RESUMO
MAK-V/Hunk is an SNF1-related serine/threonine kinase which was previously shown to be highly expressed in the mammary gland and central nervous system. In this study, we found MAK-V/Hunk is abundantly and specifically expressed in the thick ascending limbs and distal convoluted tubules (DCT) of the kidney from the embryonic stage to the adult stage. We demonstrated that dietary salt depletion significantly enhances renal MAK-V/Hunk mRNA levels compared with a normal-salt diet. To analyze the possible renal cellular function of this kinase, we employed mouse distal convoluted tubule (mDCT) cells. The results of reverse transcriptase-polymerase chain reaction and Western blot analysis revealed that MAK-V/Hunk is expressed endogenously in mDCT cells. Overexpression of MAK-V/Hunk by adenoviral gene transfer significantly inhibited the ANG II-induced stimulation of c-fos gene transcription and suppressed the ANG II-mediated increases in transforming growth factor-beta production into the medium. This phenomenon was accompanied by inhibition of ANG II-induced activation of BrdU incorporation. On the other hand, the MAK-V/Hunk knockdown by siRNA activated the ANG II-induced c-fos gene expression. In the consecutive sections stained for MAK-V/Hunk and AT(1) receptor, MAK-V/Hunk-immunopositive distal tubules expressed the AT(1) receptor. This is the first report on the intrarenal localization of MAK-V/Hunk and its cellular function in renal tubular cells.
Assuntos
Proliferação de Células , Túbulos Renais Distais/citologia , Túbulos Renais Distais/enzimologia , Proteínas Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenoviridae/genética , Animais , Dieta Hipossódica , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos , Imuno-Histoquímica , Rim/embriologia , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-fos/genética , Interferência de RNA , RNA Mensageiro/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Proteínas Recombinantes/metabolismo , Distribuição TecidualRESUMO
Activation of angiotensin II (Ang II) type 1 receptor (AT1R) signaling is reported to play an important role in cardiac hypertrophy. We previously cloned a novel molecule interacting with the AT1R, which we named ATRAP (for Ang II type 1 receptor-associated protein). Here, we report that overexpression of ATRAP significantly decreases the number of AT1R on the surface of cardiomyocytes, and also decreases the degree of p38 mitogen-activated protein kinase phosphorylation, the activity of the c-fos promoter and protein synthesis upon Ang II treatment. These results indicate that ATRAP significantly promotes downregulation of the AT1R and further attenuates certain Ang II-mediated hypertrophic responses in cardiomyocytes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Regulação para Baixo , Miócitos Cardíacos/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenoviridae/genética , Animais , Cardiomegalia/genética , Cardiomegalia/metabolismo , Tamanho Celular , Células Cultivadas , Vetores Genéticos/genética , Hipertrofia , Imunoprecipitação , Camundongos , Miócitos Cardíacos/química , Miócitos Cardíacos/citologia , Fosforilação , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptor Tipo 1 de Angiotensina/análise , Receptor Tipo 1 de Angiotensina/genética , Transcrição Gênica , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The cAMP-signaling pathway plays a crucial role in the regulation of the renin gene, but the mechanism involved remains poorly understood. We have focused our studies of renin gene regulation on the unique cAMP responsive element (huREN/CNRE, -135 to -107) in the human renin promoter. We have cloned a protein that binds to this unique CNRE and demonstrated that this protein is liver X receptor-alpha (LXRalpha), a transcriptional factor of the nuclear receptor family. Transient expression of LXRalpha in human renin-producing Calu-6 cells increased cAMP inducibility of human renin promoter. Similarly, LXRalpha-stably transfected Calu-6 cells exhibited increased cAMP inducibility of renin promoter as well as the endogenous renin gene. Site-directed mutation of huREN/CNRE, which disrupted LXRalpha binding, decreased cAMP-induced transcriptional activity of human renin promoter. Furthermore, we demonstrated that the binding of LXRalpha derived from human juxtaglomerular cells, the main production site of renin in the kidney, to the huREN/CNRE in vivo. These results suggest that LXRalpha plays an important role in the cAMP-mediated regulation of human renin gene transcription by binding to CNRE.
Assuntos
AMP Cíclico/fisiologia , Regulação da Expressão Gênica , Receptores Citoplasmáticos e Nucleares/fisiologia , Renina/genética , Elementos de Resposta/genética , Linhagem Celular , Proteínas de Ligação a DNA , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Isoquinolinas/farmacologia , Receptores X do Fígado , Mutagênese Sítio-Dirigida/genética , Naftalenos/farmacologia , Receptores Nucleares Órfãos , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Sulfonamidas/farmacologia , Transcrição GênicaRESUMO
Cholesterol crystal embolism (CCE) is caused by the shedding of cholesterol crystals into the bloodstream, and it has been recently recognized as a serious complication after vascular procedures. Our case of CCE, which was diagnosed by skin and renal biopsies, occurred in a patient with hypertension and diabetes mellitus, 3 months after coronary angiography, with the development of renal failure and blue toes. After low-density lipoprotein apheresis (LDL-A), the skin lesions, including livedo reticularis and pain from the acrocyanotic toes, dramatically improved, with partial recovery of renal function. Following the administration of low-dose corticosteroid and candesartan--an angiotensin II type 1 receptor antagonist (ARB)--the eosinophilia disappeared and renal function improved gradually with a decrease in urinary protein excretion. Therefore, a combination therapy of LDL-A, low-dose corticosteroid, and an ARB is a possible treatment for CCE, although the possibility of spontaneous recovery of renal function cannot be eliminated for this patient.