RESUMEN
We have identified a generalized arousal component in the behavior of mice. Analyzed by mathematical/statistical approaches across experiments, investigators, and mouse populations, it accounts for about 1/3 of the variance in arousal-related measures. Knockout of the gene coding for the classical estrogen receptor (ER-alpha), a ligand-activated transcription factor, greatly reduced arousal responses. In contrast, disrupting the gene for a likely gene duplication product, ER-beta, did not have these effects. A combination of mathematical and genetic approaches to arousal in an experimentally tractable mammal opens up analysis of a CNS function of considerable theoretical and practical significance.
Asunto(s)
Encéfalo/fisiología , Genética , Conducta Sexual Animal , Animales , RatonesRESUMEN
We recently found that estrogen receptor (ER) antagonists prevent high-dose estrogen from inducing the formation of new cancellous bone within the medullary cavity of mouse long bones. In the present investigation, we studied the role of specific ER subtypes in this response by examining whether this is impaired in female ERbeta(-/-) mice previously generated by targeted gene deletion. Vehicle or 17beta-estradiol (E(2)) (range 4-4,000 microg. kg(-1). day(-1)) was administered to intact female ERbeta(-/-) mice and wild-type littermates by subcutaneous injection for 28 days. The osteogenic response was subsequently assessed by histomorphometry performed on longitudinal and cross sections of the tibia. E(2) was found to cause an equivalent increase in cancellous bone formation in ERbeta(-/-) mice and littermate controls, as assessed at the proximal and distal regions of the proximal tibial metaphysis. E(2) also resulted in a similar increase in endosteal mineral apposition rate in these two genotypes, as assessed at the tibial diaphysis. In contrast, cortical area in ERbeta(-/-) mice was found to be greater than that in wild types irrespective of E(2) treatment, as was tibial bone mineral density as measured by dual-energy X-ray absorptiometry, consistent with previous reports of increased cortical bone mass in these animals. We conclude that, although ERbeta acts as a negative modulator of cortical modeling, this isoform does not appear to contribute to high-dose estrogen's ability to induce new cancellous bone formation in mouse long bones.
Asunto(s)
Estradiol/farmacología , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Receptores de Estrógenos/genética , Animales , Relación Dosis-Respuesta a Droga , Receptor beta de Estrógeno , Femenino , Eliminación de Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoblastos/efectos de los fármacos , Osteoblastos/fisiología , Receptores de Estrógenos/metabolismo , Tibia/citología , Tibia/efectos de los fármacosRESUMEN
Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are cardiac hormones that regulate blood pressure and volume, and exert their biological actions via the natriuretic peptide receptor-A gene (Npr1). Mice lacking Npr1 (Npr(-/-)) have marked cardiac hypertrophy and fibrosis disproportionate to their increased blood pressure. This study examined the relationships between ANP and BNP gene expression, immunoreactivity and fibrosis in cardiac tissue, circulating ANP levels, and ANP and BNP mRNA during embryogenesis in Npr1(-/-) mice. Disruption of the Npr1 signaling pathway resulted in augmented ANP and BNP gene and ANP protein expression in the cardiac ventricles, most pronounced for ANP mRNA in females [414 +/- 57 in Npr1(-/-) ng/mg and 124 +/- 25 ng/mg in wild-type (WT) by Taqman assay, P < 0.001]. This increased expression was highly correlated to the degree of cardiac hypertrophy and was localized to the left ventricle (LV) inner free wall and to areas of ventricular fibrosis. In contrast, plasma ANP was significantly greater than WT in male but not female Npr1(-/-) mice. Increased ANP and BNP gene expression was observed in Npr1(-/-) embryos from 16 days of gestation. Our study suggests that cardiac ventricular expression of ANP and BNP is more closely associated with local hypertrophy and fibrosis than either systemic blood pressure or circulating ANP levels.
Asunto(s)
Factor Natriurético Atrial/metabolismo , Cardiomegalia/metabolismo , Cardiomiopatías/metabolismo , Guanilato Ciclasa/deficiencia , Miocardio/metabolismo , Péptido Natriurético Encefálico/metabolismo , Receptores del Factor Natriurético Atrial/deficiencia , Animales , Factor Natriurético Atrial/sangre , Factor Natriurético Atrial/genética , Cardiomegalia/etiología , Cardiomiopatías/etiología , Cardiomiopatías/patología , Embrión de Mamíferos/metabolismo , Femenino , Fibrosis , Guanilato Ciclasa/genética , Ventrículos Cardíacos , Hipertensión/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados/genética , Péptido Natriurético Encefálico/sangre , Péptido Natriurético Encefálico/genética , ARN Mensajero/metabolismo , Receptores del Factor Natriurético Atrial/genética , Valores de ReferenciaRESUMEN
BACKGROUND: Prostanoid products of the cyclo-oxygenase (COX) pathway of arachidonic acid metabolism modulate blood pressure (BP) and sodium homeostasis. Conventional non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit both COX isoforms (COX-1 and -2), cause sodium retention, exacerbate hypertension, and interfere with the efficacy of certain anti-hypertensive agents such as angiotensin-converting enzyme (ACE) inhibitors. While a new class of NSAIDs that specifically inhibit COX-2 is now widely used, the relative contribution of the individual COX isoforms to these untoward effects is not clear. METHODS: To address this question, we studied mice with targeted disruption of the COX-1 (Ptgs1) gene. Blood pressure, renin mRNA expression, and aldosterone were measured while dietary sodium was varied. To study interactions with the renin-angiotensin system, ACE inhibitors were administered and mice with combined deficiency of COX-1 and the angiotensin II subtype 1A (AT1A) receptor were generated. RESULTS: On a regular diet, BP in COX-1-/- mice was near normal. However, during low salt feeding, BP values were reduced in COX-1-/- compared to +/+ animals, and this reduction in BP was associated with abnormal natriuresis despite appropriate stimulation of renin and aldosterone. Compared to COX-1+/+ mice, the actions of ACE inhibition were markedly accentuated in COX-1-/- mice. Sodium sensitivity and BP lowering also were enhanced in mice with combined deficiency of COX-1 and AT1A receptor. CONCLUSIONS: The absence of COX-1 is associated with sodium loss and enhanced sensitivity to ACE inhibition, suggesting that COX-1 inhibition does not cause hypertension and abnormal sodium handling associated with NSAID use.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Isoenzimas/deficiencia , Natriuresis/fisiología , Prostaglandina-Endoperóxido Sintasas/deficiencia , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Ciclooxigenasa 1 , Dieta Hiposódica , Corteza Renal/metabolismo , Proteínas de la Membrana , Ratones , Receptor de Angiotensina Tipo 1 , Receptores de Angiotensina/deficiencia , Renina/metabolismo , Sodio/deficienciaRESUMEN
Diabetic nephropathy is a major risk factor for end-stage renal disease and cardiovascular diseases and has a marked genetic component. A common variant (D allele) of the angiotensin I-converting enzyme (ACE) gene, determining higher enzyme levels, has been associated with diabetic nephropathy. To address causality underlying this association, we induced diabetes in mice having one, two, or three copies of the gene, normal blood pressure, and an enzyme level range (65-162% of wild type) comparable to that seen in humans. Twelve weeks later, the three-copy diabetic mice had increased blood pressures and overt proteinuria. Proteinuria was correlated to plasma ACE level in the three-copy diabetic mice. Thus, a modest genetic increase in ACE levels is sufficient to cause nephropathy in diabetic mice.
Asunto(s)
Diabetes Mellitus Experimental/genética , Nefropatías Diabéticas/genética , Peptidil-Dipeptidasa A/sangre , Albuminuria/genética , Animales , Presión Sanguínea/genética , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/enzimología , Nefropatías Diabéticas/enzimología , Femenino , Ratones , Ratones Endogámicos C57BL , Peptidil-Dipeptidasa A/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Highly polymorphic di- and tetranucleotide repeats in and around Npr3, a potential candidate gene for hypertension, have been identified using a novel approach. Because this chromosomal site is rich in repetitive DNA and difficult to sequence, P1 artificial chromosomes were retrofitted with a loxP transposon to map the gene sequence within a clone using a series of nested deletions. Sequences from ends of deletions 1-3 kb apart identified a (CA)(20) and a (TA)(18)-(CA)(8) repeat 8 kb upstream and within an intron of Npr3, respectively. DNA from 17 individuals was analyzed for length polymorphisms in these and eight additional repeats identified in 200 kb of working draft sequence from this region in GenBank. The sequence contigs and microsatellite repeats from GenBank were ordered using the P1-derived artificial chromosome deletion series. Several of these repeats were found to vary considerably in length in the set of genomic DNA tested. Since this site in chromosome 5p has recently been implicated in disease in studies with genetically hypertensive rats, the microsatellite markers reported here will be useful for genetic analysis and may even be implicated in the disease process in humans. We discuss how these types of data are useful for interpreting draft DNA sequence coming out of the genome projects, and the utility of deletion clones as a resource for ordering contigs and gap filling.
Asunto(s)
Guanilato Ciclasa/genética , Repeticiones de Microsatélite/genética , Receptores del Factor Natriurético Atrial/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Alelos , Cromosomas Artificiales de Bacteriófagos P1/genética , Mapeo Contig , ADN/química , ADN/genética , Elementos Transponibles de ADN/genética , Frecuencia de los Genes , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Mutagénesis Insercional , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADN , Eliminación de SecuenciaRESUMEN
Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) exert their physiological actions by binding to natriuretic peptide receptor A (NPRA), a receptor guanylate cyclase (rGC) that synthesizes cGMP in response to both ligands. The family of rGCs is rapidly expanding, and it is plausible that there might be additional, as yet undiscovered, rGCs whose function is to provide alternative signalling pathways for one or both of these peptides, particularly given the low affinity of NPRA for BNP. We have investigated this hypothesis, using a genetically modified (knockout) mouse in which the gene encoding NPRA has been disrupted. Enzyme assays and NPRA-specific Western blots performed on tissues from wild-type mice demonstrate that ANP-activated cGMP synthesis provides a good index of NPRA protein expression, which ranges from maximal in adrenal gland, lung, kidney, and testis to minimal in heart and colon. In contrast, immunoreactive NPRA is not detectable in tissues isolated from NPRA knockout animals and ANP- and BNP-stimulatable GC activities are markedly reduced in all mutant tissues. However, testis and adrenal gland retain statistically significant, high-affinity responses to BNP. This residual response to BNP cannot be accounted for by natriuretic peptide receptor B, or any other known mammalian rGC, suggesting the presence of a novel receptor in these tissues that prefers BNP over ANP.
Asunto(s)
Factor Natriurético Atrial/farmacología , Péptido Natriurético Encefálico/farmacología , Receptores del Factor Natriurético Atrial/metabolismo , Glándulas Suprarrenales/metabolismo , Animales , Western Blotting , GMP Cíclico/biosíntesis , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Masculino , Ratones , Ratones Noqueados , Péptido Natriurético Tipo-C/farmacología , Receptores del Factor Natriurético Atrial/genética , Testículo/metabolismo , Distribución TisularRESUMEN
To distinguish the contributions of Ren1(d) and Ren2 to kidney development and blood pressure homeostasis, we placed green fluorescent protein (GFP) under control of the Ren1(d) renin locus by homologous recombination in mice. Homozygous Ren1(d)-GFP animals make GFP mRNA in place of Ren1(d) mRNA in the kidney and maintain Ren2 synthesis in the juxtaglomerular (JG) cells. GFP expression provides an accurate marker of Ren1(d) expression during development. Kidneys from homozygous animals are histologically normal, although with fewer secretory granules in the JG cells. Blood pressure and circulating renin are reduced in Ren1(d)-GFP homozygotes. Acute administration of losartan decreases blood pressure further, suggesting a role for Ren2 protein in blood pressure homeostasis. These studies demonstrate that, in the absence of Ren1(d), Ren2 preserves normal kidney development and prevents severe hypotension. Chronic losartan treatment results in compensation via recruitment of both Ren1(d)- and Ren2-expressing cells along the preglomerular vessels. This response is achieved by metaplastic transformation of arteriolar smooth muscle cells, a major mechanism to control renin bioavailability and blood pressure homeostasis.
Asunto(s)
Presión Sanguínea , Riñón/embriología , Proteínas Luminiscentes/metabolismo , Renina/genética , Renina/fisiología , Antagonistas de Receptores de Angiotensina , Animales , Capilares/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Marcación de Gen , Proteínas Fluorescentes Verdes , Homeostasis , Inmunohistoquímica , Aparato Yuxtaglomerular/metabolismo , Riñón/irrigación sanguínea , Riñón/metabolismo , Losartán/farmacología , Proteínas Luminiscentes/genética , Masculino , Ratones , ARN Mensajero/biosíntesis , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Renina/inmunologíaRESUMEN
Mice lacking natriuretic peptide receptor A (NPRA) have marked cardiac hypertrophy and chamber dilatation disproportionate to their increased blood pressure (BP), suggesting, in support of previous in vitro data, that the NPRA system moderates the cardiac response to hypertrophic stimuli. Here, we have followed the changes in cardiac function in response to altered mechanical load on the heart of NPRA-null mice (Npr1-/-). Chronic treatment with either enalapril, furosemide, hydralazine, or losartan were all effective in reducing and maintaining BP at normal levels without affecting heart weight/body weight. In the reverse direction, we used transverse aortic constriction (TAC) to induce pressure overload. In the Npr1-/- mice, TAC resulted in a 15-fold increase in atrial natriuretic peptide (ANP) expression, a 55% increase in left ventricular weight/body weight (LV/BW), dilatation of the LV, and significant decline in cardiac function. In contrast, banded Npr1+/+ mice showed only a threefold increase in ANP expression, an 11% increase in LV/BW, a 0.2 mm decrease in LV end diastolic dimension, and no change in fractional shortening. The activation of mitogen-activated protein kinases that occurs in response to TAC did not differ in the Npr1+/+ and Npr1-/- mice. Taken together, these results suggest that the NPRA system has direct antihypertrophic actions in the heart, independent of its role in BP control.
Asunto(s)
Cardiomegalia/fisiopatología , Guanilato Ciclasa/fisiología , Receptores del Factor Natriurético Atrial/fisiología , Animales , Antihipertensivos/uso terapéutico , Presión Sanguínea , Cardiomegalia/tratamiento farmacológico , Cardiomegalia/metabolismo , Enalapril/uso terapéutico , Furosemida/uso terapéutico , Hidralazina/uso terapéutico , Losartán/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocardio/patología , Tamaño de los Órganos , Propranolol/uso terapéutico , Telemetría/métodos , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
The transition to pulmonary respiration following birth requires rapid alterations in the structure of the mammalian cardiovascular system. One dramatic change that occurs is the closure and remodeling of the ductus arteriosus (DA), an arterial connection in the fetus that directs blood flow away from the pulmonary circulation. A role for prostaglandins in regulating the closure of this vessel has been supported by pharmacological and genetic studies. The production of prostaglandins is dependent on two cyclooxygenases (COX-1 and COX-2), which are encoded by separate genes. We report here that the absence of either or both COX isoforms in mice does not result in premature closure of the DA in utero. However, 35% of COX-2(-/-) mice die with a patent DA within 48 h of birth. In contrast, the absence of only the COX-1 isoform does not affect closure of the DA. The mortality (35%) and patent DA incidence due to absence of COX-2 is, however, significantly increased (79%) when one copy of the gene encoding COX-1 is also inactivated. Furthermore, 100% of the mice deficient in both isoforms die with a patent DA within 12 h of birth, indicating that in COX-2-deficient mice, the contribution of COX-1 to DA closure is gene dosage-dependent. Together, these data establish roles for COX-1, and especially for COX-2, in the transition of the cardiopulmonary circulation at birth.
Asunto(s)
Conducto Arterioso Permeable/genética , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Animales , Animales Recién Nacidos , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Muerte , Conducto Arterial/patología , Conducto Arterioso Permeable/epidemiología , Femenino , Impresión Genómica , Genotipo , Isoenzimas/deficiencia , Isoenzimas/genética , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Prostaglandina-Endoperóxido Sintasas/deficiencia , Prostaglandina-Endoperóxido Sintasas/genética , Factores de TiempoRESUMEN
The inhibitory effects of estrogen (17beta-estradiol) on atherosclerosis have been well documented in numerous animal models, and epidemiological evidence supports this protective effect in humans. The detailed mechanisms for this protection are not understood, but most are thought to be mediated through estrogen receptors (ERs), of which two are known (ERalpha and ERbeta). To investigate the role of ERalpha in the atheroprotective effect of 17beta-estradiol (E2), we ovariectomized female mice that lack apoE (AAee) or lack both apoE and ERalpha (alphaalphaee), and treated half of them with E2 for three months. E2 treatment of ovariectomized AAee females dramatically reduced the size of the lesions as well as their histological complexity. Plasma cholesterol was significantly reduced in this group, although the observed extent of protection by E2 was greater than could be explained solely by the change in lipid levels. In contrast, E2 treatment of ovariectomized alphaalphaee females caused minimal reduction in lesion size and no reduction in total plasma cholesterol compared with alphaalphaee mice without E2, demonstrating that ERalpha is a major mediator of the atheroprotective effect of E2. Nevertheless, E2 treatment significantly reduced the complexity of plaques in the alphaalphaee females, although not to the same degree as in AAee females, suggesting the existence of ERalpha-independent atheroprotective effects of E2.
Asunto(s)
Apolipoproteínas E/genética , Estradiol/farmacología , Receptores de Estrógenos/fisiología , Animales , Apolipoproteínas E/deficiencia , Arteriosclerosis/sangre , Arteriosclerosis/patología , Arteriosclerosis/prevención & control , Peso Corporal , Colorantes , Preparaciones de Acción Retardada , Estradiol/administración & dosificación , Estradiol/sangre , Receptor alfa de Estrógeno , Femenino , Lípidos/sangre , Ratones , Ratones Endogámicos C57BL , Tamaño de los Órganos , Ovariectomía , Seno Aórtico , Piel/patología , Útero/patologíaRESUMEN
Adrenomedullin, a recently identified potent vasodilator, is expressed widely and has been suggested to have functions ranging from reproduction to blood pressure regulation. To elucidate these functions and define more precisely sites of Adm expression, we replaced the coding region of the Adm gene in mice with a sequence encoding enhanced green fluorescent protein while leaving the Adm promoter intact. We find that Adm(-/-) embryos die at midgestation with extreme hydrops fetalis and cardiovascular abnormalities, including overdeveloped ventricular trabeculae and underdeveloped arterial walls. These data suggest that genetically determined absence of Adm may be one cause of nonimmune hydrops fetalis in humans.
Asunto(s)
Anomalías Múltiples/genética , Muerte Fetal/genética , Corazón Fetal/anomalías , Hidropesía Fetal/genética , Péptidos/fisiología , Anomalías Múltiples/patología , Adrenomedulina , Animales , Aorta/embriología , Aorta/patología , Arterias Carótidas/embriología , Arterias Carótidas/patología , Quimera , ADN Complementario/genética , Femenino , Muerte Fetal/patología , Corazón Fetal/patología , Regulación del Desarrollo de la Expresión Génica , Marcación de Gen , Genes Reporteros , Genotipo , Edad Gestacional , Proteínas Fluorescentes Verdes , Ventrículos Cardíacos/embriología , Ventrículos Cardíacos/patología , Hidropesía Fetal/embriología , Hidropesía Fetal/patología , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Masculino , Ratones , Ratones Noqueados , Péptidos/deficiencia , Péptidos/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Male mice with a knockout of the estrogen receptor (ER)-alpha gene, a ligand-activated transcription factor, showed reduced levels of intromissions and no ejaculations whereas simple mounting behavior was not affected. In contrast, all components of sexual behaviors were intact in male mice lacking the novel ER-beta gene. Here we measure the extent of phenotype in mice that lack both ER-alpha and ER-beta genes (alphabetaERKO). alphabetaERKO male mice did not show any components of sexual behaviors, including simple mounting behavior. Nor did they show ultrasonic vocalizations during behavioral tests with receptive female mice. On the other hand, reduced aggressive behaviors of alphabetaERKO mice mimicked those of single knockout mice of ER-alpha gene (alphaERKO). They showed reduced levels of lunge and bite aggression, but rarely showed offensive attacks. Thus, either one of the ERs is sufficient for the expression of simple mounting in male mice, indicating a redundancy in function. Offensive attacks, on the other hand, depend specifically on the ER-alpha gene. Different patterns of natural behaviors require different patterns of functions by ER genes.
Asunto(s)
Agresión , Receptores de Estrógenos/fisiología , Conducta Sexual , Animales , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Recent progress has been remarkable in identifying mutations which cause diseases (mostly uncommon) that are inherited simply. Unfortunately, the common diseases of humankind with a strong genetic component, such as those affecting cardiovascular function, have proved less tractable. Their etiology is complex with substantial environmental components and strong indications that multiple genes are implicated. In this article, we consider the genetic etiology of essential hypertension. After presenting the distribution of blood pressures in the population, we propose the hypothesis that essential hypertension is the consequence of different combinations of genetic variations that are individually of little consequence. The candidate gene approach to finding relevant genes is exemplified by studies that identified potentially causative variations associated with quantitative differences in the expression of the angiotensinogen gene (AGT). Experiments to test causation directly are possible in mice, and we describe their use to establish that blood pressures are indeed altered by genetic changes in AGT expression. Tests of differences in expression of the genes coding for the angiotensin-converting enzyme (ACE) and for the natriuretic peptide receptor A are also considered, and we provide a tabulation of all comparable experiments in mice. Computer simulations are presented that resolve the paradoxical finding that while ACE inhibitors are effective, genetic variations in the expression of the ACE gene do not affect blood pressure. We emphasize the usefulness of studying animals heterozygous for an inactivating mutation and a wild-type allele, and briefly discuss a way of establishing causative links between complex phenotypes and single nucleotide polymorphisms.
Asunto(s)
Variación Genética , Hipertensión Renal/etiología , Hipertensión Renal/genética , Animales , Humanos , Carácter Cuantitativo HeredableRESUMEN
BACKGROUND: Genetic ablation of cyclooxygenase-2 (COX-2) resulted in cystic renal dysplasia and early death in adult mice. The ontologic development of the renal pathology and the biochemical and physiological abnormalities associated with the dysplasia are unknown. METHODS: Mice homozygous for a targeted deletion of COX-2 (-/-) were compared with wild-type littermates (+/+). Somatic and kidney growth and renal histology were studied at the day of birth and at a number of postnatal ages. Systolic blood pressure, urinalysis, urine osmolality, serum and urine chemistries, and inulin clearance were evaluated in adult animals. RESULTS: Beginning at postnatal day 10 (PN10), kidney growth was suppressed in -/- animals, while somatic growth and heart growth were unaffected. By PN10, -/- kidneys had thin nephrogenic cortexes and crowded, small, subcapsular glomeruli. The pathology increased with age with progressive outer cortical dysplasia, cystic subcapsular glomeruli, loss of proximal tubular mass, and tubular atrophy and cyst formation. Adult -/- kidneys had profound diffuse tubular cyst formation, outer cortical glomerular hypoplasia and periglomerular fibrosis, inner cortical nephron hypertrophy, and diffuse interstitial fibrosis. The glomerular filtration rate was reduced by more than 50% in -/- animals (6.82 +/- 0.65 mL/min/kg) compared with wild-type controls (14.7 +/- 1.01 mL/min/kg, P < 0. 001). Plasma blood urea nitrogen and creatinine were elevated in null animals compared with controls. Blood pressure, urinalysis, urine osmolality, and other plasma chemistries were unaffected by the deletion of COX-2. CONCLUSIONS: Deficiency of COX-2 results in progressive and specific renal architectural disruption and functional deterioration beginning in the final phases of nephrogenesis. Tissue-specific and time-dependent expression of COX-2 appears necessary for normal postnatal renal development and the maintenance of normal renal architecture and function.
Asunto(s)
Isoenzimas/genética , Riñón/anomalías , Riñón Displástico Multiquístico/enzimología , Riñón Displástico Multiquístico/genética , Prostaglandina-Endoperóxido Sintasas/genética , Animales , Presión Sanguínea , Nitrógeno de la Urea Sanguínea , Creatinina/sangre , Creatinina/orina , Ciclooxigenasa 2 , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Ingestión de Líquidos , Electrólitos/sangre , Electrólitos/orina , Femenino , Genotipo , Tasa de Filtración Glomerular , Inulina/farmacocinética , Riñón/enzimología , Riñón/crecimiento & desarrollo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Riñón Displástico Multiquístico/patología , Tamaño de los Órganos , Concentración Osmolar , Fenotipo , Embarazo , Urinálisis , Orina/químicaRESUMEN
Homologous recombination (gene targeting) has many desirable features for gene therapy, because it can precisely correct mutant genes and restore their normal expression, and random nonhomologous integration of DNA is infrequent in cells in which homologous recombination has occurred. There are, however, no reports of attempts to use homologous recombination to correct mutant genes in normal hematopoietic stem cells (HSCs), which are prime cells for therapy of a variety of hematological and other conditions, presumably because of their low abundance and uncertainty that homologous recombination can occur at a usable frequency in these cells. The experiments reported here encourage optimism in this respect by demonstrating targeted correction of a defective hypoxanthine phosphoribosyltransferase gene in hematopoietic progenitor cells that can form colonies in methylcellulose culture. These clonogenic cells are in the same lineage as HSCs but are more abundant and more mature and so less pluripotent. Corrected colonies were identified by their survival in selective medium after electroporation of correcting DNA into unfractionated mouse bone marrow cells and were confirmed by reverse transcription-PCR and sequencing. The observed frequency (4.4 +/- 3.3 x 10(-5) per treated clonogenic cell) is the same as in embryonic stem cells (2.3 +/- 0.4 x 10(-5)) with the same DNA and mutation. These data suggest that gene targeting to correct mutant genes eventually will prove feasible in HSCs capable of long-term bone marrow reconstitution.
Asunto(s)
Marcación de Gen , Células Madre Hematopoyéticas/metabolismo , Animales , Secuencia de Bases , ADN , Humanos , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido NucleicoRESUMEN
Two isoforms of cyclooxygenase (COX) are known, and to date most studies have implicated COX-2, rather than COX-1, as the isoform involved in colon carcinogenesis. In the present study, we show that homologous disruption of either Ptgs-1 or Ptgs-2 (genes coding for COX-1 or COX-2, respectively) reduced polyp formation in Min/+ mice by approximately 80%. Only COX-1 protein was immunohistochemically detected in normal intestinal tissue, whereas both COX-1 and variable levels of COX-2 protein were detected in polyps. Prostaglandin E2 was increased in polyps compared with normal tissue, and both COX-1 and COX-2 contributed to the PGE2 produced. The results indicate that COX-1, as well as COX-2, plays a key role in intestinal tumorigenesis and that COX-1 may also be a chemotherapeutic target for nonsteroidal anti-inflammatory drugs.
Asunto(s)
Neoplasias Intestinales/enzimología , Neoplasias Intestinales/prevención & control , Pólipos Intestinales/enzimología , Pólipos Intestinales/prevención & control , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintasas/genética , Animales , Cruzamientos Genéticos , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Dinoprostona/biosíntesis , Femenino , Neoplasias Intestinales/genética , Pólipos Intestinales/genética , Intestinos/enzimología , Isoenzimas/deficiencia , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Prostaglandina-Endoperóxido Sintasas/deficiencia , Valores de ReferenciaRESUMEN
In a previous study, it was found that a truncated erythropoietin receptor transgene (tEpoR tg) enables multilineage hematopoietic progenitor amplification after treatment with erythropoietin (epo) in vitro and in vivo. This study used competitive bone marrow (BM) repopulation to show that tEpoR tg facilitates transplantation by hematopoietic stem cells (HSC). Individual multilineage colonies, committed myeloid progenitor colonies, and lymphoid colonies (pre-B colony-forming units) were grown from the marrow of animals 6 months after they received a 50/50 mixture of transgene and wild-type BM cells. In epo-treated recipients, the transgene-bearing cells significantly outcompeted the wild-type cells (84%-100% versus 16%-0%, respectively). In recipients treated with phosphate-buffered saline, the repopulation was minimally different from the donor mixture (49%-64% transgene versus 51%-36% wild-type). The epo-induced repopulation advantage is maintained in secondary transplants. In addition, neither accelerated HSC depletion nor uncontrollable proliferation occurred during epo-stimulated serial transplants of transgene-containing BM. Thus, the tEpoR tg functions in a benign fashion in HSC and allows for a significant and controllable repopulation advantage in vivo without excessive HSC depletion relative to wild-type BM. (Blood. 2000;95:3710-3715)
Asunto(s)
Células de la Médula Ósea/citología , Trasplante de Médula Ósea/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Receptores de Eritropoyetina/fisiología , Actinas/genética , Animales , Eritropoyetina/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Receptores de Eritropoyetina/genéticaRESUMEN
We have used homologous recombination to disrupt the mouse gene coding for the NaK2Cl cotransporter (NKCC2) expressed in kidney epithelial cells of the thick ascending limb and macula densa. This gene is one of several that when mutated causes Bartter's syndrome in humans, a syndrome characterized by severe polyuria and electrolyte imbalance. Homozygous NKCC2-/- pups were born in expected numbers and appeared normal. However, by day 1 they showed signs of extracellular volume depletion (hematocrit 51%; wild type 37%). They subsequently failed to thrive. By day 7, they were small and markedly dehydrated and exhibited renal insufficiency, high plasma potassium, metabolic acidosis, hydronephrosis of varying severity, and high plasma renin concentrations. None survived to weaning. Treatment of -/- pups with indomethacin from day 1 prevented growth retardation and 10% treated for 3 weeks survived, although as adults they exhibited severe polyuria (10 ml/day), extreme hydronephrosis, low plasma potassium, high blood pH, hypercalciuria, and proteinuria. Wild-type mice treated with furosemide, an inhibitor of NaK2Cl cotransporters, have a phenotype similar to the indomethacin-rescued -/- adults except that hydronephrosis was mild. The polyuria, hypercalciuria, and proteinuria of the -/- adults and furosemide-treated wild-type mice were unresponsive to inhibitors of the renin angiotensin system, vasopressin, and further indomethacin. Thus absence of NKCC2 in the mouse causes polyuria that is not compensated elsewhere in the nephron. The NKCC2 mutant animals should be valuable for uncovering new pathophysiologic and therapeutic aspects of genetic disturbances in water and electrolyte recovery by the kidney.