RESUMEN

Renal α2-adrenoceptors have been reported to play a role in the regulation of urinary output, renin secretion, and water and sodium excretion in the kidneys. However, the distribution of α2-adrenoceptor subtypes in the kidneys remains unclear. In this study, we aimed to investigate the localization of α2-adrenoceptor subtypes in rat kidneys using 8-week-old Sprague-Dawley rats. Immunofluorescence imaging revealed that both α2A- and α2B-adrenoceptors were expressed in the basolateral, but not apical, membrane of the epithelial cells of the proximal tubules. We also found that α2A- and α2B-adrenoceptors were not expressed in the glomeruli, collecting ducts, or the descending limb of the loop of Henle and vasa recta. In contrast, α2C-adrenoceptors were found to be localized in the glomeruli and lumen of the cortical and medullary collecting ducts. These results suggest that noradrenaline may act on the basement membrane of the proximal tubules through α2A- and α2B-adrenoceptors. Moreover, noradrenaline may be involved in the regulation of glomerular filtration and proteinuria through the induction of morphological changes in mesangial cells and podocytes via α2C-adrenoceptors. In the collecting ducts, urinary noradrenaline may regulate morphological changes of the microvilli through α2C-adrenoceptors. Our findings provide an immunohistochemical basis for understanding the cellular targets of α2-adrenergic regulation in the kidneys. This may be used to devise therapeutic strategies targeting α2-adrenoceptors.

Asunto(s)

Receptores Adrenérgicos alfa 2 , Roedores , Animales , Riñón , Norepinefrina , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos alfa 2/fisiología

RESUMEN

In the present study, we investigated the renoprotective effects of long-term treatment with yohimbine, an α2-adrenoceptor inhibitor, in a 5/6 nephrectomy-induced chronic kidney disease (CKD) rat model. Male Sprague-Dawley rats were randomly allocated into the following groups: sham-operated, 5/6-nephrectomized (5/6 Nx), 5/6 Nx + low or high dose of yohimbine (0.3 or 3.0 mg/L in drinking water, respectively), and 5/6 Nx + hydralazine (250 mg/L in drinking water). The 5/6 Nx group presented with renal dysfunction, hypertension, noradrenaline overproduction, and histopathological injuries. Blood pressure decreased in both the yohimbine- and hydralazine-treated groups. Treatment with high dose of yohimbine, but not hydralazine, apparently attenuated urinary protein excretion and noradrenaline concentration of renal venous plasma. Renal fibrosis and upregulated fibrosis-related gene expression were suppressed by high dose of yohimbine. Furthermore, yohimbine, but not hydralazine, treatment ameliorated the urinary concentration ability. These findings suggest that long-term yohimbine treatment can be a useful therapeutic option to prevent the progression of CKD.

Asunto(s)

Antagonistas de Receptores Adrenérgicos alfa 2/administración & dosificación , Nefrectomía/efectos adversos , Insuficiencia Renal Crónica/prevención & control , Yohimbina/administración & dosificación , Animales , Vesícula/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Epidermólisis Ampollosa/patología , Fibrosis , Hidralazina/administración & dosificación , Masculino , Norepinefrina/metabolismo , Enfermedades Periodontales/patología , Trastornos por Fotosensibilidad/patología , Proteinuria/etiología , Proteinuria/prevención & control , Ratas Sprague-Dawley , Insuficiencia Renal Crónica/etiología , Insuficiencia Renal Crónica/patología , Insuficiencia Renal Crónica/fisiopatología

RESUMEN

Sepsis-induced acute kidney injury (AKI) is frequently observed in the intensive care unit. We previously revealed that yohimbine, an α2-adrenoceptor antagonist, has protective effects on renal ischemia/reperfusion injury-induced AKI in rats. This study aimed to investigate the renoprotective effect of yohimbine on lipopolysaccharide (LPS)-induced AKI in rats. Male Sprague Dawley rats were randomly divided into the following groups: Sham-operated group, LPS (10 mg/kg, i.p.) and LPS + yohimbine (0.1 or 0.5 mg/kg, i.p.). Kidney functional parameters of blood urea nitrogen (BUN) and plasma creatinine (Pcr) were aggravated in the LPS group. Administration of LPS decreased blood pressure. In addition, kidney injury molecule-1, inducible nitric oxide synthase (iNOS) and expression of various cytokines such as tumour necrosis factor-α, monocyte chemoattractant protein-1, and interleukin (IL)-6 were increased by LPS administration. Yohimbine treatment clearly ameliorated the damaged kidney function and low blood pressure due to LPS. Moreover, yohimbine suppressed cytokine mRNA and iNOS expression enhanced by LPS. However, anti-inflammatory cytokine IL-10 mRNA levels were augmented by yohimbine. Nuclear localization of nuclear factor-kappa B (NF-κB) in the kidney was observed 1 h after injection of LPS in rats. Yohimbine blocked the nuclear localization of NF-κB. In addition, phosphorylation of extracellular signal-regulated kinase (ERK) and cAMP response element binding protein (CREB) were enhanced with yohimbine. These results suggest that yohimbine can prevent LPS-induced sepsis associated with kidney injury by suppressing inflammatory cytokine and iNOS expression as well as enhancing IL-10 expression via ERK/CREB phosphorylation.

Asunto(s)

Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/tratamiento farmacológico , Lipopolisacáridos/farmacología , Yohimbina/farmacología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Lesión Renal Aguda/metabolismo , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Masculino , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Yohimbina/uso terapéutico

RESUMEN

Ischemia/reperfusion injury is the most common cause of acute kidney injury. We previously revealed that pre-treatment with yohimbine or JP-1302 attenuated renal ischemia/reperfusion injury by inhibition of α2C-adrenoceptor antagonist. The aim of the present study is to investigate the effects of post-treatment with JP-1302 on renal ischemia/reperfusion injury in rats. Male Sprague Dawley rats were randomly divided into four groups: sham operation, ischemia/reperfusion, pre-treatment with JP-1302 (3.0 mg/kg) and post-treatment with JP-1302 groups. In ischemia/reperfusion injury, renal functional parameters, such as blood urea nitrogen, plasma creatinine and creatinine clearance, deteriorated after reperfusion. Renal venous norepinephrine concentrations, as well as inflammatory molecules in the kidney increased after reperfusion. Both pre- and post-treatment with JP-1302 improved renal dysfunction, tissue damage, renal venous norepinephrine concentrations and inflammatory molecules expression in the kidney. In conclusion, these results suggest that post-treatment with JP-1302 protects on ischemia/reperfusion-induced acute kidney injury by suppressing cytokine upregulation via α2C-adrenoceptors.

Asunto(s)

Acridinas/farmacología , Lesión Renal Aguda/prevención & control , Antagonistas de Receptores Adrenérgicos alfa 2/farmacología , Piperazinas/farmacología , Daño por Reperfusión/tratamiento farmacológico , Acridinas/administración & dosificación , Antagonistas de Receptores Adrenérgicos alfa 2/administración & dosificación , Animales , Nitrógeno de la Urea Sanguínea , Creatinina/sangre , Citocinas/metabolismo , Esquema de Medicación , Masculino , Piperazinas/administración & dosificación , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos alfa 2/efectos de los fármacos , Receptores Adrenérgicos alfa 2/metabolismo , Daño por Reperfusión/complicaciones , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

Nephrotoxicity is a major adverse reaction of the anticancer drug, cisplatin. We investigated the renoprotective effects of the α2-adrenoceptor antagonist, yohimbine and selective α2C-adrenoceptor antagonist, JP-1302, in cisplatin-treated Sprague Dawley rats. Rats were given a single intravenous dose of 7.5 mg/kg cisplatin and then yohimbine or JP-1302 was administered intraperitoneally at 0.1 or 3 mg/kg/day, respectively, for four days. Renal functional parameters, such as blood urea nitrogen, plasma creatinine, creatinine clearance and renal venous norepinephrine concentrations were measured. Kidney tissue damage and tumour necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) mRNA levels were assessed after the animals were euthanized. Cisplatin treatment aggravated the kidney functional parameters of blood urea nitrogen, plasma creatinine and creatinine clearance. Renal venous norepinephrine concentrations were also elevated after cisplatin administration. Treatment with yohimbine or JP-1302 clearly ameliorated kidney function and cell apoptosis. These treatments suppressed elevated renal plasma norepinephrine, TNF-α, MCP-1 and cleaved caspase 3 expressions which occurred after administration of cisplatin. These results suggest that yohimbine can prevent cisplatin-induced renal toxicity associated with acute kidney injury by suppressing cytokine expression through α2C-adrenoceptors.

Asunto(s)

Acridinas/farmacología , Lesión Renal Aguda/tratamiento farmacológico , Antagonistas de Receptores Adrenérgicos alfa 2/farmacología , Cisplatino/efectos adversos , Piperazinas/farmacología , Yohimbina/farmacología , Acridinas/uso terapéutico , Lesión Renal Aguda/sangre , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/patología , Antagonistas de Receptores Adrenérgicos alfa 2/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Quimiocina CCL2/metabolismo , Modelos Animales de Enfermedad , Inyecciones Intraperitoneales , Riñón/efectos de los fármacos , Riñón/patología , Masculino , Norepinefrina/sangre , Piperazinas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos alfa 2/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Yohimbina/uso terapéutico

RESUMEN

Increases in renal sympathetic nerve activity during ischaemia and renal venous norepinephrine levels after reperfusion play important roles in the development of ischaemia/reperfusion-induced acute kidney injury. In the present study, we examined the effect of isatin, an endogenous monoamine oxidase inhibitor, on renal venous norepinephrine levels, superoxide production after reperfusion, and ischaemia/reperfusion-induced acute kidney injury. Ischaemia/reperfusion-induced acute kidney injury was accomplished by clamping the left renal artery and vein for 45min, followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal superoxide production and norepinephrine overflow were elevated and significant renal tissue damage was observed following ischaemia/reperfusion injury. Intravenous injection of isatin (10mg/kg) at 5min before ischaemia increased the renal venous plasma norepinephrine level after reperfusion and aggravated ischaemia/reperfusion-induced renal dysfunction and histological damage. The excessive superoxide production after reperfusion was significantly suppressed by isatin administration, indicating that the inhibition of oxidative deamination effectively suppressed superoxide production. These data suggest that the exacerbation effect of isatin is associated, at least in part, with increased norepinephrine levels but not with superoxide production. To the best of our knowledge, this is the first report of isatin involvement in the pathogenesis and/or development of acute kidney injury.

Asunto(s)

Lesión Renal Aguda/tratamiento farmacológico , Lesión Renal Aguda/etiología , Inhibidores de la Monoaminooxidasa/farmacología , Daño por Reperfusión/complicaciones , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Animales , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Masculino , Inhibidores de la Monoaminooxidasa/uso terapéutico , Norepinefrina/sangre , Ratas , Ratas Sprague-Dawley , Superóxidos/metabolismo

RESUMEN

Excitation of renal sympathetic nervous activity and the resulting increased levels of renal venous norepinephrine play important roles in renal ischaemia/reperfusion injury in rats. This study examined the effects of yohimbine, a non-selective α2-adrenoceptor antagonist, on renal venous norepinephrine levels and kidney function in acute kidney injury. Acute ischaemia/reperfusion-induced kidney injury was induced in rats by clamping the left renal artery and vein for 45min, followed by reperfusion, 2 weeks after a contralateral nephrectomy. Intravenous injection of yohimbine (0.1mg/kg) 5min prior to ischaemia significantly attenuated kidney injury and decreased the renal venous norepinephrine levels, as compared with vehicle-treated rats. To investigate the involvement of α2-adrenoceptor subtypes, we pre-treated with JP-1302, a selective α2C-adrenoceptor antagonist (1mg/kg). This suppressed renal venous norepinephrine levels and tumour necrosis factor-α and monocyte chemoattractant protein-1 mRNA levels after reperfusion and improved kidney function. Pre-treatment with BRL44408, a selective α2A-adrenoceptor antagonist (1mg/kg), or imiloxan, a selective α2B-adrenoceptor antagonist (1mg/kg) had no effect on renal function or tissue injury. These results suggest that yohimbine prevented ischaemia/reperfusion-induced kidney injury by inhibiting α2C-adrenoceptors and suppressing pro-inflammatory cytokine expression.

Asunto(s)

Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Citoprotección/efectos de los fármacos , Riñón/efectos de los fármacos , Receptores Adrenérgicos alfa 2/metabolismo , Daño por Reperfusión/complicaciones , Yohimbina/farmacología , Acridinas/farmacología , Lesión Renal Aguda/sangre , Lesión Renal Aguda/etiología , Antagonistas de Receptores Adrenérgicos alfa 2/farmacología , Animales , Quimiocina CCL2/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Masculino , Norepinefrina/metabolismo , Piperazinas/farmacología , Ratas , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Heat shock cognate protein 70 (Hsc70) acts as a molecular chaperone for the maintenance of intracellular proteins, which allows cancer cells to survive under proteotoxic stress. We attempted to use Hsc70 to identify key molecules in cancer cell survival. Here, we performed mass-spectrometry-based proteomics analysis utilizing affinity purification with anti-Hsc70 antibodies; as a result, 83 differentially expressed proteins were identified under stress conditions. This result implies that there was a change in the proteins with which Hsc70 interacted in response to stress. Among the proteins identified under both serum-depleted and 5-fluorouracil-treated conditions, Rab1A was identified as an essential molecule for cancer cell survival. Hsc70 interacted with Rab1A in a chaperone-dependent manner. In addition, Hsc70 knockdown decreased the level of Rab1A and increased the level of its ubiquitination under stress conditions, suggesting that Hsc70 prevented the degradation of Rab1A denatured by stress exposure. We also found that Rab1A knockdown induced cell death by inhibition of autophagosome formation. Rab1A may therefore contribute to overcoming proteotoxic insults, which allows cancer cells to survive under stress conditions. Analysis of Hsc70 interactors provided insight into changes of intracellular status. We expect further study of the Hsc70 interactome to provide a more comprehensive understanding of cancer cell physiology.

Asunto(s)

Proteínas del Choque Térmico HSC70/metabolismo , Proteínas de Unión al GTP rab1/metabolismo , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fluorouracilo/toxicidad , Regulación de la Expresión Génica , Proteínas del Choque Térmico HSC70/antagonistas & inhibidores , Proteínas del Choque Térmico HSC70/genética , Células HT29 , Humanos , Análisis por Matrices de Proteínas , Proteínas/análisis , Proteínas/metabolismo , Proteómica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al GTP rab1/antagonistas & inhibidores , Proteínas de Unión al GTP rab1/genética

RESUMEN

We have found that a series of brief renal ischemia and reperfusion (preconditioning), before the time of ischemia significantly attenuated the ischemia/reperfusion-induced acute kidney injury through endothelial nitric oxide synthase. In this study, we examined the effects of ischemic preconditioning on renal sympathetic nervous system and kidney function in ischemia/reperfusion-induced acute kidney injury with or without nitric oxide synthase inhibitor. Ischemia/reperfusion-induced acute kidney injury was made by clamping the left renal artery and vein for 45-min followed by reperfusion, 2 weeks after the contralateral nephrectomy. Ischemic preconditioning, consisting of three cycles of 2-min ischemia followed by 5-min reperfusion, was performed before the 45-min ischemia. Ischemic preconditioning suppressed the enhanced renal sympathetic nerve activity during ischemia and the elevated renal venous plasma norepinephrine level after reperfusion, and attenuated renal dysfunction and histological damage. The renoprotective effect of ischemic preconditioning was diminished by N(G)-nitro-L-arginine methyl ester (0.3 mg/kg, i.v.), a nonselective nitric oxide synthase inhibitor, 5 min before the start of ischemic preconditioning. Thus, ischemic preconditioning decreased renal sympathetic nerve activity and norepinephrine release probably through activating nitric oxide production, thereby improving ischemia/reperfusion-induced acute kidney injury.

Asunto(s)

Lesión Renal Aguda/prevención & control , Lesión Renal Aguda/fisiopatología , Precondicionamiento Isquémico , Daño por Reperfusión/complicaciones , Sistema Nervioso Simpático/fisiopatología , Lesión Renal Aguda/etiología , Lesión Renal Aguda/metabolismo , Animales , Citoprotección/efectos de los fármacos , Riñón/irrigación sanguínea , Riñón/efectos de los fármacos , Riñón/lesiones , Riñón/inervación , Masculino , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/biosíntesis , Norepinefrina/sangre , Ratas , Ratas Sprague-Dawley , Sistema Nervioso Simpático/efectos de los fármacos , Factores de Tiempo , Venas/metabolismo

RESUMEN

AIM: The molecular mechanisms by which hepatocyte nuclear factor (HNF)4α regulates fetal liver development have not been fully elucidated. We screened the downstream molecules of HNF4α during liver development and identified sodium-coupled neutral amino acid transporter (SNAT)4. The aim of this study is to investigate the regulation of SNAT4 by HNF4α and to clarify its roles in differentiating hepatocytes. METHODS: HNF4α was overexpressed in cultured liver buds using adenovirus, and suppression subtractive hybridization screening was performed. Temporal and spatial expression of SNAT4 during liver development was investigated. Regulation of SNAT4 by HNF4α was examined by promoter analyses and electrophoretic mobility shift assays (EMSA). Metabolic labeling and western blotting were carried out using primary hepatoblasts with SNAT4 overexpression. RESULTS: The expression of Slc38a4 encoding SNAT4 showed a marked perinatal increase, and was predominant among system A amino acid transporters. It was first detected in embryonic day 18.5 liver, and found in most hepatocytes after birth. Three alternative first exons were found in the SNAT4 gene. Promoter analyses using approximately 3-kb fragments corresponding to each first exon (AP1, AP2, AP3) revealed that AP1 and AP2 exhibited strong promoter activity in mouse hepatoblasts with endogenous HNF4α. Transactivation of AP2 was upregulated by HNF4α in HeLa cells without endogenous HNF4α. EMSA has demonstrated that HNF4α directly binds to cis-elements in AP2. Overexpression of SNAT4 facilitated amino acid uptake and de novo protein synthesis in primary hepatoblasts. CONCLUSION: SNAT4 functions downstream of HNF4α and plays significant roles in liver development through mechanisms of amino acid uptake and protein synthesis.

RESUMEN

Orally administered Cd is predominantly distributed to the intestine, and the majority of this mucosal Cd is bound to metallothionein (MT). MT attenuates heavy metal-induced cytotoxicity by sequestering these metals and lowering their intracellular concentrations. In addition, MT acts as an extracellular transporter of orally administered Cd to the kidney. Because of its low molecular weight, the Cd-MT complex is freely filtered at the glomerulus, and the filtered Cd-MT is then incorporated into renal proximal tubular cells. Megalin, a multiligand endocytic receptor (also known as low-density lipoprotein receptor-related protein 2 or Lrp2), acts as the receptor for Cd-MT in a renal proximal tubular cell model. Here, we used the soluble form of 39-kDa receptor-associated protein (sRAP; also known as Lrpap1), a ligand of megalin, to inhibit megalin function, and then analyzed the effect of megalin loss on Cd-MT distribution and Cd-MT-induced nephrotoxicity in an animal model. Administration of sRAP to mice caused acute loss of megalin function by removing megalin in the brush border membrane. The pre-injection of sRAP decreased renal Cd content and decreased Cd-MT-induced kidney damage. Our results demonstrate that sRAP reduces Cd-MT-induced kidney toxicity in vivo.

Asunto(s)

Endocitosis , Riñón/efectos de los fármacos , Proteína Asociada a Proteínas Relacionadas con Receptor de LDL/fisiología , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/fisiología , Metalotioneína/toxicidad , Animales , Ligandos , Masculino , Metalotioneína/farmacocinética , Ratones , Ratones Endogámicos ICR

RESUMEN

Enhanced renal sympathetic nerve activity during an ischemic period and renal venous norepinephrine overflow after reperfusion play important roles in the development of ischemic acute kidney injury. In this study, we examined the effect of 17ß-estradiol on the renal sympathetic nervous system and kidney function in ischemia/reperfusion-induced acute kidney injury in anesthetized rats. Ischemic acute kidney injury was induced by clamping the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after a contralateral nephrectomy. Intravenous injection of 17ß-estradiol (100 µg/kg) 15 min before reperfusion suppressed enhanced renal sympathetic nerve activity during renal ischemia, also suppressed renal venous norepinephrine overflow after reperfusion, and attenuated ischemia/reperfusion-induced renal dysfunction with histological damage. The above renoprotective effects of 17ß-estradiol were reversed by pretreatment with tamoxifen (5 mg/kg), an estrogen receptor antagonist, or N(G)-nitro-L-arginine methyl ester (0.3 mg/kg), a non-selective nitric oxide synthase inhibitor. These results indicate that 17ß-estradiol can suppress enhanced renal sympathetic nerve activity during renal ischemia, and its consequent effect on norepinephrine overflow from nerve endings, by nitric oxide production via estrogen receptors. These effects appear to contribute to renoprotection against ischemia/reperfusion-induced renal injury.

Asunto(s)

Estradiol/uso terapéutico , Estrógenos/uso terapéutico , Isquemia/tratamiento farmacológico , Riñón/efectos de los fármacos , Daño por Reperfusión/prevención & control , Sistema Nervioso Simpático/efectos de los fármacos , Animales , Inhibidores Enzimáticos/farmacología , Estradiol/administración & dosificación , Antagonistas de Estrógenos/farmacología , Estrógenos/administración & dosificación , Inyecciones Intravenosas , Isquemia/metabolismo , Isquemia/patología , Isquemia/fisiopatología , Riñón/irrigación sanguínea , Riñón/inervación , Riñón/metabolismo , Masculino , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Norepinefrina/sangre , Ratas , Ratas Sprague-Dawley , Insuficiencia Renal/sangre , Insuficiencia Renal/etiología , Insuficiencia Renal/patología , Insuficiencia Renal/prevención & control , Venas Renales , Daño por Reperfusión/sangre , Daño por Reperfusión/patología , Sistema Nervioso Simpático/metabolismo , Transmisión Sináptica/efectos de los fármacos , Tamoxifeno/farmacología

RESUMEN

Vitamin D receptor (VDR) is localized in nuclei and acts as a ligand-dependent transcription factor. To clarify the molecular mechanisms underlying the nuclear translocation of VDR, we utilized an in vitro nuclear transport assay using digitonin-permeabilized semi-intact cells. In this assay, recombinant whole VDR-(4-427) and a truncated mutant VDR-(4-232) lacking the carboxyl terminus of VDR were imported to nuclei even in the absence of ligand. In contrast, VDR-(91-427) lacking the amino-terminal DNA-binding domain was not imported to nuclei in the absence of ligand, and was efficiently imported in its liganded form. These results suggested that there are two distinct mechanisms underlying the nuclear transport of VDR; ligand-dependent and -independent pathways, and that the different regions of VDR are responsible for these processes. Therefore, we performed the yeast two-hybrid screening using VDR-(4-232) as the bait to explore the molecules responsible for ligand-independent nuclear translocation of VDR, and have identified importin 4 as an interacting protein. In the reconstruction experiments where transport factors were applied as recombinant proteins, recombinant importin 4 facilitated nuclear translocation of VDR regardless of its ligand, whereas importin beta failed in transporting VDR even in the presence of ligand. In conclusion, importin 4, not importin beta, is responsible for the ligand-independent nuclear translocation of VDR.

Asunto(s)

Núcleo Celular/metabolismo , Receptores de Calcitriol/metabolismo , alfa Carioferinas/fisiología , Animales , Sitios de Unión , Transporte Biológico , Células COS , Chlorocebus aethiops , ADN/metabolismo , Técnica del Anticuerpo Fluorescente , Glutatión Transferasa/genética , Células HeLa , Humanos , Modelos Estadísticos , Mutagénesis , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Receptores de Calcitriol/genética , Proteínas Recombinantes de Fusión , Proteínas Recombinantes , Saccharomyces cerevisiae , Transfección , Técnicas del Sistema de Dos Híbridos , alfa Carioferinas/química , alfa Carioferinas/genética

RESUMEN

Megalin is a multifunctional endocytic receptor that is expressed in renal proximal tubules and plays critical roles in the renal uptake of various proteins. It was hypothesized that megalin-dependent endocytosis might play a role in renal phosphate reabsorption. For addressing the short-term effects of altered megalin function, a recombinant protein for the soluble form of 39-kD receptor-associated protein (RAP) was administered intraperitoneally to 7-wk-old mice. Histidine (His)-tagged soluble RAP (amino acids 39 to 356) lacking the amino-terminal signal peptide and the carboxy-terminal endoplasmic reticulum retention signal was prepared by bacterial expression (designated His-sRAP). After the direct interaction between His-sRAP and megalin was confirmed, mice were given a single intraperitoneal administration of His-sRAP (3.5 mg/dose). Immunostaining and Western blot analyses demonstrated the uptake of His-sRAP and the accelerated internalization of megalin in proximal tubular cells 1 h after administration. In addition, internalization of the type II sodium/phosphate co-transporter (NaPi-II) was observed. The effects of three sequential administrations of His-sRAP (3.5 mg/dose, three doses at 4-h intervals) then were examined, and increased urinary excretion of low molecular weight proteins, including vitamin D-binding protein, was found, which is consistent with findings reported for megalin-deficient mice. It is interesting that urinary excretion of phosphate was also increased, and the protein level of NaPi-II in the brush border membrane was decreased. Serum concentration of 25-hydroxyvitamin D was decreased, whereas the plasma level of intact parathyroid hormone was not altered by the administration of His-sRAP. The results suggest that the His-sRAP-induced acceleration of megalin-mediated endocytosis caused phosphaturia via altered subcellular distribution of NaPi-II.

Asunto(s)

Riñón/metabolismo , Proteína Asociada a Proteínas Relacionadas con Receptor de LDL/fisiología , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/fisiología , Fosfatos/orina , Proteínas Recombinantes/administración & dosificación , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/fisiología , Proteínas Cotransportadoras de Sodio-Fosfato/metabolismo , Simportadores/metabolismo , Animales , Western Blotting , Electroforesis en Gel de Poliacrilamida , Endocitosis , Retículo Endoplásmico/metabolismo , Histidina/química , Infusiones Parenterales/métodos , Túbulos Renales/metabolismo , Ligandos , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Microscopía Fluorescente , Microvellosidades/metabolismo , Hormona Paratiroidea/metabolismo , Fosfatos/química , Unión Proteica , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Factores de Tiempo , Proteína de Unión a Vitamina D/química

RESUMEN

Gene-trap mutagenesis is based on the notion that the random insertion of a trapping vector may disturb the function of inserted genes. Here, we applied this method to murine mesenchymal ATDC5 cells, which differentiate into mature chondrocytes in the presence of insulin. As the trap vector we used pPT1-geo, which lacks its own promoter and enhancer, but contains a lacZ-neo fusion gene as a reporter and selection marker driven by the promoter of the trapped gene. After pPT1-geo was introduced into ATDC5 cells by electroporation, the neomycin-resistant clones were screened for beta-galactosidase activity. The selected clones were cultured in differentiation medium to evaluate the chondrogenic phenotype. The clones no. 6-30 and 6-175, which exhibited impaired and accelerated mineralization, respectively, were subjected to further analysis. In clone no. 6-30 in which the gene coding for the p85alpha subunit of phosphoinositide 3-kinase (PI3K) was trapped, the expression of marker genes of early chondrocytes including collagen type II, aggrecan, and PTH/PTHrP receptor was delayed. The insulin-induced stimulation of growth was reduced in clone no. 6-30 compared with the parental ATDC5 cells. Moreover, treatment of parental ATDC5 cells with a specific inhibitor of PI3K, LY294002, phenocopied clone no. 6-30, suggesting the involvement of PI3K signaling in the chondrogenic differentiation of ATDC5 cells. Clone no. 6-175 with accelerated mineralization was revealed to have a gene homologous to human KIAA0312 trapped, whose function remains unclear. Taken together, the gene-trap in ATDC5 cells might be useful to identify the molecules involved in chondrogenic differentiation.

Asunto(s)

Diferenciación Celular , Condrocitos/citología , Condrocitos/enzimología , Mutagénesis/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Cromonas/farmacología , Clonación Molecular , Inhibidores Enzimáticos/farmacología , Vectores Genéticos/genética , Insulina/farmacología , Ratones , Morfolinas/farmacología , Fenotipo , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo

RESUMEN

Dent disease is characteristic for the urinary loss of low-molecular-weight proteins and calcium, leading to renal calcification and, in some patients, chronic renal failure. This disorder is caused by loss-of-function mutations in the renal chloride channel gene, CLCN5. The animal model of this disease has demonstrated the possible role of disturbed megalin expression, which is a member of the low-density lipoprotein receptor family and is associated with renal reabsorption of a variety of proteins, in Dent disease. We examined the expression of megalin in the renal tubular epithelium of two unrelated patients with Dent disease. One patient, whose CLCN5 gene was completely deleted, showed significantly decreased staining of megalin compared with controls, while there was no change in another patient with partial deletion of the gene. These results demonstrated that mutation of CLCN5 in some patients with Dent disease may impair the expression of megalin, resulting in abnormal calcium metabolism, manifested as hypercalciuria and nephrocalcinosis.

Asunto(s)

Trastornos del Metabolismo del Calcio/metabolismo , Epitelio/metabolismo , Enfermedades Renales/metabolismo , Túbulos Renales/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/biosíntesis , Trastornos del Metabolismo del Calcio/complicaciones , Trastornos del Metabolismo del Calcio/patología , Preescolar , Canales de Cloruro/genética , Análisis Mutacional de ADN , Epitelio/patología , Técnica del Anticuerpo Fluorescente , Enfermedades Genéticas Ligadas al Cromosoma X/complicaciones , Enfermedades Genéticas Ligadas al Cromosoma X/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Humanos , Enfermedades Renales/complicaciones , Enfermedades Renales/patología , Túbulos Renales/patología , Masculino , Mutación , Proteinuria/etiología