RESUMEN
Defects in the biosynthesis of phospholipids and neutral lipids are associated with cell membrane dysfunction, disrupted energy metabolism, and diseases including lipodystrophy. In these pathways, the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) enzymes transfer a fatty acid to the sn-2 carbon of sn-1-acylglycerol-3-phosphate (lysophosphatidic acid) to form sn-1, 2-acylglycerol-3-phosphate [phosphatidic acid (PA)]. PA is a precursor for key phospholipids and diacylglycerol. AGPAT1 and AGPAT2 are highly homologous isoenzymes that are both expressed in adipocytes. Genetic defects in AGPAT2 cause congenital generalized lipodystrophy, indicating that AGPAT1 cannot compensate for loss of AGPAT2 in adipocytes. To further explore the physiology of AGPAT1, we characterized a loss-of-function mouse model (Agpat1-/-). The majority of Agpat1-/- mice died before weaning and had low body weight and low plasma glucose levels, independent of plasma insulin and glucagon levels, with reduced percentage of body fat but not generalized lipodystrophy. These mice also had decreased hepatic messenger RNA expression of Igf-1 and Foxo1, suggesting a decrease in gluconeogenesis. In male mice, sperm development was impaired, with a late meiotic arrest near the onset of round spermatid production, and gonadotropins were elevated. Female mice showed oligoanovulation yet retained responsiveness to gonadotropins. Agpat1-/- mice also demonstrated abnormal hippocampal neuron development and developed audiogenic seizures. In summary, Agpat1-/- mice developed widespread disturbances of metabolism, sperm development, and neurologic function resulting from disrupted phospholipid homeostasis. AGPAT1 appears to serve important functions in the physiology of multiple organ systems. The Agpat1-deficient mouse provides an important model in which to study the contribution of phospholipid and triacylglycerol synthesis to physiology and diseases.
Asunto(s)
1-Acilglicerol-3-Fosfato O-Aciltransferasa/genética , Glicerol-3-Fosfato O-Aciltransferasa/genética , Infertilidad/genética , Enfermedades Metabólicas/genética , Enfermedades del Sistema Nervioso/genética , Animales , Células Cultivadas , Femenino , Gluconeogénesis/genética , Metabolismo de los Lípidos/genética , Lipogénesis/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reproducción/genéticaRESUMEN
Autosomal recessive polycystic kidney disease, an inherited disorder characterized by the formation of cysts in renal collecting ducts and biliary dysgenesis, is caused by mutations of the polycystic kidney and hepatic disease 1 (PKHD1) gene. Expression of PKHD1 is tissue specific and developmentally regulated. Here, we show that a 2.0-kb genomic fragment containing the proximal promoter of mouse Pkhd1 directs tissue-specific expression of a lacZ reporter gene in transgenic mice. LacZ is expressed in renal collecting ducts beginning during embryonic development but is not expressed in extrarenal tissues. The Pkhd1 promoter contains a binding site for the transcription factor hepatocyte nuclear factor (HNF)-1ß, which is required for activity in transfected cells. Mutation of the HNF-1ß-binding site abolishes the expression of the lacZ reporter gene in renal collecting ducts. Transgenes containing the 2.0-kb promoter and 2.7 kb of additional genomic sequence extending downstream to the second exon are expressed in the kidney, intrahepatic bile ducts, and male reproductive tract. This pattern overlaps with the endogenous expression of Pkhd1 and coincides with sites of expression of HNF-1ß. We conclude that the proximal 2.0-kb promoter is sufficient for tissue-specific expression of Pkhd1 in renal collecting ducts in vivo and that HNF-1ß is required for Pkhd1 promoter activity in collecting ducts. Additional genomic sequences located from exons 1-2 or elsewhere in the gene locus are required for expression in extrarenal tissues.
Asunto(s)
Túbulos Renales Colectores/fisiología , Riñón Poliquístico Autosómico Recesivo/fisiopatología , Regiones Promotoras Genéticas/fisiología , Receptores de Superficie Celular/fisiología , Animales , Sistema Biliar/citología , Sistema Biliar/fisiología , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/fisiología , Factor Nuclear 1-beta del Hepatocito/fisiología , Túbulos Renales Colectores/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Riñón Poliquístico Autosómico Recesivo/genética , Regiones Promotoras Genéticas/genética , Receptores de Superficie Celular/genética , Sistema Urogenital/citología , Sistema Urogenital/fisiologíaRESUMEN
OBJECTIVE: To ascertain whether engineered expression of kallikreins within the kidneys, using an inducible Cre/loxP system, can ameliorate murine lupus nephritis. METHODS: In mice with a lupus-prone genetic background, we engineered the expression of tamoxifen-inducible Cre recombinase under the control of a kidney-specific promoter whose activation initiates murine kallikrein-1 expression within the kidneys. These transgenic mice were injected with either tamoxifen or vehicle at age 2 months and then were monitored for 8 months for kallikrein expression and disease. RESULTS: Elevated expression of kallikrein was detected in the kidney and urine of tamoxifen-injected mice but not in controls. At age 10 months, all vehicle-injected mice developed severe lupus nephritis, as evidenced by increased proteinuria (mean ± SD 13.43 ± 5.65 mg/24 hours), increased blood urea nitrogen (BUN) and serum creatinine levels (39.86 ± 13.45 mg/dl and 15.23 ± 6.89 mg/dl, respectively), and severe renal pathology. In contrast, the tamoxifen-injected mice showed significantly reduced proteinuria (6.6 ± 4.12 mg/24 hours), decreased BUN and serum creatinine levels (15.71 ± 8.17 mg/dl and 6.64 ± 3.39 mg/dl, respectively), and milder renal pathology. Tamoxifen-induced up-regulation of renal kallikrein expression increased nitric oxide production and dampened renal superoxide production and inflammatory cell infiltration, alluding to some of the pathways through which kallikreins may be operating within the kidneys. CONCLUSION: Local expression of kallikreins within the kidney has the capacity to dampen lupus nephritis, possibly by modulating inflammation and oxidative stress.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Túbulos Renales/fisiología , Nefritis Lúpica/genética , Estrés Oxidativo/fisiología , Calicreínas de Tejido/genética , Animales , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Integrasas/genética , Túbulos Renales/citología , Operón Lac/genética , Nefritis Lúpica/metabolismo , Nefritis Lúpica/fisiopatología , Masculino , Ratones , Ratones Congénicos , Ratones Endogámicos , Ratones Transgénicos , Óxido Nítrico/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/toxicidad , Superóxidos/metabolismo , Tamoxifeno/toxicidad , Calicreínas de Tejido/metabolismoRESUMEN
Serum and glucocorticoid-regulated kinase 2 (sgk2) is 80% identical to the kinase domain of sgk1, an important mediator of mineralocorticoid-regulated sodium (Na(+)) transport in the distal nephron of the kidney. The expression pattern and role in renal function of sgk2 are virtually uncharacterized. In situ hybridization and immunohistochemistry of rodent kidney coupled with real-time RT-PCR of microdissected rat kidney tubules showed robust sgk2 expression in the proximal straight tubule and thick ascending limb of the loop of Henle. Sgk2 expression was minimal in distal tubule cells with aquaporin-2 immunostaining but significant in proximal tubule cells with Na(+)/H(+) exchanger 3 (NHE3) immunostaining. To ascertain whether mineralocorticoids regulate expression of sgk2 in a manner similar to sgk1, we examined sgk2 mRNA expression in the kidneys of adrenalectomized rats treated with physiological doses of aldosterone together with the glucocorticoid receptor antagonist RU486. Northern blot analysis and in situ hybridization showed that, unlike sgk1, sgk2 expression in the kidney was not altered by aldosterone treatment. Based on the observation that sgk2 is expressed in proximal tubule cells that also express NHE3, we asked whether sgk2 regulates NHE3 activity. We heterologously expressed sgk2 in opossum kidney (OKP) cells and measured Na(+)/H(+) exchange activity by Na(+)-dependent cell pH recovery. Constitutively active sgk2, but not sgk1, stimulated Na(+)/H(+) exchange activity by >30%. Moreover, the sgk2-mediated increase in Na(+)/H(+) exchange activity correlated with an increase in cell surface expression of NHE3. Together, these results suggest that the pattern of expression, regulation, and role of sgk2 within the mammalian kidney are distinct from sgk1 and that sgk2 may play a previously unrecognized role in the control of transtubular Na(+) transport through NHE3 in the proximal tubule.
Asunto(s)
Proteínas Inmediatas-Precoces/biosíntesis , Proteínas Serina-Treonina Quinasas/biosíntesis , Intercambiadores de Sodio-Hidrógeno/metabolismo , Aldosterona/farmacología , Animales , Línea Celular , Regulación de la Expresión Génica , Proteínas Inmediatas-Precoces/fisiología , Hibridación in Situ , Riñón/efectos de los fármacos , Riñón/metabolismo , Túbulos Renales/metabolismo , Masculino , Ratones , Zarigüeyas , Proteínas Serina-Treonina Quinasas/fisiología , Ratas , Ratas Sprague-Dawley , Intercambiador 3 de Sodio-HidrógenoRESUMEN
Smad2 and Smad3 interact and mediate TGF-beta signaling. Although Smad3 promotes fibrosis, the role of Smad2 in fibrogenesis is largely unknown. In this study, conditional deletion of Smad2 from the kidney tubular epithelial cells markedly enhanced fibrosis in response to unilateral ureteral obstruction. In vitro, Smad2 knockdown in tubular epithelial cells increased expression of collagen I, collagen III, and TIMP-1 and decreased expression of the matrix-degrading enzyme MMP-2 in response to TGF-beta1 compared with similarly treated wild-type cells. We obtained similar results in Smad2-knockout fibroblasts. Mechanistically, Smad2 deletion promoted fibrosis through enhanced TGF-beta/Smad3 signaling, evidenced by greater Smad3 phosphorylation, nuclear translocation, promoter activity, and binding of Smad3 to a collagen promoter (COL1A2). Moreover, deletion of Smad2 increased autoinduction of TGF-beta1. Conversely, overexpression of Smad2 attenuated TGF-beta1-induced Smad3 phosphorylation and collagen I matrix expression in tubular epithelial cells. In conclusion, in contrast to Smad3, Smad2 protects against TGF-beta-mediated fibrosis by counteracting TGF-beta/Smad3 signaling.
Asunto(s)
Riñón/patología , Proteína Smad2/fisiología , Proteína smad3/fisiología , Factor de Crecimiento Transformador beta1/fisiología , Animales , Línea Celular , Colágeno Tipo I/metabolismo , Femenino , Fibrosis , Túbulos Renales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , Inhibidor Tisular de Metaloproteinasa-1/genética , Obstrucción Ureteral/patologíaRESUMEN
Highly activated microglia and followed excessive expression of inflammatory cytokines are associated with neuroexcitotoxic injuries. We use electrophysiological techniques, ELISA, western-blot, RT-PCR assay and TUNEL method to explore whether over-produced tumor necrosis factor alpha (TNFalpha) released from activated microglia results in neuronal injuries, and further causes apoptosis through increasing excitotoxicity of hippocampal neurons. Our data showed that kainic acid (KA) activated microglia highly expressed TNFalpha, mRNA and protein. KA activated microglia conditioned media ((KA-MCM) significantly enhanced the amplitude of the population spike at rat's hippocampal CA3 region. It also increased the Ca(2+) current amplitude and density in cultured hippocampal neurons, as well as the high expression of NMDAR1, iNOS, and caspase 3 mRNA and protein at both hippocampal neurons and tissues. KA-MCM also increased TUNEL-positive cells in hippocampal neurons, whereas addition of anti-TNFalpha to the KA-MCM before its application significantly reduced those effects. These studies suggest that TNFalpha derived from KA activated microglia increases excitotoxicity of hippocampal neurons, and might induce neuronal apoptosis in vitro and in vivo.
Asunto(s)
Ácido Kaínico/farmacología , Microglía/efectos de los fármacos , Neuronas/patología , Factor de Necrosis Tumoral alfa/biosíntesis , Potenciales de Acción , Animales , Apoptosis/efectos de los fármacos , Calcio/fisiología , Canales de Calcio/fisiología , Caspasa 3/biosíntesis , Caspasa 3/genética , Células Cultivadas , Medios de Cultivo Condicionados , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Microglía/metabolismo , Neuronas/fisiología , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico Sintasa de Tipo II/genética , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/biosíntesis , Receptores de N-Metil-D-Aspartato/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Mutations in 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) cause congenital generalized lipodystrophy. To understand the molecular mechanisms underlying the metabolic complications associated with AGPAT2 deficiency, Agpat2 null mice were generated. Agpat2(-/-) mice develop severe lipodystrophy affecting both white and brown adipose tissue, extreme insulin resistance, diabetes, and hepatic steatosis. The expression of lipogenic genes and rates of de novo fatty acid biosynthesis were increased approximately 4-fold in Agpat2(-/-) mouse livers. The mRNA and protein levels of monoacylglycerol acyltransferase isoform 1 were markedly increased in the livers of Agpat2(-/-) mice, suggesting that the alternative monoacylglycerol pathway for triglyceride biosynthesis is activated in the absence of AGPAT2. Feeding a fat-free diet reduced liver triglycerides by approximately 50% in Agpat2(-/-) mice. These observations suggest that both dietary fat and hepatic triglyceride biosynthesis via a monoacylglycerol pathway may contribute to hepatic steatosis in Agpat2(-/-) mice.
Asunto(s)
1-Acilglicerol-3-Fosfato O-Aciltransferasa/metabolismo , Hígado Graso/metabolismo , Resistencia a la Insulina/genética , Lipodistrofia Generalizada Congénita/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferasa/genética , Tejido Adiposo/metabolismo , Animales , Metabolismo Energético , Lipodistrofia Generalizada Congénita/genética , Ratones , Ratones Noqueados , Modelos Animales , Triglicéridos/biosíntesisRESUMEN
Polycystic kidney disease (PKD) is an inherited disorder that is characterized by the accumulation of cysts in the renal parenchyma and progressive decline in renal function. Recent studies suggest that PKD arises from abnormalities of the primary cilium. We have previously shown that kidney-specific inactivation of the ciliogenic gene Kif3a during embryonic development produces kidney cysts and renal failure. Here, we used tamoxifen-inducible, kidney-specific gene targeting to inactivate Kif3a in the postnatal mouse kidney. Kidney-specific inactivation of Kif3a in newborn mice resulted in the loss of primary cilia and produced kidney cysts primarily in the loops of Henle, whereas inactivation in adult mice did not lead to the rapid development of cysts despite a comparable loss of primary cilia. The age-dependence and locations of the cysts suggested that cyst formation required increased rates of cell proliferation. To test this possibility, we stimulated cell proliferation in the adult kidney by inducing acute kidney injury and tubular regeneration. Acute kidney injury induced cyst formation in adult Kif3a mutant mice. Analysis of pre-cystic tubules in Kif3a mutant mice showed that the loss of cilia did not stimulate cell proliferation but instead resulted in aberrant planar cell polarity as manifested by abnormalities in the orientation of cell division. We conclude that primary cilia are required for the maintenance of planar cell polarity in the mammalian kidney and that acute kidney injury exacerbates cystic disease.
Asunto(s)
Quistes/genética , Quistes/patología , Cinesinas/genética , Asa de la Nefrona/anomalías , Asa de la Nefrona/patología , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/patología , Enfermedad Aguda , Animales , Polaridad Celular , Cilios , Quistes/etiología , Humanos , Enfermedades Renales/inducido químicamente , Enfermedades Renales/complicaciones , Cinesinas/antagonistas & inhibidores , Ratones , Ratones Transgénicos , Enfermedades Renales Poliquísticas/etiologíaRESUMEN
Hepatocyte nuclear factor-1beta (HNF-1beta) is a Pit-1/Oct-1/Unc-86 (POU)/homeodomain-containing transcription factor that regulates tissue-specific gene expression in the kidney, liver, pancreas, and other epithelial organs. Mutations of HNF-1beta produce maturity-onset diabetes of the young type 5 (MODY5) and are associated with congenital cystic abnormalities of the kidney. Transgenic mice expressing mutant HNF-1beta under the control of a kidney-specific promoter develop kidney cysts and renal failure, which is similar to the phenotype of humans with MODY5. Similarly, kidney-specific deletion of HNF-1beta using Cre/loxP recombination results in renal cyst formation. HNF-1beta directly regulates the Pkhd1 promoter. HNF-1beta mutant mice show decreased expression of Pkhd1, the gene that is mutated in humans with autosomal-recessive polycystic kidney disease (ARPKD). These studies demonstrate that HNF-1beta is required for the development of the mammalian kidney. They establish a previously unrecognized link between two renal cystic diseases, MODY5 and ARPKD, and suggest that the mechanism of cyst formation in humans with mutations of HNF-1beta involves down-regulation of PKHD1 gene transcription.
Asunto(s)
Factor Nuclear 1-beta del Hepatocito/fisiología , Enfermedades Renales Quísticas/fisiopatología , Riñón/anomalías , Animales , Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 1-beta del Hepatocito/genética , Humanos , Enfermedades Renales Quísticas/congénitoRESUMEN
Hepatocyte nuclear factor-1beta (HNF-1beta) is a homeodomain-containing transcription factor that regulates tissue-specific gene expression in the kidney and other epithelial organs. Mutations of HNF-1beta produce congenital cystic abnormalities of the kidney, and previous studies showed that HNF-1beta regulates the expression of the autosomal recessive polycystic kidney disease (ARPKD) gene, Pkhd1. Here we show that the C-terminal region of HNF-1beta contains an activation domain that is functional when fused to a heterologous DNA-binding domain. An HNF-1beta deletion mutant lacking the C-terminal domain interacts with wild-type HNF-1beta, binds DNA, and functions as a dominant-negative inhibitor of a chromosomally integrated Pkhd1 promoter. The activation of the Pkhd1 promoter by wild-type HNF-1beta is stimulated by sodium butyrate or coactivators CREB (cAMP-response element)-binding protein (CBP) and P/CAF. The interaction with CBP and P/CAF requires the C-terminal domain. Expression of an HNF-1beta C-terminal deletion mutant in transgenic mice produces renal cysts, increased cell proliferation, and dilatation of the ureter similar to mice with kidney-specific inactivation of HNF-1beta. Pkhd1 expression is inhibited in cystic collecting ducts but not in non-cystic proximal tubules, despite transgene expression in this nephron segment. We conclude that the C-terminal domain of HNF-1beta is required for the activation of the Pkhd1 promoter. Deletion mutants lacking the C-terminal domain function as dominant-negative mutants, possibly by preventing the recruitment of histone acetylases to the promoter. Cyst formation correlates with inhibition of Pkhd1 expression, which argues that mutations of HNF-1beta produce kidney cysts by down-regulating the ARPKD gene, Pkhd1. Expression of HNF-1alpha in proximal tubules may protect against cystogenesis.
Asunto(s)
Proteínas de Unión al ADN/química , Enfermedades Renales Quísticas/metabolismo , Riñón/metabolismo , Receptores de Superficie Celular/fisiología , Factores de Transcripción/química , Transcripción Genética , Acetiltransferasas/metabolismo , Animales , Sitios de Unión , Butiratos/farmacología , Proliferación Celular , ADN/química , Dimerización , Regulación hacia Abajo , Células Epiteliales/citología , Eliminación de Gen , Genes Dominantes , Genes Reporteros , Células HeLa , Factor Nuclear 1-beta del Hepatocito , Histona Acetiltransferasas , Humanos , Inmunoprecipitación , Isobutiratos , Riñón/citología , Túbulos Renales/citología , Lectinas , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Mutación , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Receptores de Superficie Celular/química , TransfecciónRESUMEN
Hepatocyte nuclear factor-1beta (HNF-1beta) is a Pit-1, Oct-1/2, UNC-86 (POU)/homeodomain-containing transcription factor that regulates tissue-specific gene expression in the liver, kidney, and other organs. Humans with autosomal dominant mutations of HNF-1beta develop maturity-onset diabetes of the young type 5 (MODY5) and congenital cystic abnormalities of the kidney. Autosomal recessive polycystic kidney disease (ARPKD) is an inherited cystic disorder that produces renal failure in infants and children and is caused by mutations of PKHD1. The proximal promoter of the mouse Pkhd1 gene contains an evolutionarily conserved HNF-1-binding site that is located near a region of deoxyribonuclease hypersensitivity. HNF-1beta and the structurally related HNF-1alpha bind specifically to the Pkhd1 promoter and stimulate gene transcription. Mutations of the HNF-1 site or expression of a dominant-negative HNF-1beta mutant inhibit Pkhd1 promoter activity in transfected cells. Transgenic mice expressing a dominant-negative HNF-1beta mutant under the control of a kidney-specific promoter develop renal cysts, similarly to humans with MODY5. Pkhd1 transcripts are absent in the cells lining the cysts but are present in morphologically normal surrounding tubules. These studies identify a link between two cystic disease genes, HNF1beta (MODY5) and PKHD1 (ARPKD). HNF-1beta directly regulates the transcription of Pkhd1, and inhibition of PKHD1 gene expression may contribute to the formation of renal cysts in humans with MODY5.
Asunto(s)
Proteínas de Unión al ADN/genética , Enfermedades Renales Quísticas/genética , Mutación , Receptores de Superficie Celular/genética , Factores de Transcripción/genética , Animales , Secuencia de Bases , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Factor Nuclear 1-beta del Hepatocito , Humanos , Enfermedades Renales Quísticas/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Receptores de Superficie Celular/biosíntesis , Factores de Transcripción/metabolismoRESUMEN
Mutations in cystic kidney disease genes represent a major genetic cause of end-stage renal disease. However, the molecular cascades controlling the expression of these genes are still poorly understood. Hepatocyte Nuclear Factor 1beta (HNF1beta) is a homeoprotein predominantly expressed in renal, pancreatic and hepatic epithelia. We report here that mice with renal-specific inactivation of HNF1beta develop polycystic kidney disease. We show that renal cyst formation is accompanied by a drastic defect in the transcriptional activation of Umod, Pkhd1 and Pkd2 genes, whose mutations are responsible for distinct cystic kidney syndromes. In vivo chromatin immunoprecipitation experiments demonstrated that HNF1beta binds to several DNA elements in murine Umod, Pkhd1, Pkd2 and Tg737/Polaris genomic sequences. Our results uncover a direct transcriptional hierarchy between HNF1beta and cystic disease genes. Interestingly, most of the identified HNF1beta target gene products colocalize to the primary cilium, a crucial organelle that plays an important role in controlling the proliferation of tubular cells. This may explain the increased proliferation of cystic cells in MODY5 patients carrying autosomal dominant mutations in HNF1beta.
Asunto(s)
Regulación de la Expresión Génica , Enfermedades Renales Poliquísticas/genética , Transcripción Genética , Animales , Proliferación Celular , Cilios/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factor Nuclear 1-beta del Hepatocito , Humanos , Riñón/citología , Riñón/patología , Riñón/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Mucoproteínas/genética , Mucoproteínas/metabolismo , Enfermedades Renales Poliquísticas/metabolismo , Enfermedades Renales Poliquísticas/patología , Unión Proteica , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Canales Catiónicos TRPP , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Uréter/patología , UromodulinaRESUMEN
Ksp-cadherin is a unique, tissue-specific member of the cadherin family of cell adhesion molecules that is expressed exclusively in tubular epithelial cells in the kidney and developing genitourinary (GU) tract. Transgenic mice carrying 3425 bp of the Ksp-cadherin 5' flanking region linked to a lacZ reporter gene express beta-galactosidase exclusively in the kidney, although the expression pattern is incomplete (Am J Physiol 277: F599-F610, 1999). To further define the region that mediates tissue-specific expression, transgenic mice carrying 1341 bp or 324 bp of the 5' flanking region linked to a green fluorescent protein (GFP) reporter gene were produced. Transgenic mice carrying 1341 bp of the 5' flanking region expressed GFP in all embryonic tissues that endogenously express Ksp-cadherin, including the ureteric bud, Wolffian duct, Müllerian duct, and developing tubules in the mesonephros and metanephros. In the adult kidney, GFP was highly expressed in thick ascending limbs of Henle's loops and collecting ducts and weakly expressed in proximal tubules and Bowman's capsules. Transgenic mice carrying 324 bp of the 5' flanking region exhibited expression exclusively in tubular epithelial cells in the developing kidney and GU tract. Immunoblot analysis showed that the expression of GFP was restricted to the kidney in adult mice. Taken together, these results demonstrate that 324 bp of the Ksp-cadherin 5' flanking region is sufficient to direct epithelial-specific expression in the developing kidney and GU tract. Transgenic mice that express GFP in the mesonephros, metanephros, ureteric bud, and sex ducts may be useful for cell lineage studies.
Asunto(s)
Cadherinas/genética , Regulación del Desarrollo de la Expresión Génica , Riñón/embriología , Proteínas Luminiscentes/genética , Sistema Urogenital/embriología , Envejecimiento/fisiología , Animales , Animales Recién Nacidos/fisiología , Embrión de Mamíferos/fisiología , Genes Reporteros , Ligamiento Genético , Proteínas Fluorescentes Verdes , Riñón/fisiología , Túbulos Renales/fisiología , Ratones , Ratones Endogámicos ICR , Ratones Endogámicos , Ratones Transgénicos/genética , Regiones Promotoras Genéticas , TransgenesRESUMEN
Ksp-cadherin is a unique, tissue-specific member of the cadherin family of cell adhesion molecules that is expressed in tubular epithelial cells in the kidney and developing genitourinary (GU) tract. It has recently been shown that a 1341-bp fragment of the 5' flanking region containing the Ksp-cadherin gene promoter can recapitulate the complete expression pattern of the gene in the developing kidney and GU tract. Similar to the endogenous Ksp-cadherin gene, transgenes containing 1341 bp of the 5' flanking region are expressed in developing nephrons, ureteric bud, mesonephric tubules, Wolffian duct, and Müllerian duct. In adult mice, the expression is restricted to renal tubules. In the current study, Ksp1.3/Cre transgenic mice carrying 1329 bp of the Ksp-cadherin 5' flanking region linked to the Cre recombinase gene were produced. Adult transgenic mice expressed Cre recombinase in renal tubules, especially collecting ducts and thick ascending limbs of Henle's loops. Transgenic embryos expressed Cre recombinase in the branching ureteric bud, developing renal tubules, and sex ducts. Ksp1.3/Cre transgenic mice were crossed with mice carrying a lacZ reporter gene that is activated by Cre/lox-mediated genetic recombination. Bitransgenic progeny expressed lacZ exclusively in renal tubules, mesonephric tubules, ureteric bud, developing ureter, and Wolffian duct. These results demonstrate that Ksp1.3/Cre transgenic mice express Cre recombinase exclusively in the kidney and developing GU tract and can mediate epithelial-specific Cre/lox recombination in these tissues. Ksp1.3/Cre transgenic mice should be useful for cell lineage studies and kidney-specific gene targeting.