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.

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.

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.

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.

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.

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.

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.

En el Infarto agudo al miocardio los niveles plasmáticos de microvesículas extracelulares se elevan más precozmente que el aumento de la Troponina-I / Plasma levels of extracellular macrovesicles are elevated earlier than troponin-I in patients with myocardial infarction

Introducción: La Troponina I (TnI) plasmática es el biomarcador "Gold" estándar utilizado en diagnóstico de Infarto Agudo al Miocardio (IAM), indicando necrosis cardíaca. Las microvesículas extracelulares (MVEC), participan en comunicación celular, por lo que estudiar su distribución entregaría información respecto del evento isquémico, antesala del infarto. Objetivo: Estudiar las MVECs plasmáticas en pacientes con Síndrome Coronario Agudo (SCA) y compararlas con los niveles de TnI. Métodos: Plasma de 22 pacientes controles se recolectó 0-2hrs post-ingreso a urgencia. Plasma de 45 pacientes SCA se recolectó 0-2, 6-8 y 10-14hrs post ingreso, junto con la toma de muestra para estudio de TnI. Las MVECs plasmáticas fueron enriquecidas mediante kit comercial. La determinación de la concentración y tamaño MVECs se realizó por NTA (Nanoparticles Tracking Assay) usando el equipo Nanosight. Resultados: La concentración promedio de MVECs 0-2 hrs post ingreso fue 7,2 veces superior en plasma de pacientes con SCA vs controles y la moda del tamaño disminuyó en pacientes con SCA. La TnI no mostró diferencias significativas en 0-2 hrs post ingreso en el grupo estudiado. La concentración de las MVEC disminuyó significativamente después de 10-14 hrs post ingreso, mientras que la concentración promedio TnI se mantuvo invariable demostrando el aumento de MVECs previo al incremento de TnI. Conclusión. El aumento de MVECs previo al incremento de la TnI en pacientes infartados, sugiere que las MVECs aumentan en la fase previa del IAM, como respuesta al daño tisular. Actualmente, estudiamos el contenido molecular de las MVECs, para establecer un método diagnóstico del Síndrome Coronario Agudo basado en MVECs.

Background: Troponin I (TnI) is the gold standard used to establish the diagnosis of myocardial infarction (AMI), indicating the presence of myocardial necrosis. Extracellular micro vesicles are involved in cellular communication. Their distribution may provide information relating to the development of AMI in patients with acute coronary syndromes (ACS) Aim: to study plasma levels of ECMV compared to those of TnI in patients with ACS. Methods: The plasma levels of TnI and ECMV from 22 control patients coming to the emergency units was compared to plasma from 45 patients with ACS. Levels of both parameters were determined 0-2, 6-8 and 10-14 hours post admission. ECMVs were enriched by means of a commercial kit. Concentration and size of ECMV was determined by NTA (Nanoparticles tracking assay) using the Nanosight equipment. Results: Plasma concentration of ECMV was 7.2 times higher than that of TnI 0-2 hrs post admission. The mode of ECMV size was lower in patients with ACS. Concentration of ECMV had decreased significantly 10-14 hrs post admission, whereas the TnI levees remained stable. Conclusion: The increase in ECMV earlier than TnI in AMI suggests that ECMV are elevated in the pre-AMI phase, as a response to early tissue damage. A study of cellular content of ECMV, being carried out, may lead to develop a method for the early diagnosis of AMI in patients with ACS.

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.

[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.

Placental Aromatase Is Deficient in Placental Ischemia and Preeclampsia.

INTRODUCTION: Preeclampsia is a maternal hypertensive disorder with uncertain etiology and a leading cause of maternal and fetal mortality worldwide, causing nearly 40% of premature births delivered before 35 weeks of gestation. The first stage of preeclampsia is characterized by reduction of utero-placental blood flow which is reflected in high blood pressure and proteinuria during the second half of pregnancy. In human placenta androgens derived from the maternal and fetal adrenal glands are converted into estrogens by the enzymatic action of placental aromatase. This implies that alterations in placental steroidogenesis and, subsequently, in the functionality or bioavailability of placental aromatase may be mechanistically involved in the pathophysiology of PE. METHODS: Serum samples were collected at 32-36 weeks of gestation and placenta biopsies were collected at time of delivery from PE patients (n = 16) and pregnant controls (n = 32). The effect of oxygen tension on placental cells was assessed by incubation JEG-3 cells under 1% and 8% O2 for different time periods, Timed-mated, pregnant New Zealand white rabbits (n = 6) were used to establish an in vivo model of placental ischemia (achieved by ligature of uteroplacental vessels). Aromatase content and estrogens and androgens concentrations were measured. RESULTS: The protein and mRNA content of placental aromatase significantly diminished in placentae obtained from preeclamptic patients compared to controls. Similarly, the circulating concentrations of 17-ß-estradiol/testosterone and estrone/androstenedione were reduced in preeclamptic patients vs. controls. These data are consistent with a concomitant decrease in aromatase activity. Aromatase content was reduced in response to low oxygen tension in the choriocarcinoma JEG-3 cell line and in rabbit placentae in response to partial ligation of uterine spiral arteries, suggesting that reduced placental aromatase activity in preeclamptic patients may be associated with chronic placental ischemia and hypoxia later in gestation. CONCLUSIONS: Placental aromatase expression and functionality are diminished in pregnancies complicated by preeclampsia in comparison with healthy pregnant controls.

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

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.

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.

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.

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.

Longitudinal FGF23 and Klotho axis characterization in children treated with chronic peritoneal dialysis.

BACKGROUND: Fibroblast Growth Factor-23 (FGF23) and cofactor Klotho are key regulators of mineral metabolism in chronic kidney disease (CKD), but little is known about the mechanisms that regulate their production. This study evaluates longitudinal changes of FGF23 and Klotho levels and their regulatory factors in children on chronic peritoneal dialysis (PD). METHODS: FGF23, Klotho, 25(OH) vitamin D, 1,25-dihydroxyvitamin D and parathyroid hormone (PTH) plasma concentrations were measured during 1 year of follow-up in PD children. Anthropometric and dialytical parameters were evaluated in addition to mineral metabolism variables. RESULTS: Thirty-one patients under chronic PD were followed for 12 months. FGF23 mean plasma levels at Month 1 were significantly increased compared with controls, 215.1 ± 303.6 versus 9.4 ± 5.7 pg/mL, respectively (P < 0.001). Baseline Klotho levels were 41% lower in patients compared with controls, 132.1 ± 58 versus 320 ± 119.4 pg/mL, respectively (P < 0.001), and did not correlate with FGF23 and phosphorus levels. At Month 12, FGF23 (195 ± 300 pg/mL) and Klotho levels (130 ± 34 pg/mL) remained similar to baseline values. Log-FGF23 correlated significantly with height/age Z score (r= -0.38) and residual renal function (r = -0.44), but no correlation was found with serum phosphorus, phosphate intake, PTH and vitamin D levels. The log-FGF23 strongly correlated with calcium levels at Months 1, 6 and 12, however, this relationship was blunted if serum phosphorus was >6 mg/dL. By multiple regression analysis, calcium was the strongest variable determining FGF23 levels. CONCLUSIONS: In this longitudinal study, FGF23 levels are markedly increased, and Klotho levels are reduced in PD children compared with controls. FGF23 levels appeared to be regulated primarily by serum calcium, showing a significant correlation at each time of measurement. This relationship was lost in patients with phosphorus >6 mg/dL. These observations may have important consequences to the therapeutic management of phosphate homeostasis in CKD patients.