RESUMO
The exocyst and Ift88 are necessary for primary ciliogenesis. Overexpression of Exoc5 (OE), a central exocyst component, resulted in longer cilia and enhanced injury recovery. Mitochondria are involved in acute kidney injury (AKI). To investigate cilia and mitochondria, basal respiration and mitochondrial maximal and spare respiratory capacity were measured in Exoc5 OE, Exoc5 knockdown (KD), Exoc5 ciliary targeting sequence mutant (CTS-mut), control Madin-Darby canine kidney (MDCK), Ift88 knockout (KO), and Ift88 rescue cells. In Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells, these parameters were decreased. In Exoc5 OE and Ift88 rescue cells they were increased. Reactive oxygen species were higher in Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells compared with Exoc5 OE, control, and Ift88 rescue cells. By electron microscopy, mitochondria appeared abnormal in Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells. A metabolomics screen of control, Exoc5 KD, Exoc5 CTS-mut, Exoc5 OE, Ift88 KO, and Ift88 rescue cells showed a marked increase in tryptophan levels in Exoc5 CTS-mut (113-fold) and Exoc5 KD (58-fold) compared with control cells. A 21% increase was seen in Ift88 KO compared with rescue cells. In Exoc5 OE compared with control cells, tryptophan was decreased 59%. To determine the effects of ciliary loss on AKI, we generated proximal tubule-specific Exoc5 and Ift88 KO mice. These mice had loss of primary cilia, decreased mitochondrial ATP synthase, and increased tryptophan in proximal tubules with greater injury following ischemia-reperfusion. These data indicate that cilia-deficient renal tubule cells are primed for injury with mitochondrial defects in tryptophan metabolism.NEW & NOTEWORTHY Mitochondria are centrally involved in acute kidney injury (AKI). Here, we show that cilia-deficient renal tubule cells both in vitro in cell culture and in vivo in mice are primed for injury with mitochondrial defects and aberrant tryptophan metabolism. These data suggest therapeutic strategies such as enhancing ciliogenesis or improving mitochondrial function to protect patients at risk for AKI.
Assuntos
Injúria Renal Aguda , Cílios , Mitocôndrias , Triptofano , Animais , Cílios/metabolismo , Cílios/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Cães , Triptofano/metabolismo , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Injúria Renal Aguda/genética , Células Madin Darby de Rim Canino , Espécies Reativas de Oxigênio/metabolismo , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Camundongos , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/deficiência , Camundongos KnockoutRESUMO
The exocyst is a highly conserved protein complex found in most eukaryotic cells and is associated with many functions, including protein translocation in the endoplasmic reticulum, vesicular basolateral targeting, and ciliogenesis in the kidney. To investigate the exocyst functions, here we exchanged proline for alanine in the highly conserved VXPX ciliary targeting motif of EXOC5 (exocyst complex component 5), a central exocyst gene/protein, and generated stable EXOC5 ciliary targeting sequence-mutated (EXOC5CTS-m) Madin-Darby canine kidney (MDCK) cells. The EXOC5CTS-m protein was stable and could bind other members of the exocyst complex. Culturing stable control, EXOC5-overexpressing (OE), Exoc5-knockdown (KD), and EXOC5CTS-m MDCK cells on Transwell filters, we found that primary ciliogenesis is increased in EXOC5 OE cells and inhibited in Exoc5-KD and EXOC5CTS-m cells. Growing cells in collagen gels until the cyst stage, we noted that EXOC5-OE cells form mature cysts with single lumens more rapidly than control cysts, whereas Exoc5-KD and EXOC5CTS-m MDCK cells failed to form mature cysts. Adding hepatocyte growth factor to induce tubulogenesis, we observed that EXOC5-OE cell cysts form tubules more efficiently than control MDCK cell cysts, EXOC5CTS-m MDCK cell cysts form significantly fewer tubules than control cell cysts, and Exoc5-KD cysts did not undergo tubulogenesis. Finally, we show that EXOC5 mRNA almost completely rescues the ciliary phenotypes in exoc5-mutant zebrafish, unlike the EXOC5CTS-m mRNA, which could not efficiently rescue the phenotypes. Taken together, these results indicate that the exocyst, acting through the primary cilium, is necessary for renal ciliogenesis, cystogenesis, and tubulogenesis.
Assuntos
Cílios/fisiologia , Cistos/patologia , Túbulos Renais/crescimento & desenvolvimento , Rim/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , DNA Complementar/genética , Cães , Técnicas de Silenciamento de Genes , Humanos , Nefropatias/patologia , Células Madin Darby de Rim Canino , Mutagênese Sítio-Dirigida , Ligação Proteica , Transporte Proteico , RNA Mensageiro/metabolismo , Proteínas de Transporte Vesicular/genética , Peixe-ZebraRESUMO
The intrarenal renin angiotensin system (RAS) is activated in polycystic kidney disease. We have recently shown in the Pkd1 mouse that Gen 2 antisense oligonucleotide (ASO), which suppresses angiotensinogen (Agt) synthesis, is efficacious in slowing kidney cyst formation compared with lisinopril. The aim of this current study was to determine 1) if unilateral nephrectomy accelerates cystogenesis in Pkd1 mice (as previously shown in cilia knockout mice) and 2) whether Agt ASO can slow the progression in this accelerated cystic mouse model. Adult Pkd1 conditional floxed allele mice expressing cre were administered tamoxifen, resulting in global knockout of Pkd1. Three weeks after tamoxifen injection, mice underwent left unilateral nephrectomy. Mice were then treated with Agt ASO (75 mg/kg per week) or aliskiren (20 mg/kg per day)+Agt ASO or control for 8 wk. Unilateral nephrectomy accelerated kidney cyst formation compared with nonnephrectomized mice. Both Agt ASO and Aliskiren+Agt ASO treatments significantly reduced plasma and urinary Agt levels. Blood pressure was lowest in Aliskiren+Agt ASO mice among all treatment groups, and the control group had the highest blood pressure. All mice developed significant kidney cysts at 8 wk after nephrectomy, but Agt ASO and Aliskiren+Agt ASO groups had fewer kidney cysts than controls. Renal pAkt, pS6 levels, and apoptosis were significantly suppressed in those receiving Agt ASO compared with controls. These results indicate that suppressing Agt using an ASO slowed the progression of accelerated cystic kidney disease induced by unilateral nephrectomy in Pkd1 mice by suppressing intrarenal RAS, mammalian target of rapamycin pathway, and cell proliferation.
Assuntos
Amidas/farmacologia , Angiotensinogênio/metabolismo , Fumaratos/farmacologia , Rim/efeitos dos fármacos , Rim Policístico Autossômico Dominante/prevenção & controle , Sistema Renina-Angiotensina/efeitos dos fármacos , Renina/antagonistas & inibidores , Canais de Cátion TRPP/metabolismo , Angiotensinogênio/genética , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Receptores ErbB/metabolismo , Feminino , Predisposição Genética para Doença , Rim/metabolismo , Rim/patologia , Masculino , Camundongos Knockout , Nefrectomia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Fenótipo , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/patologia , Renina/metabolismo , Sistema Renina-Angiotensina/genética , Serina-Treonina Quinases TOR/metabolismo , Canais de Cátion TRPP/deficiência , Canais de Cátion TRPP/genética , Fatores de TempoRESUMO
Activation of the intrarenal renin angiotensin system (RAS) is believed to play an important role in the development of hypertension and cystogenesis in autosomal dominant polycystic kidney disease (ADPKD). Results of clinical studies testing RAS inhibitors in slowing the progression of cystic disease in ADPKD are inconclusive, and we hypothesized that current RAS inhibitors do not adequately suppress intrarenal RAS. For this study, we compared a novel Gen 2 antisense oligonucleotide (ASO) that inhibits angiotensinogen (Agt) synthesis to lisinopril in adult conditional Pkd1 systemic-knockout mice, a model of ADPKD. Six weeks after Pkd1 global gene knockout, the mice were treated with Agt-ASO (66 mg/kg/wk), lisinopril (100 mg/kg/d), PBS (control), or scrambled ASO (66 mg/kg/wk) for 10 wk, followed by tissue collection. Agt ASO resulted in significant reduction in plasma, liver, and kidney Agt, and increased kidney renin compared with control treatments. Kidneys from Agt-ASO-treated mice were not as enlarged and showed reduced cystic volume compared with lisinopril or control treatments. Blood pressure was better controlled with lisinopril than with Agt-ASO. Agt-ASO suppressed cell proliferation in both cystic and noncystic cells compared with lisinopril and control treatments. However, Agt-ASO did not reduce cell proliferation in liver, which indicates that Agt-ASO targets cell signaling pathways that specifically suppresses cystogenesis in the kidney. These data suggest that Agt-ASO effectively attenuates intrarenal RAS and therefore can be a novel and effective agent for treating ADPKD.
Assuntos
Angiotensinogênio/metabolismo , Doenças Renais Policísticas/metabolismo , Canais de Cátion TRPP/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Angiotensinogênio/biossíntese , Animais , Feminino , Rim/efeitos dos fármacos , Rim/metabolismo , Lisinopril/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doenças Renais Policísticas/genética , Canais de Cátion TRPP/genéticaRESUMO
The mechanism for early hypertension in polycystic kidney disease (PKD) has not been elucidated. One potential pathway that may contribute to the elevation in blood pressure in PKD is the activation of the intrarenal renin-angiotensin-system (RAS). For example, it has been shown that kidney cyst and cystic fluid contains renin, angiotensin II (AngII), and angiotensinogen (Agt). Numerous studies suggest that ciliary dysfunction plays an important role in PKD pathogenesis. However, it is unknown whether the primary cilium affects the intrarenal RAS in PKD. The purpose of this study was to determine whether loss of cilia or polycystin 1 (PC1) increases intrarenal RAS in mouse model of PKD. Adult Ift88 and Pkd1 conditional floxed allele mice with or without cre were administered tamoxifen to induce global knockout of the gene. Three months after tamoxifen injection, kidney tissues were examined by histology, immunofluorescence, western blot, and mRNA to assess intrarenal RAS components. SV40 immortalized collecting duct cell lines from hypomorphic Ift88 mouse were used to assess intrarenal RAS components in collecting duct cells. Mice without cilia and PC1 demonstrated increased kidney cyst formation, systolic blood pressure, prorenin, and kidney and urinary angiotensinogen levels. Interestingly immunofluorescence study of the kidney revealed that the prorenin receptor was localized to the basolateral membrane of principal cells in cilia (-) but not in cilia (+) kidneys. Collecting duct cAMP responses to AngII administration was greater in cilia (-) vs. cilia (+) cells indicating enhanced intrarenal RAS activity in the absence of cilia. These data suggest that in the absence of cilia or PC1, there is an upregulation of intrarenal RAS components and activity, which may contribute to elevated blood pressure in PKD.
RESUMO
Polycystic kidney disease (PKD) is a common genetic disorder leading to cyst formation in the kidneys and other organs that ultimately results in kidney failure and death. Currently, there is no therapy for slowing down or stopping the progression of PKD. In this study, we identified the disintegrin metalloenzyme 17 (ADAM17) as a key regulator of cell proliferation in kidney tissues of conditional knockout Ift88(-/-) mice and collecting duct epithelial cells from Ift88°(rpk) mice, animal models of autosomal recessive polycystic kidney disease (ARPKD). Using Western blotting, an enzyme activity assay, and a growth factor-shedding assay in the presence or absence of the specific ADAM17 inhibitor TMI-005, we show that increased expression and activation of ADAM17 in the cystic kidney and in collecting duct epithelial cells originating from the Ift88°(rpk) mice (designated as PKD cells) lead to constitutive shedding of several growth factors, including heparin-binding EGF-like growth factor (HB-EGF), amphiregulin, and transforming growth factor-α (TGF-α). Increased growth factor shedding induces activation of the EGFR/MAPK/ERK pathway and maintains higher cell proliferation rate in PKD cells compared with control cells. PKD cells also displayed increased lactate formation and extracellular acidification indicative of aerobic glycolysis (Warburg effect), which was blocked by ADAM17 inhibition. We propose that ADAM17 is a key promoter of cellular proliferation in PKD cells by activating the EGFR/ERK axis and a proproliferative glycolytic phenotype.
Assuntos
Proteínas ADAM/fisiologia , Proliferação de Células/fisiologia , Células Epiteliais/patologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Glicólise/fisiologia , Túbulos Renais Coletores/patologia , Doenças Renais Policísticas/fisiopatologia , Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/efeitos dos fármacos , Proteína ADAM17 , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Receptores ErbB/fisiologia , Feminino , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/fisiologia , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Morfolinas/farmacologia , Fenótipo , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia , Fator de Crescimento Transformador alfa/fisiologia , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genéticaRESUMO
BACKGROUND: The disintegrin and metalloenzyme ADAM17 participates in numerous inflammatory and proliferative diseases, and its pathophysiological role was implicated in kidney fibrosis, polycystic kidney disease and other chronic kidney diseases. At present, we have little understanding how the enzyme activity is regulated. In this study we wanted to characterize the role of α5ß1 integrin in ADAM17 activity regulation during G protein-coupled receptor (GPCR) stimulation. METHODOLOGY/PRINCIPAL FINDINGS: We showed previously that the profibrotic GPCR agonist serotonin (5-HT) induced kidney mesangial cell proliferation through ADAM17 activation and heparin-binding epidermal growth factor (HB-EGF) shedding. In the present studies we observed that in unstimulated mesangial cell lysates α5ß1 integrin co-precipitated with ADAM17 and that 5-HT treatment of the cells induced dissociation of α5ß1 integrin from ADAM17. Using fluorescence immunostaining and in situ proximity ligation assay, we identified the perinuclear region as the localization of the ADAM17/α5ß1 integrin interaction. In cell-free assays, we showed that purified α5ß1 integrin and ß1 integrin dose-dependently bound to and inhibited activity of recombinant ADAM17. We provided evidence that the conformation of the integrin determines its ADAM17-binding ability. To study the effect of ß1 integrin on ADAM17 sheddase activity, we employed alkaline phosphatase-tagged HB-EGF. Overexpression of ß1 integrin lead to complete inhibition of 5-HT-induced HB-EGF shedding and silencing ß1 integrin by siRNA significantly increased mesangial cells ADAM17 responsiveness to 5-HT. CONCLUSIONS/SIGNIFICANCE: Our data show for the first time that ß1 integrin has an important physiological role in ADAM17 activity regulation. We suggest that regulating α5ß1 integrin binding to ADAM17 could be an attractive therapeutic target in chronic kidney diseases.
Assuntos
Proteínas ADAM/metabolismo , Integrina alfa5beta1/metabolismo , Células Mesangiais/enzimologia , Proteína ADAM17 , Animais , Células Cultivadas , Ativação Enzimática , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Integrina beta1/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células Mesangiais/metabolismo , Complexos Multiproteicos/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistas , Tiofenos/farmacologiaRESUMO
A novel tissue-specific shRNA delivery system has been developed using cre-lox technology. Conditionally silenced pSico vector containing oligonucleotides of CD44shRNA and tissue-specific promoter-driven Cre-recombinase expression vector are packaged into transferrin-coated nanoparticles that can deliver shRNA into specific tumors. This system has strong potential in cancer therapy.
Assuntos
Terapia Genética/métodos , Receptores de Hialuronatos/genética , Ácido Hialurônico/metabolismo , Neoplasias/terapia , Interferência de RNA , RNA Interferente Pequeno/genética , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Neoplasias do Colo/terapia , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Receptores de Hialuronatos/metabolismo , Integrases/metabolismo , Masculino , Nanopartículas , Neoplasias/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/terapia , TransfecçãoRESUMO
We showed that renal proximal tubular cells (RPTC) can proliferate and migrate following plating and oxidant or mechanical injury in the absence of exogenous growth factors; however, the mechanisms of this response remain unclear. We examined whether epidermal growth factor receptor (EGFR) signaling is activated following plating and mechanical injury and mediates RPTC proliferation and migration. EGFR, Akt [a target of phosphoinositide-3-kinase (PI3K)], and ERK1/2 were activated after plating and mechanical injury, and their phosphorylation was further enhanced by addition of exogenous EGF. Inactivation of the EGFR with the selective inhibitor AG-1478 completely blocked phosphorylation of EGFR, Akt, and ERK1/2 and blocked cell proliferation and migration after plating and injury. Inhibition of PI3K with LY-294002 blocked Akt phosphorylation and proliferation, whereas U-0126 blocked ERK1/2 phosphorylation but had no effect on proliferation. Furthermore, p38 was phosphorylated following mechanical injury and the p38 inhibitor SB-203580 blocked p38 phosphorylation and cell migration. In contrast, neither PI3K nor ERK1/2 inhibition blocked cell migration. These results show that EGFR activation is required for RPTC proliferation and migration and that proliferation is mediated by PI3K, whereas migration is mediated by p38.