RESUMO
Tubular cell necrosis is a key histological feature of acute kidney injury (AKI). Necroptosis is a type of programed necrosis, which is executed by mixed lineage kinase domain-like protein (MLKL) upon its binding to the plasma membrane. Emerging evidence indicates that necroptosis plays a critical role in the development of AKI. However, it is unclear whether renal tubular cells undergo necroptosis in vivo and how the necroptotic pathway is regulated during AKI. Repulsive guidance molecule (RGM)-b is a member of the RGM family. Our previous study demonstrated that RGMb is highly expressed in kidney tubular epithelial cells, but its biological role in the kidney has not been well characterized. In the present study, we found that RGMb reduced membrane-associated MLKL levels and inhibited necroptosis in cultured cells. During ischemia/reperfusion injury (IRI) or oxalate nephropathy, MLKL was induced to express on the apical membrane of proximal tubular (PT) cells. Specific knockout of Rgmb in tubular cells (Rgmb cKO) increased MLKL expression at the apical membrane of PT cells and induced more tubular cell death and more severe renal dysfunction compared with wild-type mice. Treatment with the necroptosis inhibitor Necrostatin-1 or GSK'963 reduced MLKL expression on the apical membrane of PT cells and ameliorated renal function impairment after IRI in both wild-type and Rgmb cKO mice. Taken together, our results suggest that proximal tubular cell necroptosis plays an important role in AKI, and that RGMb protects against AKI by inhibiting MLKL membrane association and necroptosis in proximal tubular cells.
Assuntos
Injúria Renal Aguda/prevenção & controle , Apoptose , Túbulos Renais/patologia , Necrose , Proteínas do Tecido Nervoso/fisiologia , Proteínas Quinases/metabolismo , Traumatismo por Reperfusão/complicações , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/patologia , Animais , Moléculas de Adesão Celular Neuronais , Proteínas Ligadas por GPI , Túbulos Renais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Substâncias Protetoras/farmacologia , Proteínas Quinases/genéticaRESUMO
Hepcidin, the main regulator of iron homeostasis, is repressed when erythropoiesis is acutely stimulated by erythropoietin (EPO) to favor iron supply to maturing erythroblasts. Erythroferrone (ERFE) has been identified as the erythroid regulator that inhibits hepcidin in stress erythropoiesis. A powerful hepcidin inhibitor is the serine protease matriptase-2, encoded by TMPRSS6, whose mutations cause iron refractory iron deficiency anemia. Because this condition has inappropriately elevated hepcidin in the presence of high EPO levels, a role is suggested for matriptase-2 in EPO-mediated hepcidin repression. To investigate the relationship between EPO/ERFE and matriptase-2, we show that EPO injection induces Erfe messenger RNA expression but does not suppress hepcidin in Tmprss6 knockout (KO) mice. Similarly, wild-type (WT) animals, in which the bone morphogenetic protein-mothers against decapentaplegic homolog (Bmp-Smad) pathway is upregulated by iron treatment, fail to suppress hepcidin in response to EPO. To further investigate whether the high level of Bmp-Smad signaling of Tmprss6 KO mice counteracts hepcidin suppression by EPO, we generated double KO Bmp6-Tmprss6 KO mice. Despite having Bmp-Smad signaling and hepcidin levels that are similar to WT mice under basal conditions, double KO mice do not suppress hepcidin in response to EPO. However, pharmacologic downstream inhibition of the Bmp-Smad pathway by dorsomorphin, which targets the BMP receptors, improves the hepcidin responsiveness to EPO in Tmprss6 KO mice. We concluded that the function of matriptase-2 is dominant over that of ERFE and is essential in facilitating hepcidin suppression by attenuating the BMP-SMAD signaling.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Eritropoetina/farmacologia , Hepcidinas/metabolismo , Fígado/metabolismo , Proteínas de Membrana/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais/fisiologia , Proteínas Smad/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/genética , Hepcidinas/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Serina Endopeptidases/genética , Proteínas Smad/genéticaRESUMO
Liver disease due to alpha-1-antitrypsin deficiency (A1ATD) is associated with hepatic iron overload in a subgroup of patients. The underlying cause for this association is unknown. The aim of the present study was to define the genetics of this correlation and the effect of alpha-1-antitrypsin (A1AT) on the expression of the iron hormone hepcidin. Full exome and candidate gene sequencing were carried out in a family with A1ATD and hepatic iron overload. Regulation of hepcidin expression by A1AT was studied in primary murine hepatocytes. Cells co-transfected with hemojuvelin (HJV) and matriptase-2 (MT-2) were used as a model to investigate the molecular mechanism of this regulation. Observed familial clustering of hepatic iron overload with A1ATD suggests a genetic cause, but genotypes known to be associated with hemochromatosis were absent. Individuals homozygous for the A1AT Z-allele with environmental or genetic risk factors such as steatosis or heterozygosity for the HAMP non-sense mutation p.Arg59* presented with severe hepatic siderosis. In hepatocytes, A1AT induced hepcidin mRNA expression in a dose-dependent manner. Experiments in overexpressing cells show that A1AT reduces cleavage of the hepcidin inducing bone morphogenetic protein co-receptor HJV via inhibition of the membrane-bound serine protease MT-2. The acute-phase protein A1AT is an inducer of hepcidin expression. Through this mechanism, A1ATD could be a trigger of hepatic iron overload in genetically predisposed individuals or patients with environmental risk factors for hepatic siderosis.
Assuntos
Hepcidinas/biossíntese , Sobrecarga de Ferro/genética , Deficiência de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Adulto , Idoso , Animais , Células Cultivadas , Progressão da Doença , Feminino , Proteínas Ligadas por GPI/metabolismo , Células HEK293 , Hemocromatose/genética , Hemocromatose/metabolismo , Proteína da Hemocromatose , Hepatócitos/metabolismo , Humanos , Sobrecarga de Ferro/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Serina Endopeptidases/metabolismo , alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/metabolismoRESUMO
Circulating levels of fibroblast growth factor 23 (FGF23) are elevated in patients with chronic kidney disease (CKD), but the mechanisms are poorly understood. Here we tested whether inflammation and iron deficiency regulate FGF23. In wild-type mice, acute inflammation induced by single injections of heat-killed Brucella abortus or interleukin-1ß (IL-1ß) decreased serum iron within 6 h, and was accompanied by significant increases in osseous Fgf23 mRNA expression and serum levels of C-terminal FGF23, but no changes in intact FGF23. Chronic inflammation induced by repeated bacteria or IL-1ß injections decreased serum iron, increased osseous Fgf23 mRNA, and serum C-terminal FGF23, but modestly increased biologically active, intact FGF23 serum levels. Chronic iron deficiency mimicked chronic inflammation. Increased osseous FGF23 cleavage rather than a prolonged half-life of C-terminal FGF23 fragments accounted for the elevated C-terminal FGF23 but near-normal intact FGF23 levels in inflammation. IL-1ß injection increased Fgf23 mRNA and C-terminal FGF23 levels similarly in wildtype and Col4a3(ko) mice with CKD but markedly increased intact FGF23 levels only in the CKD mice. Inflammation increased Fgf23 transcription by activating Hif1α signaling. Thus, inflammation and iron deficiency stimulate FGF23 production. Simultaneous upregulation of FGF23 cleavage in osteocytes maintains near-normal levels of biologically active, intact circulating FGF23, whereas downregulated or impaired FGF23 cleavage may contribute to elevated intact serum FGF23 in CKD.
Assuntos
Fêmur/metabolismo , Fatores de Crescimento de Fibroblastos/sangue , Inflamação/sangue , Ferro/sangue , RNA Mensageiro/metabolismo , Insuficiência Renal Crônica/sangue , Animais , Autoantígenos/genética , Linhagem Celular , Colágeno Tipo IV/genética , Desferroxamina/farmacologia , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Interleucina-1beta/farmacologia , Deficiências de Ferro , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/efeitos dos fármacos , Insuficiência Renal Crônica/metabolismo , Sideróforos/farmacologia , Transcrição GênicaRESUMO
Bone morphogenetic protein (BMP) signaling plays an important role in spermatogenesis and follicle development. Our previous studies have shown that repulsive guidance molecule b (RGMb, also known as Dragon) is a coreceptor that enhances BMP2 and BMP4 signaling in several cell types and that RGMb is expressed in spermatocytes and spermatids in the testis and in oocytes of the secondary follicles in the ovary. Here, we demonstrated that specific deletion of Rgmb in germ cells in the testis and ovary did not alter Smad1/5/8 phosphorylation, gonadal structures, and fertility. In addition, ovaries from postnatal global Rgmb knockout mice showed similar structures to the wild-type ovaries. Our results suggest that RGMb is not essential for normal gonadal function.
Assuntos
Proteínas do Tecido Nervoso/fisiologia , Folículo Ovariano/crescimento & desenvolvimento , Testículo/fisiologia , Animais , Moléculas de Adesão Celular Neuronais , Feminino , Fertilidade , Proteínas Ligadas por GPI/metabolismo , Masculino , Camundongos Knockout , Proteínas Musculares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/intoxicação , Folículo Ovariano/anatomia & histologia , Testículo/anatomia & histologiaRESUMO
In this issue of Blood, Poli et al demonstrate that heparin analogs engineered to minimize their anticoagulant properties can potently downregulate hepcidin production in vitro and in vivo, and may potentially be used to treat the anemia of inflammation.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Heparina/análogos & derivados , Hepcidinas/genética , Animais , Feminino , Heparina/farmacologia , HumanosRESUMO
Iron is a micronutrient essential for almost all organisms: bacteria, plants, and animals. It is a metal that exists in multiple redox states, including the divalent ferrous (Fe(2+)) and the trivalent ferric (Fe(3+)) species. The multiple oxidation states of iron make it excellent for electron transfer, allowing iron to be selected during evolution as a cofactor for many proteins involved in central cellular processes including oxygen transport, mitochondrial respiration, and DNA synthesis. However, the redox cycling of ferrous and ferric iron in the presence of H2O2, which is physiologically present in the cells, also leads to the production of free radicals (Fenton reaction) that can attack and damage lipids, proteins, DNA, and other cellular components. To meet the physiological needs of the body, but to prevent cellular damage by iron, the amount of iron in the body must be tightly regulated. Here we review how the liver is the central conductor of systemic iron balance and show that this central role is related to the secretion of a peptide hormone hepcidin by hepatocytes. We then review how the liver receives and integrates the many signals that report the body's iron needs to orchestrate hepcidin production and maintain systemic iron homeostasis.
Assuntos
Ferro/metabolismo , Fígado/metabolismo , Animais , Transporte Biológico , Hepcidinas/genética , Hepcidinas/metabolismo , Homeostase , Humanos , Transdução de SinaisRESUMO
BACKGROUND: Atherosclerotic lesions grow via the accumulation of leukocytes and oxidized lipoproteins in the vessel wall. Leukocytes can attenuate or augment atherosclerosis through the release of cytokines, chemokines, and other mediators. Deciphering how leukocytes develop, oppose, and complement each other's function and shape the course of disease can illuminate our understanding of atherosclerosis. Innate response activator (IRA) B cells are a recently described population of granulocyte macrophage colony-stimulating factor-secreting cells of hitherto unknown function in atherosclerosis. METHODS AND RESULTS: Here, we show that IRA B cells arise during atherosclerosis in mice and humans. In response to a high-cholesterol diet, IRA B cell numbers increase preferentially in secondary lymphoid organs via Myd88-dependent signaling. Mixed chimeric mice lacking B cell-derived granulocyte macrophage colony-stimulating factor develop smaller lesions with fewer macrophages and effector T cells. Mechanistically, IRA B cells promote the expansion of classic dendritic cells, which then generate interferon γ-producing T helper-1 cells. This IRA B cell-dependent T helper-1 skewing manifests in an IgG1-to-IgG2c isotype switch in the immunoglobulin response against oxidized lipoproteins. CONCLUSIONS: Granulocyte macrophage colony-stimulating factor-producing IRA B cells alter adaptive immune processes and shift the leukocyte response toward a T helper-1-associated milieu that aggravates atherosclerosis.
Assuntos
Imunidade Adaptativa , Aterosclerose/imunologia , Linfócitos B/imunologia , Imunidade Inata , Ativação Linfocitária/imunologia , Células Th1/imunologia , Imunidade Adaptativa/genética , Animais , Aterosclerose/genética , Aterosclerose/patologia , Linfócitos B/patologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Colesterol na Dieta/administração & dosagem , Colesterol na Dieta/efeitos adversos , Técnicas de Cocultura , Humanos , Imunidade Inata/genética , Ativação Linfocitária/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Quimera por Radiação , Células Th1/patologiaRESUMO
BACKGROUND: There currently is a need for a non-invasive measure of renal fibrosis. We aim to explore whether shear wave elastography (SWE)-derived estimates of tissue stiffness may serve as a non-invasive biomarker that can distinguish normal and abnormal renal parenchymal tissue. METHODS: Participants with CKD (by estimated GFR) and healthy volunteers underwent SWE. Renal elasticity was estimated as Young's modulus (YM) in kilopascals (kPa). Univariate Wilcoxon rank-sum tests were used. RESULTS: Twenty-five participants with CKD (median GFR 38 mL/min; quartile 1, quartile 3 28, 42) and 20 healthy controls without CKD underwent SWE performed by a single radiologist. CKD was associated with increased median YM (9.40 [5.55, 22.35] vs. 4.40 [3.68, 5.70] kPa; p = 0.002) and higher median intra-subject inter-measurement estimated YM's variability (4.27 [2.89, 9.90] vs. 1.51 [1.21, 2.05] kPa; p < 0.001). CONCLUSIONS: SWE-derived estimates of renal stiffness and intra-subject estimated stiffness variability are higher in patients with CKD than in healthy controls. Renal fibrosis is a plausible explanation for the observed difference in YM. Further studies are required to determine the relationship between YM, estimated renal stiffness, and renal fibrosis severity.
Assuntos
Módulo de Elasticidade , Rim/diagnóstico por imagem , Insuficiência Renal Crônica/diagnóstico por imagem , Adulto , Idoso , Estudos de Casos e Controles , Estudos Transversais , Elasticidade , Técnicas de Imagem por Elasticidade , Feminino , Fibrose , Humanos , Rim/patologia , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Insuficiência Renal Crônica/patologiaRESUMO
Hepcidin is a key regulator of systemic iron homeostasis. Hepcidin deficiency induces iron overload, whereas hepcidin excess induces anemia. Mutations in the gene encoding hemojuvelin (HFE2, also known as HJV) cause severe iron overload and correlate with low hepcidin levels, suggesting that hemojuvelin positively regulates hepcidin expression. Hemojuvelin is a member of the repulsive guidance molecule (RGM) family, which also includes the bone morphogenetic protein (BMP) coreceptors RGMA and DRAGON (RGMB). Here, we report that hemojuvelin is a BMP coreceptor and that hemojuvelin mutants associated with hemochromatosis have impaired BMP signaling ability. Furthermore, BMP upregulates hepatocyte hepcidin expression, a process enhanced by hemojuvelin and blunted in Hfe2-/- hepatocytes. Our data suggest a mechanism by which HFE2 mutations cause hemochromatosis: hemojuvelin dysfunction decreases BMP signaling, thereby lowering hepcidin expression.
Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana/fisiologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Sequência de Aminoácidos , Animais , Proteína Morfogenética Óssea 2 , Células CHO , Cricetinae , Proteínas Ligadas por GPI , Proteína da Hemocromatose , Hepcidinas , Humanos , Fígado/citologia , Fígado/metabolismo , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação , Reação em Cadeia da PolimeraseRESUMO
PURPOSE: Iron overload accelerates bone loss in mice lacking the bone morphogenetic protein 6 (Bmp6) gene, which is the key endogenous regulator of hepcidin, iron homeostasis gene. We investigated involvement of other BMPs in preventing haemochromatosis and subsequent osteopenia in Bmp6-/- mice. METHODS: Iron-treated wild-type (WT) and Bmp6-/- mice were analysed for hepcidin messenger RNA (mRNA) and tissue and blood BMP levels by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), immunohistochemistry, Western blot, enzyme-linked immunosorbent assay (ELISA) and proximity extension assay. BMPs labeled with technetium-99m were used in pharmacokinetic studies. RESULTS: In WT mice, 4 h following iron challenge, liver Bmp6 and hepcidin expression were increased, while expression of other Bmps was not affected. In parallel, we provided the first evidence that BMP6 circulates in WT mice and that iron increased the BMP6 serum level and the specific liver uptake of (99m)Tc-BMP6. In Bmp6-/- mice, iron challenge led to blunted activation of liver Smad signaling and hepcidin expression with a delay of 24 h, associated with increased Bmp5 and Bmp7 expression and increased Bmp2, 4, 5 and 9 expression in the duodenum. Liver Bmp7 expression and increased circulating BMP9 eventually contributed to the late hepcidin response. This was further supported by exogenous BMP7 therapy resulting in an effective hepcidin expression followed by a rapid normalisation of plasma iron values and restored osteopenia in Bmp6-/- mice. CONCLUSION: In Bmp6-/- mice, iron activated endogenous compensatory mechanisms of other BMPs that were not sufficient for preventing hemochromatosis and bone loss. Administration of exogenous BMP7 was effective in correcting the plasma iron level and bone loss, indicating that BMP6 is an essential but not exclusive in vivo regulator of iron homeostasis.
Assuntos
Doenças Ósseas Metabólicas/tratamento farmacológico , Proteínas Morfogenéticas Ósseas/metabolismo , Sobrecarga de Ferro/tratamento farmacológico , Animais , Western Blotting , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/farmacologia , Ensaio de Imunoadsorção Enzimática , Feminino , Hepcidinas/metabolismo , Homeostase/fisiologia , Imuno-Histoquímica , Ferro/metabolismo , Fígado/metabolismo , Masculino , Espectrometria de Massas , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
Dragon is one of the three members of the repulsive guidance molecule (RGM) family, i.e. RGMa, RGMb (Dragon), and RGMc (hemojuvelin). We previously identified the RGM members as bone morphogenetic protein (BMP) co-receptors that enhance BMP signaling. Our previous studies found that Dragon is highly expressed in the tubular epithelial cells of mouse kidneys. However, the roles of Dragon in renal epithelial cells are yet to be defined. We now show that overexpression of Dragon increased cell death induced by hypoxia in association with increased cleaved poly(ADP-ribose) polymerase and cleaved caspase-3 levels in mouse inner medullary collecting duct (IMCD3) cells. Dragon also inhibited E-cadherin expression but did not affect epithelial-to-mesenchymal transition induced by TGF-ß in IMCD3 cells. Previous studies suggest that the three RGM members can function as ligands for the receptor neogenin. Interestingly, our present study demonstrates that the Dragon actions on apoptosis and E-cadherin expression in IMCD3 cells were mediated by the neogenin receptor but not through the BMP pathway. Dragon expression in the kidney was up-regulated by unilateral ureteral obstruction in mice. Compared with wild-type mice, heterozygous Dragon knock-out mice exhibited 45-66% reduction in Dragon mRNA expression, decreased epithelial apoptosis, and increased tubular E-cadherin expression and had attenuated tubular injury after unilateral ureteral obstruction. Our results suggest that Dragon may impair tubular epithelial integrity and induce epithelial apoptosis both in vitro and in vivo.
Assuntos
Apoptose , Caderinas/biossíntese , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Túbulos Renais Proximais/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Obstrução Ureteral/metabolismo , Animais , Caderinas/genética , Caspase 3/genética , Caspase 3/metabolismo , Moléculas de Adesão Celular Neuronais , Hipóxia Celular/genética , Linhagem Celular , Células Epiteliais/patologia , Proteínas Ligadas por GPI , Túbulos Renais Proximais/patologia , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Obstrução Ureteral/genética , Obstrução Ureteral/patologiaRESUMO
During inflammation, the liver becomes resistant to growth hormone (GH) actions, leading to downregulation of the GH target gene IGF-I and activation of catabolism. Proinflammatory cytokines IL-6, TNF-α, and IL-1ß are critically involved in the pathogenesis of hepatic GH resistance. However, the mechanisms used by endogenous IL-6, TNF-α, and IL-1ß to inhibit the hepatic GH-IGF-I pathway during inflammation are not fully understood. Here, we show that TNF-α and IL-1ß inhibited GH receptor (GHR) expression but had minor effects on the downstream suppressor of cytokine signaling (SOCS)3, while IL-6 induced SOCS3 expression but had no effect on GHR expression in Huh-7 cells. Consistent with the in vitro observations, neutralization of TNF-α and IL-1ß in mouse models of inflammation did not significantly alter SOCS3 expression stimulated by inflammation but restored GHR and IGF-I expression suppressed by inflammation. Neutralization of IL-6 did not alter inflammation-suppressed GHR expression but drastically reduced the inflammation-stimulated SOCS3 expression and restored IGF-I expression. Interestingly, when the GH-IGF-I pathway was turned off by maximal inhibition of GHR expression, IL-6 and SOCS3 were no longer able to regulate IGF-I expression. Taken together, our results suggest that TNF-α/IL-1ß and IL-6 use distinct mechanisms to induce hepatic GH resistance, with TNF-α and IL-1ß acting primarily on GHR and IL-6 acting primarily on SOCS3. IL-6 action may be superseded by factors such as TNF-α and IL-1ß that inhibit GHR expression.
Assuntos
Hormônio do Crescimento/metabolismo , Interleucina-1beta/farmacologia , Interleucina-6/farmacologia , Fígado/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Células Cultivadas , Humanos , Inflamação/genética , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Moléculas de Adesão de Célula Nervosa/genética , Receptores da Somatotropina/genética , Receptores da Somatotropina/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Vasopressin (VP) binds to the vasopressin type 2 receptor (V2R) to trigger physiological effects including body fluid homeostasis and blood pressure regulation. Signaling is terminated by receptor downregulation involving clathrin-mediated endocytosis and V2R degradation. We report here that both native and epitope-tagged V2R are internalized from the plasma membrane of LLC-PK1 kidney epithelial cells in the presence of another ligand, transferrin (Tf). The presence of iron-saturated Tf (holo-Tf; 4 h) reduced V2R binding sites at the cell surface by up to 33% while iron-free (apo-Tf) had no effect. However, no change in green fluorescent protein-tagged V2R distribution was observed in the presence of bovine serum albumin, atrial natriuretic peptide, or ANG II. Conversely, holo-Tf did not induce the internalization of another G protein-coupled receptor, the parathyroid hormone receptor. In contrast to the effect of VP, Tf did not increase intracellular cAMP or modify aquaporin-2 distribution in these cells, although addition of VP and Tf together augmented VP-induced V2R internalization. Tf receptor coimmunoprecipitated with V2R, suggesting that they interact closely, which may explain the additive effect of VP and Tf on V2R endocytosis. Furthermore, Tf-induced V2R internalization was abolished in cells expressing a dominant negative dynamin (K44A) mutant, indicating the involvement of clathrin-coated pits. We conclude that Tf can induce heterologous downregulation of the V2R and this might desensitize VP target cells without activating downstream V2R signaling events. It also provides new insights into urine-concentrating defects observed in rat models of hemochromatosis.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Rim/efeitos dos fármacos , Receptores de Vasopressinas/metabolismo , Transferrina/farmacologia , Vasopressinas/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Rim/citologia , Rim/metabolismo , Receptores de Vasopressinas/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , SuínosRESUMO
Mutations in transmembrane protease, serine 6 (TMPRSS6), encoding matriptase-2, are responsible for the familial anemia disorder iron-refractory iron deficiency anemia (IRIDA). Patients with IRIDA have inappropriately elevated levels of the iron regulatory hormone hepcidin, suggesting that TMPRSS6 is involved in negatively regulating hepcidin expression. Hepcidin is positively regulated by iron via the bone morphogenetic protein (BMP)-SMAD signaling pathway. In this study, we investigated whether BMP6 and iron also regulate TMPRSS6 expression. Here we demonstrate that, in vitro, treatment with BMP6 stimulates TMPRSS6 expression at the mRNA and protein levels and leads to an increase in matriptase-2 activity. Moreover, we identify that inhibitor of DNA binding 1 is the key element of the BMP-SMAD pathway to regulate TMPRSS6 expression in response to BMP6 treatment. Finally, we show that, in mice, Tmprss6 mRNA expression is stimulated by chronic iron treatment or BMP6 injection and is blocked by injection of neutralizing antibody against BMP6. Our results indicate that BMP6 and iron not only induce hepcidin expression but also induce TMPRSS6, a negative regulator of hepcidin expression. Modulation of TMPRSS6 expression could serve as a negative feedback inhibitor to avoid excessive hepcidin increases by iron to help maintain tight homeostatic balance of systemic iron levels.
Assuntos
Anemia Ferropriva/metabolismo , Proteína Morfogenética Óssea 6/metabolismo , Ferro/metabolismo , Proteínas de Membrana/metabolismo , Serina Endopeptidases/metabolismo , Anemia Ferropriva/fisiopatologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Hepcidinas , Humanos , Neoplasias Hepáticas , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Serina Endopeptidases/genética , Transdução de Sinais/fisiologiaRESUMO
Anemia of chronic inflammation (ACI) is the most frequent anemia in hospitalized patients and is associated with significant morbidity. A major underlying mechanism of ACI is the retention of iron within cells of the reticuloendothelial system (RES), thus making the metal unavailable for efficient erythropoiesis. This reticuloendothelial iron sequestration is primarily mediated by excess levels of the iron regulatory peptide hepcidin down-regulating the functional expression of the only known cellular iron export protein ferroportin resulting in blockade of iron egress from these cells. Using a well-established rat model of ACI, we herein provide novel evidence for effective treatment of ACI by blocking endogenous hepcidin production using the small molecule dorsomorphin derivative LDN-193189 or the protein soluble hemojuvelin-Fc (HJV.Fc) to inhibit bone morphogenetic protein-Smad mediated signaling required for effective hepcidin transcription. Pharmacologic inhibition of hepcidin expression results in mobilization of iron from the RES, stimulation of erythropoiesis and correction of anemia. Thus, hepcidin lowering agents are a promising new class of pharmacologic drugs to effectively combat ACI.
Assuntos
Anemia/tratamento farmacológico , Peptídeos Catiônicos Antimicrobianos/antagonistas & inibidores , Inflamação/tratamento farmacológico , Pirazóis/farmacologia , Pirimidinas/farmacologia , Anemia/etiologia , Anemia/genética , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Células Cultivadas , Doença Crônica , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Proteínas Ligadas por GPI , Expressão Gênica/efeitos dos fármacos , Proteína da Hemocromatose , Hepcidinas , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Inflamação/complicações , Inflamação/genética , Proteínas de Membrana/imunologia , Ratos , Ratos Endogâmicos Lew , Indução de RemissãoRESUMO
Hemojuvelin is a critical regulator of hepcidin expression and can be cleaved by proteases to form soluble hemojuvelin. Soluble hemojuvelin has been recently identified in human serum but the presence and quantity of soluble hemojuvelin in mouse serum is unknown. We developed a two-site enzyme-linked immunosorbent assay using a monoclonal anti-hemojuvelin as the capture antibody and a biotinylated polyclonal anti-hemojuvelin as the detection antibody to quantify the levels of soluble hemojuvelin in mouse serum. We validated this assay using cell-conditioned media and serum from Hemojuvelin-null and Bone morphogenetic protein 6-null mice. We also used this validated assay to measure serum soluble hemojuvelin concentrations in mice receiving an acute low iron or high iron treatment. This two-site enzyme-linked immunosorbent assay was highly specific for mouse hemojuvelin, with a lower limit of detection at 13.2-26.8 ng/mL of soluble hemojuvelin in mouse serum. The median serum soluble hemojuvelin concentration in wild-type C57BL/6J mice was 57.9 ± 22 ng/mL, which is 4- to 20-fold less than that reported in healthy human volunteers. After acute low iron diet treatment in these mice, serum soluble hemojuvelin levels were increased and correlated with lowered serum iron levels and decreased hepatic hepcidin expression. An acute high iron diet in wild-type mice or chronically iron-overloaded Bone morphogenetic protein 6-null mice did not significantly lower serum soluble hemojuvelin concentrations. Here we report reliable quantitation of mouse serum soluble hemojuvelin using a novel and validated enzyme-linked immunosorbent assay. This assay may provide a useful tool to elucidate the source and physiological role of serum soluble hemojuvelin in hepcidin regulation and iron metabolism using well-established mouse models of iron-related disorders.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Proteínas de Membrana/sangue , Animais , Linhagem Celular , Meios de Cultivo Condicionados , Feminino , Proteínas Ligadas por GPI , Proteína da Hemocromatose , Humanos , Ferro/sangue , Masculino , Camundongos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Anemia is a common complication of chronic kidney disease (CKD) that negatively impacts the quality of life and is associated with numerous adverse outcomes. Excess levels of the iron regulatory hormone hepcidin are thought to contribute to anemia in CKD patients by decreasing iron availability from the diet and from body stores. Adenine treatment in rats has been proposed as an animal model of anemia of CKD with high hepcidin levels that mirrors the condition in human patients. METHODS: We developed a modified adenine-induced kidney disease model with a higher survival rate than previously reported models, while maintaining persistent kidney disease and anemia. We then tested whether the small molecule bone morphogenetic protein (BMP) inhibitor LDN-193189, which was previously shown to lower hepcidin levels in rodents, mobilized iron into the plasma and improved iron-restricted erythropoiesis in this model. RESULTS: Adenine-treated rats exhibited increased hepatic hepcidin mRNA, decreased serum iron, increased spleen iron content, low hemoglobin (Hb) and inappropriately low erythropoietin (EPO) levels relative to the degree of anemia. LDN-193189 administration to adenine-treated rats lowered hepatic hepcidin mRNA, mobilized stored iron into plasma and increased Hb content of reticulocytes. CONCLUSIONS: Our data suggest that hepcidin lowering agents may provide a new therapeutic strategy to improve iron availability for erythropoiesis in CKD.
Assuntos
Adenina/toxicidade , Anemia Ferropriva/tratamento farmacológico , Modelos Animais de Doenças , Eritrócitos/efeitos dos fármacos , Hepcidinas/metabolismo , Ferro/metabolismo , Nefropatias/complicações , Pirazóis/farmacologia , Pirimidinas/farmacologia , Anemia Ferropriva/sangue , Anemia Ferropriva/etiologia , Animais , Anti-Infecciosos/antagonistas & inibidores , Anti-Infecciosos/metabolismo , Western Blotting , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Ensaio de Imunoadsorção Enzimática , Eritrócitos/citologia , Eritrócitos/metabolismo , Eritropoese/efeitos dos fármacos , Hepcidinas/antagonistas & inibidores , Humanos , Nefropatias/sangue , Nefropatias/induzido quimicamente , Masculino , RNA Mensageiro/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Repulsive guidance molecule (RGM) family members RGMa, RGMb/Dragon, and RGMc/hemojuvelin were found recently to act as bone morphogenetic protein (BMP) coreceptors that enhance BMP signaling activity. Although our previous studies have shown that hemojuvelin regulates hepcidin expression and iron metabolism through the BMP pathway, the role of the BMP signaling mediated by Dragon remains largely unknown. We have shown previously that Dragon is expressed in neural cells, germ cells, and renal epithelial cells. In this study, we demonstrate that Dragon is highly expressed in macrophages. Studies with RAW264.7 and J774 macrophage cell lines reveal that Dragon negatively regulates IL-6 expression in a BMP ligand-dependent manner via the p38 MAPK and Erk1/2 pathways but not the Smad1/5/8 pathway. We also generated Dragon knockout mice and found that IL-6 is upregulated in macrophages and dendritic cells derived from whole lung tissue of these mice compared with that in respective cells derived from wild-type littermates. These results indicate that Dragon is an important negative regulator of IL-6 expression in immune cells and that Dragon-deficient mice may be a useful model for studying immune and inflammatory disorders.
Assuntos
Regulação da Expressão Gênica/imunologia , Interleucina-6/antagonistas & inibidores , Interleucina-6/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Animais , Moléculas de Adesão Celular Neuronais , Linhagem Celular , Modelos Animais de Doenças , Regulação para Baixo/imunologia , Proteínas Ligadas por GPI , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Interleucina-6/biossíntese , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
Anemia is a common feature of CKD associated with poor outcomes. The current management of patients with anemia in CKD is controversial, with recent clinical trials demonstrating increased morbidity and mortality related to erythropoiesis stimulating agents. Here, we examine recent insights into the molecular mechanisms underlying anemia of CKD. These insights hold promise for the development of new diagnostic tests and therapies that directly target the pathophysiologic processes underlying this form of anemia.