex as compared to the kidney medulla. The protective effect of IPC was associated with amelioration of oxidative stress, tubular damage, and the induction of markers of Treg lymphocytes activity in the cortical region. Further studies are needed to evaluate if lower tubular cell stress/damage after I/R may explain the preferential induction of Treg response in the kidney cortex induced by IPC.

Emerging new role of NFAT5 in inducible nitric oxide synthase in response to hypoxia in mouse embryonic fibroblast cells.

We previously described the protective role of the nuclear factor of activated T cells 5 (NFAT5) during hypoxia. Alternatively, inducible nitric oxide synthase (iNOS) is also induced by hypoxia. Some evidence indicates that NFAT5 is essential for the expression of iNOS in Toll-like receptor-stimulated macrophages and that iNOS inhibition increases NFAT5 expression in renal ischemia-reperfusion. Here we studied potential NFAT5 target genes stimulated by hypoxia in mouse embryonic fibroblast (MEF) cells. We used three types of MEF cells associated with NFAT5 gene: NFAT5 wild type (MEF-NFAT5+/+), NFAT5 knockout (MEF-NFAT5-/-), and NFAT5 dominant-negative (MEF-NFAT5Δ/Δ) cells. MEF cells were exposed to 21% or 1% O2 in a time course curve of 48 h. We found that, in MEF-NFAT5+/+ cells exposed to 1% O2, NFAT5 was upregulated and translocated into the nuclei, and its transactivation domain activity was induced, concomitant with iNOS, aquaporin 1 (AQP-1), and urea transporter 1 (UTA-1) upregulation. Interestingly, in MEF-NFAT5-/- or MEF-NFAT5Δ/Δ cells, the basal levels of iNOS and AQP-1 expression were strongly downregulated, but not for UTA-1. The upregulation of AQP-1, UTA-1, and iNOS by hypoxia was blocked in both NFAT5-mutated cells. The iNOS induction by hypoxia was recovered in MEF-NFAT5-/- MEF cells, when recombinant NFAT5 protein expression was reconstituted, but not in MEF-NFAT5Δ/Δ cells, confirming the dominant-negative effect of MEF-NFAT5Δ/Δ cells. We did not see the rescue effect on AQP-1 expression. This work provides novel and relevant information about the signaling pathway of NFAT5 during responses to oxygen depletion in mammalian cells and suggests that the expression of iNOS induced by hypoxia is dependent on NFAT5.

l-NIL prevents the ischemia and reperfusion injury involving TLR-4, GST, clusterin, and NFAT-5 in mice.

On renal ischemia-reperfusion (I/R) injury, recruitment of neutrophils during the inflammatory process promotes local generation of oxygen and nitrogen reactive species, which, in turn, are likely to exacerbate tissue damage. The mechanism by which inducible nitric oxide synthase (iNOS) is involved in I/R has not been elucidated. In this work, the selective iNOS inhibitor l- N6-(1-iminoethyl)lysine (l-NIL) and the NOS substrate l-arginine were employed to understand the role of NOS activity on the expression of particular target genes and the oxidative stress elicited after a 30-min of bilateral renal ischemia, followed by 48-h reperfusion in Balb/c mice. The main findings of the present study were that pharmacological inhibition of iNOS with l-NIL during an I/R challenge of mice kidney decreased renal injury, prevented tissue loss of integrity, and improved renal function. Several novel findings regarding the molecular mechanism by which iNOS inhibition led to these protective effects are as follows: 1) a prevention of the I/R-related increase in expression of Toll-like receptor 4 (TLR-4), and its downstream target, IL-1ß; 2) reduced oxidative stress following the I/R challenge; noteworthy, this study shows the first evidence of glutathione S-transferase (GST) inactivation following kidney I/R, a phenomenon fully prevented by iNOS inhibition; 3) increased expression of clusterin, a survival autophagy component; and 4) increased expression of nuclear factor of activated T cells 5 (NFAT-5) and its target gene aquaporin-1. In conclusion, prevention of renal damage following I/R by the pharmacological inhibition of iNOS with l-NIL was associated with the inactivation of proinflammatory pathway triggered by TLR-4, oxidative stress, renoprotection (autophagy inactivation), and NFAT-5 signaling pathway.

Oxidative damage and nitric oxide synthase induction by surgical uteroplacental circulation restriction in the rabbit fetal heart.

OBJECTIVE: This study investigated the role of oxidative damage and nitric oxide (NO) synthases in the fetal heart using a model of intrauterine growth restriction induced by uteroplacental circulation restriction (UCR). METHODS: New Zealand white rabbits kept under 12-h light cycles, with food and water provided ad libitum, were subjected at day 25 of pregnancy to 40-50% uteroplacental artery ligation. We analyzed the gene expression of enzymes linked to nitric oxide synthesis (iNOS, eNOS, HO-1, and ARG-2), hypoxia inducible factor 1 alpha (HIF-1α), and the state of oxidative stress (protein carbonyl levels) in fetal heart homogenates. Additionally, we studied the histological morphology of the fetal heart. RESULTS: We found that fetal growth restriction was associated with a significant reduction in heart weight but a normal heart/body weight ratio in UCR animals. Hematoxylin and eosin staining showed normal left and right ventricular thickness but increased vessel dilatation with hyperemia in the hearts of the UCR group. We observed HIF-1α, eNOS, p-eNOS, and iNOS induction concomitant with intensified protein carbonyl levels but observed no changes in HO-1 or ARG-2 expression, suggesting increased NO and oxidative stress in the hearts of UCR animals. CONCLUSION: Uteroplacental circulation restriction increased NO-linked enzymes, oxidative damage, and dilated coronary vessels in fetal hearts. © 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Nitric oxide synthase and changes in oxidative stress levels in embryonic kidney observed in a rabbit model of intrauterine growth restriction.

OBJECTIVE: This work aimed to study the effect of uteroplacental circulation restriction on endothelial kidney damage in a fetal rabbit model. METHODS: New Zealand rabbits were subjected to 40% to 50% of uteroplacental artery ligation at day 25 of pregnancy. After 5 days, surviving fetuses were harvested by cesarean section. The gene and protein expressions of selected enzymes associated with nitric oxide production and oxidative stress were analyzed in fetal kidney homogenates. RESULTS: The placenta weight (6.06 ± 0.27, p < 0.0319) and fetal body (19.90 ± 1.03, p < 0.0001) were significantly reduced in the uteroplacental circulation restriction group. The kidneys from restricted fetuses presented a mild vascular congestion and glomerular capillary congestion, without inflammation or hypertrophy. We found endothelial nitric oxide synthase phosphorylation inhibition (0.23 ± 0.13, p < 0.012) and arginase-2 (0.29 ± 0.14, p < 0.023) protein induction in fetal kidneys of the circulation restriction group. Finally, the kidneys from circulation-restricted fetuses showed increased inducible nitric oxide synthase messenger RNA (mRNA) (2.68 ± 0.24, p < 0.01) and reduced heme oxygenase-1 mRNA (23 ± 1.3, p < 0.003), with increased reactive oxygen species (1.69 ± 0.09, p < 0.001) and nitrotyrosine protein (1.74 ± 0.28, p < 0.003) levels, without changes in Nox mRNA. CONCLUSION: We describe significant deregulation of vascular activity and oxidative damage in kidneys of fetal rabbits that have been exposed to restriction of the uterine circulation. © 2016 John Wiley & Sons, Ltd.

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 up-regulated 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.

[Incidence and consequences of acute kidney injury among patients admitted to critical care units]. / Incidencia de la injuria renal aguda en unidad de paciente crítico y su mortalidad a 30 días y un año.

BACKGROUND: Acute Kidney Injury (AKI) increases morbidity, mortality and hospital stay in critical patients units (CPU). AIM: To determine the incidence and mortality of AKI in CPU. MATERIAL AND METHODS: Review of electronic medical records of 1,769 patients aged 61 ± 20 years (47% males) discharged from a CPU during one year. Acute Kidney Injury diagnosis and severity was established using the Acute Kidney Injury Network (AKIN) criteria. RESULTS: A history of hypertension and Diabetes Mellitus was present in 44 and 22% of patients, respectively. APACHE II and SOFA scores were 14.6 ± 6.8 and 3.6 ± 2.1 respectively. AKI incidence was 28.9% (stage I, 16.7%, stage II, 5.3% and stage III, 6.9%). Mortality during the first 30 days and during the first year was 8.1 and 20.0% respectively. Patients with stage III AKI had the highest mortality (23.8 and 40.2% at 30 days and one year respectively). Compared with patients without AKI, the Odds ratio for mortality at 30 days and one year of patients with AKI stage III was 3.7 and 2.5, respectively. CONCLUSIONS: Thirty percent of patients admitted to UPC develop an AKI, which influences 30 days and one year mortality.

Phospholipase C-gamma1 is involved in signaling the activation by high NaCl of the osmoprotective transcription factor TonEBP/OREBP.

High NaCl elevates activity of the osmoprotective transcription factor TonEBP/OREBP by increasing its phosphorylation, transactivating activity, and localization to the nucleus. We investigated the possible role in this activation of phospholipase C-gamma1 (PLC-gamma1), which has a predicted binding site at TonEBP/OREBP-phospho-Y143. We find the following. (i) Activation of TonEBP/OREBP transcriptional activity by high NaCl is reduced in PLC-gamma1 null cells and in HEK293 cells in which PLC-gamma1 is knocked down by a specific siRNA. (ii) High NaCl increases phosphorylation of TonEBP/OREBP at Y143. (iii) Wild-type PLC-gamma1 coimmunoprecipitates with wild-type TonEBP/OREBP but not TonEBP/OREBP-Y143A, and the coimmunoprecipitation is increased by high NaCl. (iv) PLC-gamma1 is part of the protein complex that associates with TonEBP/OREBP at its DNA binding site. (v) Knockdown of PLC-gamma1 or overexpression of a PLC-gamma1-SH3 deletion mutant reduces high NaCl-dependent TonEBP/OREBP transactivating activity. (vi) Nuclear localization of PLC-gamma1 is increased by high NaCl. (vii) High NaCl-induced nuclear localization of TonEBP/OREBP is reduced if cells lack PLC-gamma1, if PLC-gamma1 mutated in its SH2C domain is overexpressed, or if Y143 in TonEBP/OREBP is mutated to alanine. (viii) Expression of recombinant PLC-gamma1 restores nuclear localization of wild-type TonEBP/OREBP in PLC-gamma1 null cells but not of TonEBP/OREBP-Y143A. (ix) The PLC-gamma1 phospholipase inhibitor U72133 inhibits nuclear localization of TonEBP/OREBP but not the increase of its transactivating activity. We conclude that, when NaCl is elevated, TonEBP/OREBP becomes phosphorylated at Y143, resulting in binding of PLC-gamma1 to that site, which contributes to TonEBP/OREBP transcriptional activity, transactivating activity, and nuclear localization.

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 Drug-Induced Renal Injury Consortium (DIRECT) study (NCT02159209), an international, multicenter, observational cohort study of enriched clinically adjudicated DI-AKI cases. Cases met the primary inclusion criteria if the patient was exposed to at least 1 nephrotoxic drug for a minimum of 24 hours prior to 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: A total of 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%), nonsteroidal antiinflammatory 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 (SCr) trends, and contrast media as significant predictors of DI-AKI with good performance (ROC AUC 0.86). Conclusion: 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.

IL-17A levels increase in the infarcted region of the left ventricle in a rat model of myocardial infarction.

Th17 cells, a recently described subtype of CD4+ effector lymphocytes, have been linked to cell-mediated autoimmune and inflammatory diseases as well as to cardiovascular diseases. However, the participation of IL-17A in myocardial ischemic injury has not been clearly defined. We therefore conducted the present study to evaluate IL-17A and Th17-related cytokine levels in a rat model of myocardial infarction (MI). MI was induced in male Sprague Dawley rats by coronary artery ligation. Controls were sham-operated (Sh) or non-operated (C). Blood and samples from the left ventricle (LV) were collected at weeks 1 and 4 post-MI. At week 1, MI animals exhibited increased IL-6, IL-23 and TGF-ß mRNA levels with no apparent change in IL-17 mRNA or protein levels in whole LV. Only TGF-ß mRNA remained elevated at week 4 post-MI. However, further analysis revealed that IL-17A mRNA and protein levels as well as IL-6 and IL-23 mRNA were indeed increased in the infarcted region, though not in the remote non infarcted region of the LV, except for IL-23 mRNA. The increased expression of IL-17A and Th17-related cytokines in the infarcted region of LV, suggests that this proinflammatory pathway might play a role in early stages of post MI cardiac remodelling.

Aminoguanidine Prevents the Oxidative Stress, Inhibiting Elements of Inflammation, Endothelial Activation, Mesenchymal Markers, and Confers a Renoprotective Effect in Renal Ischemia and Reperfusion Injury.

Oxidative stress produces macromolecules dysfunction and cellular damage. Renal ischemia-reperfusion injury (IRI) induces oxidative stress, inflammation, epithelium and endothelium damage, and cessation of renal function. The IRI is an inevitable process during kidney transplantation. Preliminary studies suggest that aminoguanidine (AG) is an antioxidant compound. In this study, we investigated the antioxidant effects of AG (50 mg/kg, intraperitoneal) and its association with molecular pathways activated by IRI (30 min/48 h) in the kidney. The antioxidant effect of AG was studied measuring GSSH/GSSG ratio, GST activity, lipoperoxidation, iNOS, and Hsp27 levels. In addition, we examined the effect of AG on elements associated with cell survival, inflammation, endothelium, and mesenchymal transition during IRI. AG prevented lipid peroxidation, increased GSH levels, and recovered the GST activity impaired by IRI. AG was associated with inhibition of iNOS, Hsp27, endothelial activation (VE-cadherin, PECAM), mesenchymal markers (vimentin, fascin, and HSP47), and inflammation (IL-1ß, IL-6, Foxp3, and IL-10) upregulation. In addition, AG reduced kidney injury (NGAL, clusterin, Arg-2, and TFG-ß1) and improved kidney function (glomerular filtration rate) during IRI. In conclusion, we found new evidence of the antioxidant properties of AG as a renoprotective compound during IRI. Therefore, AG is a promising compound to treat the deleterious effect of renal IRI.

Urinary Extracellular Vesicles as a Source of NGAL for Diabetic Kidney Disease Evaluation in Children and Adolescents With Type 1 Diabetes Mellitus.

Background: Tubular damage has a role in Diabetic Kidney Disease (DKD). We evaluated the early tubulointerstitial damage biomarkers in type-1 Diabetes Mellitus (T1DM) pediatric participants and studied the correlation with classical DKD parameters. Methods: Thirty-four T1DM and fifteen healthy participants were enrolled. Clinical and biochemical parameters [Glomerular filtration Rate (GFR), microalbuminuria (MAU), albumin/creatinine ratio (ACR), and glycated hemoglobin A1c (HbA1c)] were evaluated. Neutrophil gelatinase-associated lipocalin (NGAL), Hypoxia-inducible Factor-1α (HIF-1α), and Nuclear Factor of Activated T-cells-5 (NFAT5) levels were studied in the supernatant (S) and the exosome-like extracellular vesicles (E) fraction from urine samples. Results: In the T1DM, 12% had MAU >20 mg/L, 6% ACR >30 mg/g, and 88% had eGFR >140 ml/min/1.72 m2. NGAL in the S (NGAL-S) or E (NGAL-E) fraction was not detectable in the control. The NGAL-E was more frequent (p = 0.040) and higher (p = 0.002) than NGAL-S in T1DM. The T1DM participants with positive NGAL had higher age (p = 0.03), T1DM evolution (p = 0.03), and serum creatinine (p = 0.003) than negative NGAL. The NGAL-E correlated positively with tanner stage (p = 0.0036), the median levels of HbA1c before enrollment (p = 0.045) and was independent of ACR, MAU, and HbA1c at the enrollment. NFAT5 and HIF-1α levels were not detectable in T1DM or control. Conclusion: Urinary exosome-like extracellular vesicles could be a new source of early detection of tubular injury biomarkers of DKD in T1DM patients.

Association between p53 codon 72 genetic polymorphism and tobacco use and lung cancer risk.

Lung cancer (LCa) is the leading cause of death by cancer in men. Genetic and environmental factors play a synergistic role in its etiology. We explore in 111 lung cancer cases and 133 unrelated noncancer controls the gene-environment interaction (G x E) between p53cd72 polymorphism variants and smoking and the effect on LCa risk in two kinds of case-control designs. We assessed the interaction odds ratio (IOR) using an adjusted unconditional logistic model. We found a significant and positive interaction association between Pro* allele carriers and smoking habits in both case-control and case-only designs: IOR = 3.90 (95% confidence interval [CI] = 1.10-13.81) and 3.05 (95% CI = 1.63-5.72), respectively. These exploratory results suggest a synergistic effect of the smoking habit and the susceptibility of the Pro allele on lung cancer risk compared with each risk factor alone.

Joint effect among p53, CYP1A1, GSTM1 polymorphism combinations and smoking on prostate cancer risk: an exploratory genotype-environment interaction study.

AIM: To assess the role of several genetic factors in combination with an environmental factor as modulators of prostate cancer risk. We focus on allele variants of low-penetrance genes associated with cell control, the detoxification processes and smoking. METHODS: In a case-control study we compared people carrying p53cd72 Pro allele, CYP1A1 M1 allele and GSTM1 null genotypes with their prostate cancer risk. RESULTS: The joint risk for smokers carrying Pro* and M1*, Pro* and GSTM1null or GSTM1 null and CYP1A1 M1* variants was significantly higher (odds ratio [OR]: 13.13, 95% confidence interval [CI]: 2.41-71.36; OR: 3.97, 95% CI: 1.13-13.95 and OR: 6.87, 95% CI: 1.68-27.97, respectively) compared with that for the reference group, and for non-smokers was not significant. OR for combinations among p53cd72, GSTM1 and CYP1A1 M1 in smokers were positively and significantly associated with prostate cancer risk compared with non-smokers and compared with the putative lowest risk group (OR: 8.87, 95% CI: 1.25-62.71). CONCLUSION: Our results suggest that a combination of p53cd72, CYP1A1, GSTM1 alleles and smoking plays a significant role in modified prostate cancer risk on the study population, which means that smokers carrying susceptible genotypes might have a significantly higher risk than those carrying non-susceptible genotypes.

Spironolactone inhibits the activity of the Na+/H+ exchanger in the aorta of mineralocorticoid-induced hypertensive rats.

INTRODUCTION: Aldosterone can induce changes in the expression or activity of Na(+)/H(+) exchanger isoform 1 (NHE-1) in vascular smooth muscle cells. We aimed to clarify whether chronic mineralocorticoid receptor activation exerts an effect on the activity of NHE-1 in the aorta of mineralocorticoid-induced hypertensive rats. METHODS: Uninephrectomized male Sprague-Dawley rats received subcutaneously 10 mg/week of desoxycorticosterone (DOCA) with or without 20 mg/kg of spironolactone, or vehicle alone (n = 20). After four weeks of treatment, the animals were sacrificed; the aorta was excised for subsequent studies, including histological analysis, RT-PCR, Western blot, measurement of NHE-1 activity and vascular contractility in the presence or absence of the selective NHE-1 inhibitor ethyl-isopropyl amiloride (EIPA). RESULTS: Chronic DOCA treatment increased the NHE-1 activity, systolic and diastolic blood pressure, and aortic wall thickness. All these effects were prevented by co-treatment with Spironolactone (p < 0.05). Phenylephrine-induced vascular contractility was significantly reduced in the DOCA group when EIPA was added in the media (p < 0.05). No significant differences in NHE-1 mRNA or protein levels were detected between groups. CONCLUSIONS: Chronic DOCA administration induced functional and morphological alterations in the rat aorta that are partially explained by enhanced NHE-1 activity and prevented by spironolactone. However, we did not observe changes in the NHE-1 transcript or protein levels, suggesting that the effect may be due to post-transcriptional modifications induced by mineralocorticoid receptor activation.

Intravenous administration of bone marrow-derived mesenchymal stem cells induces a switch from classical to atypical symptoms in experimental autoimmune encephalomyelitis.

Potent immunosuppressive and regenerative properties of mesenchymal stem cells (MSCs) position them as a novel therapy for autoimmune diseases. This research examines the therapeutic effect of MSCs administration at different disease stages in experimental autoimmune encephalomyelitis (EAE). Classical and atypical scores of EAE, associated with Th1 and Th17 response, respectively, and also Treg lymphocytes, were evaluated. MSCs administration at the onset (EAE+MSConset) induced an important amelioration of the clinical signs and less lasting effect at the peak of EAE (EAE+MSCpeak). No effect was observed when MSCs were applied after EAE stabilization (EAE+MSClate). Surprisingly, EAE atypical signs were detected in EAE+MSCpeak and EAE+MSClate mice. However, no correlation was found in Th17/Th1 ratio. Interestingly, regardless of time administration, MSCs significantly reduced IL-6 and also T-bet, RORγT, and Foxp3 mRNA levels in brain samples of EAE mice. The downregulation of IL-6 could restore the well-functioning of the blood-brain barrier of EAE mice, correlated with a decreased number of brain infiltrating leukocytes. These results suggest that the inflammatory status is important to be considered for administering MSCs in autoimmune pathologies, leading to a further research to clarify the effect of MSCs for multiple sclerosis.