Insuficiencia suprarrenal secundaria: una causa subestimada de hiponatremia euvolemica

RESUMEN Introducción: La hiponatremia por insuficiencia suprarenal secundaria es subestimada tratamiento inapropiados. Objetivos: Describir las características clínicas y bioquímicas de pacientes con hiponatremia por insuficiencia suprarrenal secundaria y sus causas. Materiales y Metodos: Revisión retrospectiva de historias clínicas de pacientes consultantes a un hospital de tercer nivel entre Enero 2015 a Septiembre 2017 con hiponatremia y bioquímica de insuficiencia suprarenal secundaria. Los hallazgos fueron comparados con los reportados por estudios previamente publicados. Resultados: Todos los pacientes con insuficiencia suprarrenal secundaria se presentaron con hiponatremia euvolemica hipotónica. 54.5% eran mujeres, la edad promedio fue 57 años. Solo 1 paciente tuvo hiponatremia leve. La mediana de la concentración de cortisol fue 2.8 mcg/dL (RIQ 1.75-3.25 mcg/dL) y la de ACTH fue de 7.7 pg/nL (RIQ 4.5-9.5 pg/nL). Todos los pacientes tuvieron densidad urinaria alta indistinguible del SSIDH. El hipogonadismo hipogonadotrópico y el hipotiroidismo central fueron las alteraciones de ejes hipofisarios mas comúnmente asociados. La presencia de hipoglicemia, hipotensión e hipercaliemia fue baja. La causa más frecuente fue silla turca vacía. Conclusiones: La hiponatremia hipotonica euvolémica es una presentación común de insuficiencia suprarrenal secundaria y no suele acompañarse de otras manifestaciones de deficiencia de glucocorticoides. Es clínica y bioquímicamente indistinguible del SSIDH. Un bajo umbral de sospecha y la medición de cortisol serico matutino es esencial en estos pacientes para evitar un diagnostico y manejo inapropiados.

ABSTRACT Introduction: Hyponatremia due to secondary adrenal insufficiency is frequently underestimated and underdiagnosed. This paper underscores the importance of an adequate evaluation of euvolemic hyponatremia to avoid an inappropriate treatment and diagnosis. Objectives: To describe the clinical and biochemical characteristics of patients with hyponatremia due to secondary adrenal insufficiency and its causes. Materials and Methods: A retrospective review of the clinical records of patients presenting to a third level hospital between January 2015 to September 2017 with hyponatremia and a biochemical profile of secondary adrenal insufficiency. Findings were compared with previously published reports. Results: All patients with secondary adrenal insufficiency presented with hypotonic euvolemic hyponatremia. 54.5% of patients were females, median age was 57 years. Only 1 patient had mild hyponatremia. Cortisol median concentration was 2.8 mcg/dL (IQR 1.75-3.25 mcg/dL) and median ACTH concentration was 7.7 pg/nL (IQR 4.5-9.5 pg/nL). All the patients had high urinary density and features indistinguishable from SIADH. Hypogonadotropic hypogonadism and central hypothyroidism were the most commonly accompanying hypophyseal axis. Hypoglycemia, hypotension, and hyperkalemia were infrequent findings in these patients. The most frequent etiology identified was empty sella syndrome. Conclusions: Euvolemic hypotonic hyponatremia is a common presentation of secondary adrenal insufficiency and is often not accompanied with other manifestations of glucocorticoid deficiency. This disease is clinical and biochemical indistinguishable from SIADH. A low threshold for suspicion and a serum morning cortisol measurement in these patients is essential to avoid an inappropriate diagnosis and management.

Stem cells and regenerative medicine in domestic and companion animals: a multispecies perspective.

Since their original isolation, the majority of the work on embryonic stem cells (ESC) has been carried out in mice. While the mouse is an outstanding model for basic research, it also has considerable limitations for translational work, especially in the area of regenerative medicine. This is due to a combination of factors that include physiological and size differences when compared to humans. In contrast, domestic animal species, such as swine, and companion animal species, such as dogs, provide unique opportunities to develop regenerative medicine protocols that can then be utilized in humans. Unfortunately, at present, the state of knowledge related to, and availability of, ESC from domestic animals vary among species such as pig, horse, dog and cat, and without exception lags significantly behind the mouse and human. It is clear that much still needs to be discovered. The 'stem cell-like' cell lines being reported are still not satisfactorily used in regenerative medicine, due to reasons such as heterogeneity and chromosomal instability. As a result, investigators have searched for alternate source of cells that can be used for regenerative medicine. This approach has uncovered a range of adult stem cells and adult progenitor cells that have utility in both human and veterinary medicine. Here, we review a range of stem cells, from ESC to induced pluripotent stem cells, and discuss their potential application in the field of regenerative medicine.

Differentially expressed microRNAs and affected biological pathways revealed by modulated modularity clustering (MMC) analysis of human preeclamptic and IUGR placentas.

INTRODUCTION: This study focuses on the implementation of modulated modularity clustering (MMC) a new cluster algorithm for the identification of molecular signatures of preeclampsia and intrauterine growth restriction (IUGR), and the identification of affected microRNAs METHODS: Eighty-six human placentas from normal (40), growth-restricted (27), and preeclamptic (19) term pregnancies were profiled using Illumina Human-6 Beadarrays. MMC was utilized to generate modules based on similarities in placental transcriptome. Gene Set Enrichment Analysis (GSEA) was used to predict affected microRNAs. Expression levels of these candidate microRNAs were investigated in seventy-one human term placentas as follows: control (29); IUGR (26); and preeclampsia (16). RESULTS: MMC identified two modules, one representing IUGR placentas and one representing preeclamptic placentas. 326 differentially expressed genes in the module representing IUGR and 889 differentially expressed genes in a module representing preeclampsia were identified. Functional analysis of molecular signatures associated with IUGR identified P13K/AKT, mTOR, p70S6K, apoptosis and IGF-1 signaling as being affected. Analysis of variance of GSEA-predicted microRNAs indicated that miR-194 was significantly down-regulated both in preeclampsia (p = 0.0001) and IUGR (p = 0.0304), and miR-149 was significantly down-regulated in preeclampsia (p = 0.0168). DISCUSSION: Implementation of MMC, allowed identification of genes disregulated in IUGR and preeclampsia. The reliability of MMC was validated by comparing to previous linear modeling analysis of preeclamptic placentas. CONCLUSION: MMC allowed the elucidation of a molecular signature associated with preeclampsia and a subset of IUGR samples. This allowed the identification of genes, pathways, and microRNAs affected in these diseases.

Expression of antisense of insulin-like growth factor-2 receptor RNA non-coding (AIRN) during early gestation in cattle.

The insulin-like growth factor type 2 receptor (IGF2R) regulates fetal growth by removing IGF2 from circulation. In mice, expression of the Igf2r gene is only imprinted after implantation and is associated with expression of the antisense non-coding (nc)RNA, Airn. The objectives of this study were, first, to determine if bovine AIRN was expressed during developmentally important stages of gestation, and second, to determine if expression of bAIRN was affected by method of embryo production. Control reactions confirmed that sequence verified bAIRN PCR amplicons resulted from RNA within the sample and not from genomic DNA contamination. IGF2R mRNA was expressed in all fetal liver samples at Days 35-55 and 70 of gestation as well as in 8 of 9 Day 15 conceptuses, 10 of 10 Day 18 conceptuses, and in all day 7 blastocyst pools. bAIRN was expressed in all samples of fetal liver at Days 35-55 and 70 of gestation. The proportion of conceptuses that expressed bAIRN increased from 1 of 9 at Day 15 of gestation to 8 of 10 at Day 18 of gestation. No bAIRN was expressed in any blastocyst pools. The relative level of bAIRN was greater (P<0.05) in fetal liver from embryos produced in vivo compared to that from embryos produced in vitro. In summary bAIRN was not expressed in blastocyst-stage embryos, was expressed in an increasing proportion of embryos around the time of maternal recognition of pregnancy and was expressed following implantation. Furthermore, relative levels of bAIRN in bovine fetal liver can be altered by method of embryo production.

Transcriptional profiling of human placentas from pregnancies complicated by preeclampsia reveals disregulation of sialic acid acetylesterase and immune signalling pathways.

The placenta plays an important role as a regulator of fetal nutrition and growth throughout development and placental factors contribute to gestational abnormalities such as preeclampsia. This study describes the genome-wide gene expression profiles of a large (n = 60) set of human placentas in order to uncover gene expression patterns associated with preeclampsia. In addition to confirming changes in expression of soluble factors associated with preeclampsia such as sFLT1 (soluble fms-like tyrosine kinase-1), sENG (soluble endoglin), and INHA (inhibin alpha), we also find changes in immune-associated signaling pathways, offering a potential upstream explanation for the shallow trophoblast invasion and inadequate uterine remodeling typically observed in pathogenesis of preeclampsia. Notably, we also find evidence of preeclampsia-associated placental upregulation of sialic acid acetylesterase (SIAE), a gene functionally associated with autoimmune diseases.

Evaluation of the Serratia marcescens nuclease (NucA) as a transgenic cell ablation system in porcine.

The efficiency of the Serratia marcescens nuclease encoded by the NucA gene, with or without a nuclear localization signal (NLS), and the commonly used diphtheria toxin A (DTA) were compared for their ability to ablate cells in culture. Constructs containing the test genes driven by the beta-actin promoter coupled with enhancer elements from the cytomegalovirus promoter and rabbit beta-globin gene (pCAG) and the blasticidin resistance gene driven by the phosphoglycerate kinase (PGK) promoter were generated and electroporated into porcine fetal fibroblasts. Three independent replicates were completed. Following blasticidin selection, the number of surviving colonies was counted to assess the efficiency of the toxic gene. Both NucA and DTA proved to be effective in killing porcine fibroblasts compared to controls. However, the efficiency of cell ablation was significantly higher with DTA than with NucA or NucANLS (p < 0.05). Gene expression analysis of surviving colonies indicated that survival is related to low or absent expression of the toxic genes. These results indicate that the NucA gene, while capable of mammalian cell ablation, is less efficient than DTA.

Functional genomic approaches for the study of fetal/placental development in swine with special emphasis on imprinted genes.

This chapter describes the application of functional genomic approaches to the study of imprinted genes in swine. While there are varied definitions of "functional genomics", in general they focus on the application of DNA microarrays, single nucleotide polymorphism (SNP) arrays, and other high coverage genomic analyses, and their combination with downstream methods of gene modification such as silencing RNA (siRNA) and viral and non-viral transfection. Between the initial data acquisition and the actual genetic manipulation of the system lies bioinformatics, where massive amounts of data are analyzed to extract meaningful information. This area is in constant flux with an increased emphasis on detection of affected pathways and processes rather than generation of simple affected gene lists. We will expand on each of these points and describe how we have used these technologies for the study of imprinted genes in swine. First we will introduce the biological question to provide context for the discussion of the functional genomic approaches and the types of information they generate.

Development of fetuses from in vitro-produced and cloned bovine embryos.

The establishment of in vitro fertilization and culture systems for mammalian embryos has facilitated the application of embryo technologies in research, industry, and clinical settings. Furthermore, the advent of cloning by nuclear transfer has significantly enhanced the potential for genetic modification of livestock. Based on studies in cattle, sheep, and mice, it has become apparent that embryos produced using these systems can differ in morphology and developmental potential compared with embryos produced in vivo. Referred to as "large offspring syndrome," these abnormalities in the development of fetuses, placentas, and offspring are particularly evident following transfer of cloned embryos, but they also occur in pregnancies from embryos produced using in vitro culture alone. The objective of this review is to examine the effects of in vitro production and cloning on bovine embryo and fetal development. Literature pertaining to preimplantation embryo, conceptus, and fetal development, as well as gene expression occurring at each of these three stages, is reviewed. Physiologic and genetic mechanisms that contribute to large offspring syndrome also are discussed.

Stable transfection of the bovine NRAMP1 gene into murine RAW264.7 cells: effect on Brucella abortus survival.

Genetically based natural resistance to brucellosis in cattle provides for novel strategies to control zoonotic diseases. Bovine NRAMP1, the homologue of a murine gene (Bcg), has been identified as a major candidate for controlling the in vivo resistant phenotype. We developed an in vitro model for expression of resistance- and susceptibility-associated alleles of bovine NRAMP1 as stable transgenes under the regulatory control of the bovine NRAMP1 promoter in the murine RAW264.7 macrophage cell line (Bcg(s)) to analyze the regulation of the NRAMP1 gene and its role in macrophage function. We demonstrated that the 5'-flanking region of bovine NRAMP1, despite the lack of TATA and CAAT boxes, has a functional promoter capable of driving the expression of a transgene in murine macrophages. A polymorphism within a microsatellite in the 3' untranslated region critically affects the expression of bovine NRAMP1 and the control of in vitro replication of Brucella abortus but not Salmonella enterica serovar Dublin. We did not observe any differences in the production of NO by resting or gamma interferon (IFN-gamma)- and IFN-gamma-lipopolysaccharide (LPS)-treated transfected cell lines, yet the resistant transfected cell lines produced significantly less NO than other cell lines, following stimulation with LPS at 24 and 48 h.

Cloning to reproduce desired genotypes.

Cloned sheep, cattle, goats, pigs and mice have now been produced using somatic cells for nuclear transplantation. Animal cloning is still very inefficient with on average less than 10% of the cloned embryos transferred resulting in a live offspring. However successful cloning of a variety of different species and by a number of different laboratory groups has generated tremendous interest in reproducing desired genotypes. Some of these specific genotypes represent animal cell lines that have been genetically modified. In other cases there is a significant demand for cloning animals characterized by their inherent genetic value, for example prize livestock, household pets and rare or endangered species. A number of different variables may influence the ability to reproduce a specific genotype by cloning. These include species, source of recipient ova, cell type of nuclei donor, treatment of donor cells prior to nuclear transfer, and the techniques employed for nuclear transfer. At present, there is no solid evidence that suggests cloning will be limited to only a few specific animals, and in fact, most data collected to date suggests cloning will be applicable to a wide variety of different animals. The ability to reproduce any desired genotype by cloning will ultimately depend on the amount of time and resources invested in research.

Genetic basis of susceptibility to environmentally induced neural tube defects.

Neural tube defects (NTDs) are among the most common of all human congenital defects, with multifactorial etiologies comprising both environmental and genetic components. Several murine model systems have been developed in an effort to elucidate genetic factors regulating expression of NTDs. Strain-dependent differences in susceptibility to teratogenic insults and altered patterns of gene expression observed within the neuroepithelium of affected embryos support the hypothesis that subtle genetic changes can result in NTDs. Since several affected genes are folate-regulated, transgenic knockout mice lacking a functional folate receptor were developed. Nullizygous embryos died in utero with significant morphological defects, supporting the critical role of folic acid in early embryogenesis. While epidemiological studies have not established an association between polymorphisms in the human folate receptor gene and NTDs, it is known that folate supplementation reduces infant NTD risk. Continued efforts are therefore necessary to reveal the mechanism by which folate works and the nature of the gene(s) responsible for human NTDs.

Pathologic findings and association of Mycobacterium bovis infection with the bovine NRAMP1 gene in cattle from herds with naturally occurring tuberculosis.

OBJECTIVE: To determine necropsy and Mycobacterium bovis culture results in cattle from herds with tuberculosis, the role of the bovine NRAMP1 gene in resistance and susceptibility to infection with M bovis, and the association between magnitude of the tuberculous lesions and various types of M bovis isolates. ANIMALS: 61 cattle from herds with tuberculosis in Texas and Mexico. PROCEDURE: 61 cattle were evaluated by necropsy; 59 had positive and 2 had negative caudal fold tuberculin intradermal test (CFT) results. Thirty-three cattle with positive CFT results were genotyped to evaluate polymorphism of the 3' untranslated region of the bovine NRAMP1 gene, using single-stranded conformational analysis, 9 were resistant to M bovis with no tuberculous lesions and negative M bovis culture results, and 24 were susceptible with tuberculous lesions and positive M bovis culture results. Isolates of M bovis were analyzed by restriction fragment length polymorphism (RFLP) on the basis of IS6110 sequences and direct-repeat fingerprinting patterns. RESULTS: 21 (35.6%; 21/59) cattle with positive CFT results had tuberculous lesions or positive culture results; in addition, 1 of 2 cattle with negative CFT results had tuberculous lesions and positive culture results. Tuberculous lesions were most common in the thorax (35/63; 55.5%) and lymphoid tissues of the head (10/63; 15.9%). Tuberculous lesions varied from 1 to 11/animal; 8 of 21 (38.1%) had solitary lesions. Associations were not found between resistance or susceptibility to infection with M bovis and polymorphism in the NRAMP1 gene or between the magnitude of the lesions and various RFLP types of M bovis isolates. CONCLUSIONS AND CLINICAL RELEVANCE: The NRAMP1 gene does not determine resistance and susceptibility to infection with M bovis in cattle.

Effects of protease inhibitors and antioxidants on In vitro survival of porcine primordial germ cells.

One of the problems associated with in vitro culture of primordial germ cells (PGCs) is the large loss of cells during the initial period of culture. This study characterized the initial loss and determined the effectiveness of two classes of apoptosis inhibitors, protease inhibitors, and antioxidants on the ability of porcine PGCs to survive in culture. Results from electron microscopic analysis and in situ DNA fragmentation assay indicated that porcine PGCs rapidly undergo apoptosis when placed in culture. Additionally, alpha(2)-macroglobulin, a protease inhibitor and cytokine carrier, and N:-acetylcysteine, an antioxidant, increased the survival of PGCs in vitro. While other protease inhibitors tested did not affect survival of PGCs, all antioxidants tested improved survival of PGCs (P: < 0.05). Further results indicated that the beneficial effect of the antioxidants was critical only during the initial period of culture. Finally, it was determined that in short-term culture, in the absence of feeder layers, antioxidants could partially replace the effect(s) of growth factors and reduce apoptosis. Collectively, these results indicate that the addition of alpha(2)-macroglobulin and antioxidants can increase the number of PGCs in vitro by suppressing apoptosis.

Targeted modification of the domestic animal genome.

While the technique of homologous recombination, or gene targeting, has led to the generation of transgenic mice of great value to biomedical research, similar approaches are only being developed in other species. With the exception of recent reports on the generation of gene-targeted sheep, the technology in domestic animals is still in its infancy (45). The development of techniques for generating large animals with deleted or modified genes will result in the generation of animals of great value to society. While the technical difficulties to achieve gene targeting in domestic species are significant, they are not insurmountable. Potential applications in both the bovine and porcine species are described with particular emphasis on the generation of cattle resistant to bovine spongiform encephalopathy (BSE) and pigs that can be of use in xenotransplantation.

Effects of growth factors and feeder cells on porcine primordial germ cells in vitro.

As embryonic stem (ES) cells are not available in swine, embryonic germ (EG) cells derived from primordial germ cells (PGCs) are an alternate source of pluripotent embryonic cells for genetic modification through homologous recombination. Although morphological and biochemical characteristics are similar between ES and EG cells, culture conditions are quite different. To optimize the culture condition for the establishment of porcine EG cells, porcine PGCs were cultured in vitro with various combinations of growth factors (leukemia inhibitory factor [LIF], stem cell factor [SCF], and basic fibroblast growth factor [bFGF]) and on different kinds of feeder cells (STO, TM(4), Sl/Sl(4) m220, porcine embryonic fibroblasts, and COS-7 cells). Optimal results were obtained when all three growth factors (LIF, SCF, and bFGF) were present in the media. Also, feeder cells expressing membrane-bound SCF are required for survival and establishment of porcine EG cells. Therefore, a combination of growth factors and proper feeder cells are critical for the establishment of undifferentiated porcine EG cells.

Mice lacking the folic acid-binding protein Folbp1 are defective in early embryonic development.

Periconceptional folic acid supplementation reduces the occurrence of several human congenital malformations, including craniofacial, heart and neural tube defects. Although the underlying mechanism is unknown, there may be a maternal-to-fetal folate-transport defect or an inherent fetal biochemical disorder that is neutralized by supplementation. Previous experiments have identified a folate-binding protein (Folbp1) that functions as a membrane receptor to mediate the high-affinity internalization and delivery of folate to the cytoplasm of the cell. In vitro, this receptor facilitates the accumulation of cellular folate a thousand-fold relative to the media, suggesting that it may be essential in cytoplasmic folate delivery in vivo. The importance of an adequate intracellular folate pool for normal embryogenesis has long been recognized in humans and experimental animals. To determine whether Folbp1 is involved in maternal-to-fetal folate transport, we inactivated Folbp1 in mice. We also produced mice lacking Folbp2, another member of the folate receptor family that is GPI anchored but binds folate poorly. Folbp2-/- embryos developed normally, but Folbp1-/- embryos had severe morphogenetic abnormalities and died in utero by embryonic day (E) 10. Supplementing pregnant Folbp1+/- dams with folinic acid reversed this phenotype in nullizygous pups. Our results suggest that Folbp1 has a critical role in folate homeostasis during development, and that functional defects in the human homologue (FOLR1) of Folbp1 may contribute to similar defects in humans.

Lack of class I major histocompatibility antigens on trophoblast of periimplantation blastocysts and term placenta in the pig.

In this study, the pattern of expression of class I major histocompatibility (MHC) antigens and mRNA on periimplantation blastocysts and term placental tissue was determined for the pig. Class I MHC antigens could not be detected immunohistochemically either on extra-embryonic membranes or on the embryonic portion of Day 14, 16, 22, and 25 blastocysts. Nor could class I MHC antigens be detected on the outer trophoblast epithelium and inner endodermal surface of the chorioallantoic membrane or on the outer and inner surfaces of the amnion at term. However, MHC class I antigens were detected on the vascular mesoderm found in both the chorion and amnion at term, and in Day 25 extra-embryonic membranes. Uterine endometrial cells and tissues and maternal peripheral blood leukocytes stained strongly for class I MHC antigens. There was a large difference in the intensity of class I MHC mRNA signal, detected by Northern blot analysis, in embryo/fetus-derived tissues compared to that in maternal tissues. The embryos appeared to express even less class I MHC mRNA than did the extra-embryonic membranes. In addition, in situ hybridization of Day 16 blastocysts indicated class I MHC mRNA to be ubiquitously expressed at low levels in embryos and extra-embryonic tissues compared to uterine endometrial tissue controls. Taken together, these results indicate that class I MHC antigens are either not expressed on the surface of the extra-embryonic/fetal membranes during gestation in the pig or are expressed at very low levels, and that specific mRNA is expressed at correspondingly low levels.