RESUMEN
Polycystic kidney disease (PKD) is a ciliopathy characterized by fluid-filled epithelial cysts in the kidney. Although it is well established that the primary cilium is essential for hedgehog (HH) signaling and HH signaling is abnormally activated in multiple PKD models, the mechanism and function of HH activation in PKD pathogenesis remain incompletely understood. Here we used a transgenic HH reporter mouse line to identify the target tissue of HH signaling in Arl13f/f;Ksp-Cre mutant kidney, in which the cilia biogenesis gene Arl13b is specifically deleted in epithelial cells of the distal nephron. In addition, we used a co-culture system to dissect cross-talk between epithelial and mesenchymal cells in the absence of expanding cysts. Finally, we treated Arl13bf/f;Ksp-Cre mice with the GLI inhibitor GANT61 and analyzed its impact on PKD progression in this model. We found that deletion of Arl13b in epithelial cells in the mouse kidney, in vivo, led to non-cell-autonomous activation of the HH pathway in the interstitium. In vitro, when co-cultured with mesenchymal cells, Arl13b-/- epithelial cells produced more sonic hedgehog in comparison to cells expressing Arl13b. Reciprocally, HH signaling was activated in mesenchymal cells co-cultured with Arl13b-/- epithelial cells. Finally, whole body inhibition of the HH pathway by GANT61 reduced the number of proliferating cells, inhibited cyst progression and fibrosis and preserved kidney function in Arl13bf/f;Ksp-Cre mice. Our results reveal non-cell-autonomous activation of HH signaling in the interstitium of the Arl13bf/f;Ksp-Cre kidney and suggest that abnormal activation of the HH pathway contributes to disease progression.
Asunto(s)
Ciliopatías , Quistes , Enfermedades Renales Quísticas , Ratones , Animales , Proteínas Hedgehog/metabolismo , Enfermedades Renales Quísticas/genética , Modelos Animales de Enfermedad , Ratones Transgénicos , Ciliopatías/genética , Progresión de la EnfermedadRESUMEN
Steatotic livers represent a potentially underutilized resource to increase the donor graft pool; however, 1 barrier to the increased utilization of such grafts is the heterogeneity in the definition and the measurement of macrovesicular steatosis (MaS). Digital imaging software (DIS) may better standardize definitions to study posttransplant outcomes. Using HALO, a DIS, we analyzed 63 liver biopsies, from 3 transplant centers, transplanted between 2016 and 2018, and compared macrovesicular steatosis percentage (%MaS) as estimated by transplant center, donor hospital, and DIS. We also quantified the relationship between DIS characteristics and posttransplant outcomes using log-linear regression for peak aspartate aminotransferase, peak alanine aminotransferase, and total bilirubin on postoperative day 7, as well as logistic regression for early allograft dysfunction. Transplant centers and donor hospitals overestimated %MaS compared with DIS, with better agreement at lower %MaS and less agreement for higher %MaS. No DIS analyzed liver biopsies were calculated to be >20% %MaS; however, 40% of liver biopsies read by transplant center pathologists were read to be >30%. Percent MaS read by HALO was positively associated with peak aspartate aminotransferase (regression coefficient= 1.04 1.08 1.12 , p <0.001), peak alanine aminotransferase (regression coefficient = 1.04 1.08 1.12 , p <0.001), and early allograft dysfunction (OR= 1.10 1.40 1.78 , p =0.006). There was no association between HALO %MaS and total bilirubin on postoperative day 7 (regression coefficient = 0.99 1.01 1.04 , p =0.3). DIS provides reproducible quantification of steatosis that could standardize MaS definitions and identify phenotypes associated with good clinical outcomes to increase the utilization of steatite livers.
Asunto(s)
Hígado Graso , Procesamiento de Imagen Asistido por Computador , Trasplante de Hígado , Humanos , Alanina Transaminasa , Aspartato Aminotransferasas , Bilirrubina , Biopsia , Hígado Graso/diagnóstico por imagen , Hígado Graso/patología , Hígado/diagnóstico por imagen , Hígado/patología , Trasplante de Hígado/métodos , Programas Informáticos , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
Primary cilia are hairlike extensions of the plasma membrane of most mammalian cells that serve specialized signaling functions. To traffic properly to cilia, multiple cilia proteins rely on palmitoylation, the post-translational attachment of a saturated 16-carbon lipid. However, details regarding the mechanism of how palmitoylation affects cilia protein localization and function are unknown. Herein, we investigated the protein ADP-ribosylation factor-like GTPase 13b (ARL13b) as a model palmitoylated ciliary protein. Using biochemical, cellular, and in vivo studies, we found that ARL13b palmitoylation occurs in vivo in mouse kidneys and that it is required for trafficking to and function within cilia. Myristoylation, a 14-carbon lipid, is shown to largely substitute for palmitoylation with regard to cilia localization of ARL13b, but not with regard to its function within cilia. The functional importance of palmitoylation results in part from a dramatic increase in ARL13b stability, which is not observed with myristoylation. Additional results show that blockade of depalmitoylation slows the degradation of ARL13b that occurs during cilia resorption, raising the possibility that the sensitivity of ARL13b stability to palmitoylation may be exploited by the cell to accelerate degradation of ARL13b by depalmitoylating it. Together, the results show that palmitoylation plays a unique and critical role in controlling the localization, stability, abundance, and thus function of ARL13b. Pharmacological manipulation of protein palmitoylation may be a strategy to alter cilia function.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Lipoilación/fisiología , Factores de Ribosilacion-ADP/genética , Animales , Cilios/enzimología , Cilios/genética , Estabilidad de Enzimas/fisiología , Células HEK293 , Humanos , Ratones , Transporte de Proteínas/fisiologíaRESUMEN
The gene for ADP ribosylation factor-like GTPase 13B (Arl13b) encodes a small GTPase essential for cilia biogenesis in multiple model organisms. Inactivation of arl13b in zebrafish leads to a number of phenotypes indicative of defective cilia, including cystic kidneys. In mouse, null mutation in Arl13b results in severe patterning defects in the neural tube and defective Hedgehog signaling. Human mutations of ARL13B lead to Joubert syndrome, a ciliopathy. However, patients with mutated ARL13B do not develop kidney cysts. To investigate whether Arl13b has a role in ciliogenesis in mammalian kidney and whether loss of function of Arl13b leads to cystic kidneys in mammals, we generated a mouse model with kidney-specific conditional knockout of Arl13b Deletion of Arl13b in the distal nephron at the perinatal stage led to a cilia biogenesis defect and rapid kidney cyst formation. Additionally, we detected misregulation of multiple pathways in the cystic kidneys of this model. Moreover, valproic acid, a histone deacetylase inhibitor that we previously showed slows cyst progression in a mouse cystic kidney model with neonatal inactivation of Pkd1, inhibited the early rise of Wnt7a expression, ameliorated fibrosis, slowed cyst progression, and improved kidney function in the Arl13b mutant mouse. Finally, in rescue experiments in zebrafish, all ARL13B allele combinations identified in patients with Joubert syndrome provided residual Arl13b function, supporting the idea that the lack of cystic kidney phenotype in human patients with ARL13B mutations is explained by the hypomorphic nature of the mutations.
Asunto(s)
Factores de Ribosilacion-ADP/genética , Anomalías Múltiples/genética , Cerebelo/anomalías , Anomalías del Ojo/genética , Eliminación de Gen , Enfermedades Renales Quísticas/genética , Mutación , Retina/anomalías , Animales , Humanos , Ratones , Pez CebraRESUMEN
Cytokines are known to shape the tumor microenvironment and although progress has been made in understanding their role in carcinogenesis, much remains to learn regarding their role in tumor growth and progression. We have identified granulocyte colony-stimulating factor (G-CSF) as one such cytokine, showing that G-CSF is linked with metastasis in human gastrointestinal tumors and neutralizing G-CSF in a mouse model of colitis-associated cancer is protective. Here, we set out to identify the role of G-CSF and its receptor, G-CSFR, in CD4+ and CD8+ T cell responses in the tumor microenvironment. MC38 colon cancer cells were injected into WT, G-CSFR-/- mice, or Rag2-/- mice. Flow cytometry, Real Time PCR and Multiplex cytokine array analysis were used for in vitro T cell phenotype analysis. Adoptive transfer of WT or G-CSFR-/- CD4+ of CD8+ T cells were performed. Mouse tumor size, cytokine expression, T cell phenotype, and cytotoxic activity were analyzed. We established that in G-CSFR-/- mice, tumor growth of MC38 colon cancer cells is significantly decreased. T cell phenotype and cytokine production were also altered, as both in vitro and in vivo approaches revealed that the G-CSF/G-CSFR stimulate IL-10-producing, FoxP3-expressing CD4+ and CD8+ T cells, whereas G-CSFR-/- T cells exhibit increased IFNγ and IL-17A production, leading to increased cytotoxic activity in the tumor microenvironment. Furthermore, peritumoral injection of recombinant IFNγ or IL-17A inhibited colon and pancreas tumor growth compared to controls. Taken together, our data reveal an unknown mechanism by which G-CSF, through its receptor G-CSFR, promotes an inhibitory Treg phenotype that limits tumor immune responses and furthermore suggest that targeting this cytokine/receptor axis could represent a novel therapeutic approach for gastrointestinal, and likely other tumors with high expression of these factors.
Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Neoplasias del Colon/metabolismo , Citotoxicidad Inmunológica , Factor Estimulante de Colonias de Granulocitos/metabolismo , Linfocitos Infiltrantes de Tumor/metabolismo , Receptores del Factor Estimulante de Colonias/metabolismo , Linfocitos T Reguladores/metabolismo , Animales , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Técnicas de Cocultivo , Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Femenino , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Receptores del Factor Estimulante de Colonias/deficiencia , Receptores del Factor Estimulante de Colonias/genética , Transducción de Señal , Linfocitos T Reguladores/inmunología , Carga Tumoral , Microambiente TumoralRESUMEN
The zebrafish kidney has been used effectively for studying kidney development, repair and disease. New gene editing capability makes it a more versatile in vivo vertebrate model system to investigate renal epithelial cells in their native environment. In this chapter we focus on dissecting gene function in basic cellular biology of renal epithelial cells, including lumen formation and cell polarity, in intact zebrafish embryos.
Asunto(s)
Embrión no Mamífero/citología , Células Epiteliales/citología , Edición Génica/métodos , Inmunohistoquímica/métodos , Riñón/citología , Proteínas de Pez Cebra/genética , Animales , Secuencia de Bases , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Polaridad Celular , Cilios/metabolismo , Cilios/ultraestructura , Embrión no Mamífero/anatomía & histología , Embrión no Mamífero/metabolismo , Células Epiteliales/metabolismo , Femenino , Riñón/embriología , Riñón/metabolismo , Masculino , Organogénesis/genética , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Pez Cebra , Proteínas de Pez Cebra/deficienciaRESUMEN
A major risk factor for colon cancer growth and progression is chronic inflammation. We have shown that the MAPK-activated protein kinase 2 (MK2) pathway is critical for colon tumor growth in colitis-associated and spontaneous colon cancer models. This pathway is known to regulate expression of the tumor-promoting cytokines, IL-1, IL-6, and TNF-α. However, little is known about the ability of MK2 to regulate chemokine production. This is the first study to demonstrate this pathway also regulates the chemokines, MCP-1, Mip-1α, and Mip-2α (MMM). We show that these chemokines induce tumor cell growth and invasion in vitro and that MK2 inhibition suppresses tumor cell production of chemokines and reverses the resulting pro-tumorigenic effects. Addition of MMM to colon tumors in vivo significantly enhances tumor growth in control tumors and restores tumor growth in the presence of MK2 inhibition. We also demonstrate that MK2 signaling is critical for chemokine expression and macrophage influx to the colon tumor microenvironment. MK2 signaling in macrophages was essential for inflammatory cytokine/chemokine production, whereas MK2-/- macrophages or MK2 inhibition suppressed cytokine expression. We show that addition of bone marrow-derived macrophages to the tumor microenvironment enhances tumor growth in control tumors and restores tumor growth in tumors treated with MK2 inhibitors, while addition of MK2-/- macrophages had no effect. This is the first study to demonstrate the critical role of the MK2 pathway in chemokine production, macrophage influx, macrophage function, and tumor growth.
Asunto(s)
Quimiotaxis/inmunología , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Citocinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular/inmunología , Proliferación Celular , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Citocinas/genética , Modelos Animales de Enfermedad , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Activación de Macrófagos/inmunología , Macrófagos/efectos de los fármacos , Ratones , Ratones Noqueados , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/efectos de los fármacos , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The kidneys are a crucial pair of organs that are responsible for filtering the blood to remove waste, maintain electrolyte and water homeostasis, and regulate blood pressure. There are a number of factors, both genetic and environmental, that can impair the function of the kidneys resulting in significant morbidity and mortality for millions of people affected by kidney disease worldwide. The zebrafish, Danio rerio, has emerged as an attractive vertebrate model in the study of kidney development and disease and has proven to be a powerful tool in the advancement of how kidney development occurs in vertebrates and how the kidney repairs itself after injury. Zebrafish share significant similarities in kidney development and composition of nephrons, the functional unit of the kidney. This makes the zebrafish a very promising model to study the mechanisms by which renal developmental defects occur. Furthermore, zebrafish are ideally suited for the study of how vertebrate kidneys respond to injury and have provided researchers with invaluable information on repair processes after kidney injury. Importantly, zebrafish have profound potential for discovering treatment modalities and, in fact, studies in zebrafish models have provided leads for therapeutics for human patients suffering from kidney disease and kidney injury. Here, we discuss the similarities and differences in zebrafish and mammalian kidney models, and highlight some of the major contributions the zebrafish has made in the understanding of kidney development and disease.
Asunto(s)
Enfermedades Renales/patología , Riñón/embriología , Organogénesis , Pez Cebra/fisiología , Animales , Humanos , Regeneración , Células Madre/citologíaRESUMEN
Mutation of the X-linked oral-facial-digital syndrome type 1 (OFD1) gene is embryonic lethal in males and results in craniofacial malformations and adult onset polycystic kidney disease in females. While the OFD1 protein localizes to centriolar satellites, centrosomes and basal bodies, its cellular function and how it relates to cystic kidney disease is largely unknown. Here, we demonstrate that OFD1 is assembled into a protein complex that is localized to the primary cilium and contains the epidermal growth factor receptor (EGFR) and domain organizing flotillin proteins. This protein complex, which has similarity to a basolateral adhesion domain formed during cell polarization, also contains the polycystin proteins that when mutant cause autosomal dominant polycystic kidney disease (ADPKD). Importantly, in human ADPKD cells where mutant polycystin-1 fails to localize to cilia, there is a concomitant loss of localization of polycystin-2, OFD1, EGFR and flotillin-1 to cilia. Together, these data suggest that polycystins are necessary for assembly of a novel flotillin-containing ciliary signaling complex and provide a molecular rationale for the common renal pathologies caused by OFD1 and PKD mutations.