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Mesangial cells offer structural support to the glomerular tuft and regulate glomerular capillary flow through their contractile capabilities. These cells undergo phenotypic changes, such as proliferation and mesangial expansion, resulting in abnormal glomerular tuft formation and reduced capillary loops. Such adaptation to the changing environment is commonly associated with various glomerular diseases, including diabetic nephropathy and glomerulonephritis. Thrombin-induced mesangial remodeling was found in diabetic patients, and expression of the corresponding protease-activated receptors (PARs) in the renal mesangium was reported. However, the functional PAR-mediated signaling in mesangial cells was not examined. This study investigated protease-activated mechanisms regulating mesangial cell calcium waves that may play an essential role in the mesangial proliferation or constriction of the arteriolar cells. Our results indicate that coagulation proteases like thrombin induce synchronized oscillations in cytoplasmic Ca2+ concentration of mesangial cells. The oscillations required PAR1 GPCRs-related activation, but not a PAR4, and were further mediated presumably through store-operated calcium entry and TRPC3 channel activity. Understanding thrombin signaling pathways and their relation to mesangial cells' contractile or synthetic (proliferative) phenotype may play a role in the development of chronic kidney disease and requires further investigation.
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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.
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Lesión Renal Aguda , Cilios , Mitocondrias , Triptófano , Animales , Cilios/metabolismo , Cilios/patología , Mitocondrias/metabolismo , Mitocondrias/patología , Perros , Triptófano/metabolismo , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Lesión Renal Aguda/genética , Células de Riñón Canino Madin Darby , Especies Reactivas de Oxígeno/metabolismo , Túbulos Renales/metabolismo , Túbulos Renales/patología , Ratones , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/deficiencia , Ratones NoqueadosRESUMEN
BACKGROUND: Podocytes have a remarkable ability to recover from injury; however, little is known about the recovery mechanisms involved in this process. We recently showed that formoterol, a long-acting ß2-adrenergic receptor (ß2-AR) agonist, induced mitochondrial biogenesis (MB) in podocytes and led to renoprotection in mice. However, it is not clear whether this effect was mediated by formoterol acting through the ß2-AR or if it occurred through "off-target" effects. METHODS: We genetically deleted the ß2-AR specifically in murine podocytes and used these mice to determine whether formoterol acting through the podocyte ß2-AR alone is sufficient for recovery of renal filtration function following injury. The podocyte-specific ß2-AR knockout mice (ß2-ARfl/fl/PodCre) were generated by crossing ß2-AR floxed mice with podocin Cre (B6.Cg-Tg(NPHS2-cre)295Lbh/J) mice. These mice were then subjected to both acute and chronic glomerular injury using nephrotoxic serum (NTS) and adriamycin (ADR), respectively. The extent of injury was evaluated by measuring albuminuria and histological and immunostaining analysis of the murine kidney sections. RESULTS: A similar level of injury was observed in ß2-AR knockout and control mice; however, the ß2-ARfl/fl/PodCre mice failed to recover in response to formoterol. Functional evaluation of the ß2-ARfl/fl/PodCre mice following injury plus formoterol showed similar albuminuria and glomerular injury to control mice that were not treated with formoterol. CONCLUSIONS: These results indicate that the podocyte ß2-AR is a critical component of the recovery mechanism and may serve as a novel therapeutic target for treating podocytopathies.
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Agonistas de Receptores Adrenérgicos beta 2 , Doxorrubicina , Fumarato de Formoterol , Ratones Noqueados , Podocitos , Receptores Adrenérgicos beta 2 , Animales , Podocitos/metabolismo , Podocitos/efectos de los fármacos , Podocitos/patología , Receptores Adrenérgicos beta 2/metabolismo , Ratones , Fumarato de Formoterol/farmacología , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Doxorrubicina/farmacología , Doxorrubicina/toxicidad , Masculino , Ratones Endogámicos C57BL , Albuminuria/metabolismo , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patologíaRESUMEN
We have previously shown that the long-acting ß2-adrenergic receptor (ß2-AR) agonist formoterol induced recovery from acute kidney injury in mice. To determine whether formoterol protected against diabetic nephropathy, the most common cause of end-stage kidney disease (ESKD), we used a high-fat diet (HFD), a murine type 2 diabetes model, and streptozotocin, a murine type 1 diabetes model. Following formoterol treatment, there was a marked recovery from and reversal of diabetic nephropathy in HFD mice compared with those treated with vehicle alone at the ultrastructural, histological, and functional levels. Similar results were seen after formoterol treatment in mice receiving streptozotocin. To investigate effects in humans, we performed a competing risk regression analysis with death as a competing risk to examine the association between Veterans with chronic kidney disease (CKD) and chronic obstructive pulmonary disease (COPD), who use ß2-AR agonists, and Veterans with CKD but no COPD, and progression to ESKD in a large national cohort of Veterans with stage 4 CKD between 2011 and 2013. Veterans were followed until 2016 or death. ESKD was defined as the initiation of dialysis and/or receipt of kidney transplant. We found that COPD was associated with a 25.6% reduction in progression from stage 4 CKD to ESKD compared with no COPD after adjusting for age, diabetes, sex, race-ethnicity, comorbidities, and medication use. Sensitivity analysis showed a 33.2% reduction in ESKD in Veterans with COPD taking long-acting formoterol and a 20.8% reduction in ESKD in Veterans taking other ß2-AR agonists compared with those with no COPD. These data indicate that ß2-AR agonists, especially formoterol, could be a treatment for diabetic nephropathy and perhaps other forms of CKD.NEW & NOTEWORTHY Diabetic nephropathy is the most common cause of ESKD. Formoterol, a long-acting ß2-adrenergic receptor (ß2-AR) agonist, reversed diabetic nephropathy in murine models of type 1 and 2 diabetes. In humans, there was an association with protection from progression of CKD in patients with COPD, by means of ß2-AR agonist intake, compared with those without COPD. These data indicate that ß2-AR agonists, especially formoterol, could be a new treatment for diabetic nephropathy and other forms of CKD.
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Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Nefropatías Diabéticas , Fallo Renal Crónico , Enfermedad Pulmonar Obstructiva Crónica , Humanos , Animales , Ratones , Nefropatías Diabéticas/tratamiento farmacológico , Agonistas de Receptores Adrenérgicos beta 2/uso terapéutico , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Estreptozocina , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Fumarato de Formoterol/uso terapéutico , Fallo Renal Crónico/tratamiento farmacológico , Fallo Renal Crónico/etiología , Receptores Adrenérgicos/uso terapéuticoRESUMEN
Angiotensin receptor blockers (ARBs) are the first-line treatment for hypertension; they act by inhibiting signaling through the angiotensin 1 receptor (AT1R). Recently, a novel biased AT1R agonist, TRV120027 (TRV), which selectively activates the ß-arrestin cascade and blocks the G-protein-coupled receptor pathway has been proposed as a potential blood pressure medication. Here, we explored the effects of TRV and associated ß-arrestin signaling in podocytes, essential cells of the kidney filter. We used human podocyte cell lines to determine ß-arrestin's involvement in calcium signaling and cytoskeletal reorganization and Dahl SS rats to investigate the chronic effects of TRV administration on glomerular health. Our experiments indicate that the TRV-activated ß-arrestin pathway promotes the rapid elevation of intracellular Ca2+ in a dose-dependent manner. Interestingly, the amplitude of ß-arrestin-mediated Ca2+ influx was significantly higher than the response to similar Ang II concentrations. Single-channel analyses show rapid activation of transient receptor potential canonical (TRPC) channels following acute TRV application. Furthermore, the pharmacological blockade of TRPC6 significantly attenuated the ß-arrestin-mediated Ca2+ influx. Additionally, prolonged activation of the ß-arrestin pathway in podocytes resulted in pathological actin cytoskeleton rearrangements, higher apoptotic cell markers, and augmented glomerular damage. TRV-activated ß-arrestin signaling in podocytes may promote TRPC6 channel-mediated Ca2+ influx, foot process effacement, and apoptosis, possibly leading to severe defects in glomerular filtration barrier integrity and kidney health. Under these circumstances, the potential therapeutic application of TRV for hypertension treatment requires further investigation to assess the balance of the benefits versus possible deleterious effects and off-target damage.
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Hipertensión , Enfermedades Renales , Podocitos , Ratas , Animales , Humanos , Podocitos/metabolismo , Canal Catiónico TRPC6/metabolismo , Calcio/metabolismo , beta-Arrestinas/metabolismo , Antagonistas de Receptores de Angiotensina/farmacología , Ratas Endogámicas Dahl , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Enfermedades Renales/metabolismo , Hipertensión/metabolismo , Canales Catiónicos TRPC/metabolismo , Canales Catiónicos TRPC/farmacologíaRESUMEN
Background: The primary cilium protrudes from the cell surface and functions as a mechanosensor. Recently, we found that water intake restriction shortens the primary cilia of renal tubular cells, and a blockage of the shortening disturbs the ability of the kidneys to concentrate urine. Here, we investigate whether high water intake (HWI) alters primary cilia length, and if so, what is its underlying mechanism and its role on kidney urine production. Methods: Experimental mice were given free access to normal water (normal water intake) or 3% sucrose-containing water for HWI for 2 days. Some mice were administered with U0126 (10 mg/kg body weight), an inhibitor of MEK kinase, from 2 days before HWI, daily. The primary cilium length and urine amount and osmolality were investigated. Results: HWI-induced diluted urine production and primary cilium elongation in renal tubular cells. HWI increased the expression of α-tubulin acetyltransferase 1 (αTAT1), leading to the acetylation of α-tubulins, a core protein of the primary cilia. HWI also increased phosphorylated ERK1/2 (p-ERK1/2) and exocyst complex component 5 (EXOC5) expression in the kidneys. U0126 blocked HWI-induced increases in αTAT1, p-ERK1/2, and EXOC5 expression. U0126 inhibited HWI-induced α-tubulin acetylation, primary cilium elongation, urine amount increase, and urine osmolality decrease. Conclusion: These results show that increased water intake elongates the primary cilia via ERK1/2 activation and that ERK inhibition prevents primary cilium elongation and diluted urine production. These data suggest that the elongation of primary cilium length is associated with the production of diluted urine.
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BACKGROUND: The primary cilium, a microtubule-based cellular organelle present in certain kidney cells, functions as a mechano-sensor to monitor fluid flow in addition to various other biological functions. In kidneys, the primary cilia protrude into the tubular lumen and are directly exposed to pro-urine flow and components. However, their effects on urine concentration remain to be defined. Here, we investigated the association between primary cilia and urine concentration. METHODS: Mice either had free access to water (normal water intake, NWI) or were not allowed access to water (water deprivation, WD). Some mice received tubastatin, an inhibitor of histone deacetylase 6 (HDAC6), which regulates the acetylation of α-tubulin, a core protein of microtubules. RESULTS: WD decreased urine output and increased urine osmolality, concomitant with apical plasma membrane localization of aquaporin 2 (AQP2) in the kidney. After WD, compared with after NWI, the lengths of primary cilia in renal tubular epithelial cells were shortened and HDAC6 activity increased. WD induced deacetylation of α-tubulin without altering α-tubulin levels in the kidney. Tubastatin prevented the shortening of cilia through increasing HDAC6 activity and consequently increasing acetylated α-tubulin expression. Furthermore, tubastatin prevented the WD-induced reduction of urine output, urine osmolality increase, and apical plasma membrane localization of AQP2. CONCLUSIONS: WD shortens primary cilia length through HDAC6 activation and α-tubulin deacetylation, while HDAC6 inhibition blocks the WD-induced changes in cilia length and urine output. This suggests that cilia length alterations are involved, at least in part, in the regulation of body water balance and urine concentration.
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Primary cilia on kidney tubular cells play crucial roles in maintaining structure and physiological function. Emerging evidence indicates that the absence of primary cilia, and their length, are associated with kidney diseases. The length of primary cilia in kidney tubular epithelial cells depends, at least in part, on oxidative stress and extracellular signal-regulated kinase 1/2 (ERK) activation. Hydrogen sulfide (H2S) is involved in antioxidant systems and the ERK signaling pathway. Therefore, in this study, we investigated the role of H2S in primary cilia elongation and the downstream pathway. In cultured Madin-Darby Canine Kidney cells, the length of primary cilia gradually increased up to 4 days after the cells were grown to confluent monolayers. In addition, the expression of H2S-producing enzyme increased concomitantly with primary cilia length. Treatment with NaHS, an exogenous H2S donor, accelerated the elongation of primary cilia whereas DL-propargylglycine (a cystathionine γ-lyase inhibitor) and hydroxylamine (a cystathionine-ß-synthase inhibitor) delayed their elongation. NaHS treatment increased ERK activation and Sec10 and Arl13b protein expression, both of which are involved in cilia formation and elongation. Treatment with U0126, an ERK inhibitor, delayed elongation of primary cilia and blocked the effect of NaHS-mediated primary cilia elongation and Sec10 and Arl13b upregulation. Finally, we also found that H2S accelerated primary cilia elongation after ischemic kidney injury. These results indicate that H2S lengthens primary cilia through ERK activation and a consequent increase in Sec10 and Arl13b expression, suggesting that H2S and its downstream targets could be novel molecular targets for regulating primary cilia.
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Phosphorylation (activation) and dephosphorylation (deactivation) of the slit diaphragm proteins NEPHRIN and NEPH1 are critical for maintaining the kidney epithelial podocyte actin cytoskeleton and, therefore, proper glomerular filtration. However, the mechanisms underlying these events remain largely unknown. Here we show that NEPHRIN and NEPH1 are novel receptor proteins for hepatocyte growth factor (HGF) and can be phosphorylated independently of the mesenchymal epithelial transition receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Furthermore, we demonstrate SH2 domain-containing protein tyrosine phosphatase-2-dependent dephosphorylation of these proteins. To establish HGF as a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report high-affinity interactions of NEPHRIN and NEPH1 with HGF, although NEPHRIN binding was 20-fold higher than that of NEPH1. In addition, using molecular modeling we constructed peptides that were used to map specific HGF-binding regions in the extracellular domains of NEPHRIN and NEPH1. Finally, using an in vitro model of cultured podocytes and an ex vivo model of Drosophila nephrocytes, as well as chemically induced injury models, we demonstrated that HGF-induced phosphorylation of NEPHRIN and NEPH1 is centrally involved in podocyte repair. Taken together, this is the first study demonstrating a receptor-based function for NEPHRIN and NEPH1. This has important biological and clinical implications for the repair of injured podocytes and the maintenance of podocyte integrity.
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Factor de Crecimiento de Hepatocito/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Línea Celular , Tasa de Filtración Glomerular/fisiología , Factor de Crecimiento de Hepatocito/fisiología , Humanos , Uniones Intercelulares/metabolismo , Riñón/patología , Glomérulos Renales/metabolismo , Proteínas de la Membrana/genética , Ratones , Péptidos/metabolismo , Fosforilación , Podocitos/metabolismo , Unión Proteica/fisiología , Transducción de Señal/fisiologíaRESUMEN
To characterize the mechanisms by which the highly conserved exocyst trafficking complex regulates eye physiology in zebrafish and mice, we focused on Exoc5 (also known as sec10), a central exocyst component. We analyzed both exoc5 zebrafish mutants and retinal pigmented epithelium (RPE)-specific Exoc5 knockout mice. Exoc5 is present in both the non-pigmented epithelium of the ciliary body and in the RPE. In this study, we set out to establish an animal model to study the mechanisms underlying the ocular phenotype and to establish if loss of visual function is induced by postnatal RPE Exoc5-deficiency. Exoc5-/- zebrafish had smaller eyes, with decreased number of melanocytes in the RPE and shorter photoreceptor outer segments. At 3.5 days post-fertilization, loss of rod and cone opsins were observed in zebrafish exoc5 mutants. Mice with postnatal RPE-specific loss of Exoc5 showed retinal thinning associated with compromised visual function and loss of visual photoreceptor pigments. Abnormal levels of RPE65 together with a reduced c-wave amplitude indicate a dysfunctional RPE. The retinal phenotype in Exoc5-/- mice was present at 20 weeks, but was more pronounced at 27 weeks, indicating progressive disease phenotype. We previously showed that the exocyst is necessary for photoreceptor ciliogenesis and retinal development. Here, we report that exoc5 mutant zebrafish and mice with RPE-specific genetic ablation of Exoc5 develop abnormal RPE pigmentation, resulting in retinal cell dystrophy and loss of visual pigments associated with compromised vision. Together, these data suggest that exocyst-mediated signaling in the RPE is required for RPE structure and function, indirectly leading to photoreceptor degeneration.
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Células Fotorreceptoras/patología , Degeneración Retiniana , Epitelio Pigmentado de la Retina/patología , Proteínas de Transporte Vesicular/fisiología , Trastornos de la Visión/patología , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Fotorreceptoras/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Trastornos de la Visión/metabolismo , Pez CebraRESUMEN
Myosin 1c (Myo1c) is an unconventional myosin that modulates signaling pathways involved in tissue injury and repair. In this study, we observed that Myo1c expression is significantly upregulated in human chronic liver disease such as nonalcoholic steatohepatitis (NASH) and in animal models of liver fibrosis. High throughput data from the GEO-database identified similar Myo1c upregulation in mice and human liver fibrosis. Notably, transforming growth factor-ß1 (TGF-ß1) stimulation to hepatic stellate cells (HSCs), the liver pericyte and key cell type responsible for the deposition of extracellular matrix, upregulates Myo1c expression, whereas genetic depletion or pharmacological inhibition of Myo1c blunted TGF-ß-induced fibrogenic responses, resulting in repression of α-smooth muscle actin (α-SMA) and collagen type I α 1 chain (Col1α1) mRNA. Myo1c deletion also decreased fibrogenic processes such as cell proliferation, wound healing response, and contractility when compared with vehicle-treated HSCs. Importantly, phosphorylation of mothers against decapentaplegic homolog 2 (SMAD2) and mothers against decapentaplegic homolog 3 (SMAD3) were significantly blunted upon Myo1c inhibition in GRX cells as well as Myo1c knockout (Myo1c-KO) mouse embryonic fibroblasts (MEFs) upon TGF-ß stimulation. Using the genetic Myo1c-KO mice, we confirmed that Myo1c is critical for fibrogenesis, as Myo1c-KO mice were resistant to carbon tetrachloride (CCl4)-induced liver fibrosis. Histological and immunostaining analysis of liver sections showed that deposition of collagen fibers and α-SMA expression were significantly reduced in Myo1c-KO mice upon liver injury. Collectively, these results demonstrate that Myo1c mediates hepatic fibrogenesis by modulating TGF-ß signaling and suggest that inhibiting this process may have clinical application in treating liver fibrosis.NEW & NOTEWORTHY The incidences of liver fibrosis are growing at a rapid pace and have become one of the leading causes of end-stage liver disease. Although TGF-ß1 is known to play a prominent role in transforming cells to produce excessive extracellular matrix that lead to hepatic fibrosis, the therapies targeting TGF-ß1 have achieved very limited clinical impact. This study highlights motor protein myosin-1c-mediated mechanisms that serve as novel regulators of TGF-ß1 signaling and fibrosis.
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Fibroblastos/metabolismo , Cirrosis Hepática/metabolismo , Hígado/metabolismo , Miosina Tipo I/metabolismo , Animales , Cadena alfa 1 del Colágeno Tipo I , Fibroblastos/patología , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Hígado/patología , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Ratones , Miosina Tipo I/genética , Fosforilación , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Dietary vitamin A/all-trans retinol/ROL plays a critical role in human vision. ROL circulates bound to the plasma retinol-binding protein (RBP4) as RBP4-ROL. In the eye, the STRA6 membrane receptor binds to circulatory RBP4 and internalizes ROL. STRA6 is, however, not expressed in systemic tissues, where there is high affinity RBP4 binding and ROL uptake. We tested the hypothesis that the second retinol binding protein 4 receptor 2 (Rbpr2), which is highly expressed in systemic tissues of zebrafish and mouse, contains a functional RBP4 binding domain, critical for ROL transport. As for STRA6, modeling and docking studies confirmed three conserved RBP4 binding residues in zebrafish Rbpr2. In cell culture studies, disruption of the RBP4 binding residues on Rbpr2 almost completely abolished uptake of exogenous vitamin A. CRISPR-generated rbpr2-RBP4 domain zebrafish mutants showed microphthalmia, shorter photoreceptor outer segments, and decreased opsins, which were attributed to impaired ocular retinoid content. Injection of WT-Rbpr2 mRNA into rbpr2 mutant or all-trans retinoic acid treatment rescued the mutant eye phenotypes. In conclusion, zebrafish Rbpr2 contains a putative extracellular RBP4-ROL ligand-binding domain, critical for yolk vitamin A transport to the eye for ocular retinoid production and homeostasis, for photoreceptor cell survival.
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Supervivencia Celular/fisiología , Ojo/metabolismo , Homeostasis/fisiología , Retinoides/metabolismo , Vitamina A/sangre , Animales , Proteínas Portadoras/metabolismo , Hígado/metabolismo , Proteínas de la Membrana/metabolismo , Células Fotorreceptoras/metabolismo , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
Non-syndromic mitral valve prolapse (MVP) is the most common heart valve disease affecting 2.4% of the population. Recent studies have identified genetic defects in primary cilia as causative to MVP, although the mechanism of their action is currently unknown. Using a series of gene inactivation approaches, we define a paracrine mechanism by which endocardially-expressed Desert Hedgehog (DHH) activates primary cilia signaling on neighboring valve interstitial cells. High-resolution imaging and functional assays show that DHH de-represses smoothened at the primary cilia, resulting in kinase activation of RAC1 through the RAC1-GEF, TIAM1. Activation of this non-canonical hedgehog pathway stimulates α-smooth actin organization and ECM remodeling. Genetic or pharmacological perturbation of this pathway results in enlarged valves that progress to a myxomatous phenotype, similar to valves seen in MVP patients. These data identify a potential molecular origin for MVP as well as establish a paracrine DHH-primary cilium cross-talk mechanism that is likely applicable across developmental tissue types.
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Cilios/metabolismo , Proteínas Hedgehog/metabolismo , Válvula Mitral/embriología , Actinas/metabolismo , Animales , Matriz Extracelular/metabolismo , Enfermedades de las Válvulas Cardíacas , Proteínas Hedgehog/fisiología , Ratones , Prolapso de la Válvula Mitral/genética , Prolapso de la Válvula Mitral/metabolismo , Músculo Liso/metabolismo , Músculo Liso/fisiología , Miocitos del Músculo Liso/metabolismo , Neuropéptidos/metabolismo , Fenotipo , Transducción de Señal , Factores de Transcripción/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The recently proposed idea of "urocrine signaling" hypothesizes that small secreted extracellular vesicles (EVs) contain proteins that transmit signals to distant cells. However, the role of renal primary cilia in EV production and content is unclear. We previously showed that the exocyst, a highly conserved trafficking complex, is necessary for ciliogenesis; that it is present in human urinary EVs; that knockdown (KD) of exocyst complex component 5 (EXOC5), a central exocyst component, results in very short or absent cilia; and that human EXOC5 overexpression results in longer cilia. Here, we show that compared with control Madin-Darby canine kidney (MDCK) cells, EXOC5 overexpression increases and KD decreases EV numbers. Proteomic analyses of isolated EVs from EXOC5 control, KD, and EXOC5-overexpressing MDCK cells revealed significant alterations in protein composition. Using immunoblotting to specifically examine the expression levels of ADP-ribosylation factor 6 (ARF6) and EPS8-like 2 (EPS8L2) in EVs, we found that EXOC5 KD increases ARF6 levels and decreases EPS8L2 levels, and that EXOC5 overexpression increases EPS8L2. Knockout of intraflagellar transport 88 (IFT88) confirmed that the changes in EV number/content were due to cilia loss: similar to EXOC5, the IFT88 loss resulted in very short or absent cilia, decreased EV numbers, increased EV ARF6 levels, and decreased Eps8L2 levels compared with IFT88-rescued EVs. Compared with control animals, urine from proximal tubule-specific EXOC5-KO mice contained fewer EVs and had increased ARF6 levels. These results indicate that perturbations in exocyst and primary cilia affect EV number and protein content.
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Cilios/metabolismo , Exocitosis , Vesículas Extracelulares/metabolismo , Riñón/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Factor 6 de Ribosilación del ADP , Animales , Células Cultivadas , Perros , Humanos , Células de Riñón Canino Madin Darby/metabolismo , Ratones , Ratones Noqueados , Proteínas de Transporte Vesicular/deficienciaRESUMEN
BACKGROUND: Bicuspid aortic valve (BAV) disease is a congenital defect that affects 0.5% to 1.2% of the population and is associated with comorbidities including ascending aortic dilation and calcific aortic valve stenosis. To date, although a few causal genes have been identified, the genetic basis for the vast majority of BAV cases remains unknown, likely pointing to complex genetic heterogeneity underlying this phenotype. Identifying genetic pathways versus individual gene variants may provide an avenue for uncovering additional BAV causes and consequent comorbidities. METHODS: We performed genome-wide association Discovery and Replication Studies using cohorts of 2131 patients with BAV and 2728 control patients, respectively, which identified primary cilia genes as associated with the BAV phenotype. Genome-wide association study hits were prioritized based on P value and validated through in vivo loss of function and rescue experiments, 3-dimensional immunohistochemistry, histology, and morphometric analyses during aortic valve morphogenesis and in aged animals in multiple species. Consequences of these genetic perturbations on cilia-dependent pathways were analyzed by Western and immunohistochemistry analyses, and assessment of aortic valve and cardiac function were determined by echocardiography. RESULTS: Genome-wide association study hits revealed an association between BAV and genetic variation in human primary cilia. The most associated single-nucleotide polymorphisms were identified in or near genes that are important in regulating ciliogenesis through the exocyst, a shuttling complex that chaperones cilia cargo to the membrane. Genetic dismantling of the exocyst resulted in impaired ciliogenesis, disrupted ciliogenic signaling and a spectrum of cardiac defects in zebrafish, and aortic valve defects including BAV, valvular stenosis, and valvular calcification in murine models. CONCLUSIONS: These data support the exocyst as required for normal ciliogenesis during aortic valve morphogenesis and implicate disruption of ciliogenesis and its downstream pathways as contributory to BAV and associated comorbidities in humans.
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Estenosis de la Válvula Aórtica/patología , Válvula Aórtica/anomalías , Cilios/fisiología , Cardiopatías Congénitas/patología , Enfermedades de las Válvulas Cardíacas/patología , Animales , Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Estenosis de la Válvula Aórtica/genética , Enfermedad de la Válvula Aórtica Bicúspide , Estudios de Casos y Controles , Cilios/patología , Frecuencia de los Genes , Estudio de Asociación del Genoma Completo , Genotipo , Cardiopatías Congénitas/genética , Enfermedades de las Válvulas Cardíacas/genética , Enfermedades de las Válvulas Cardíacas/metabolismo , Humanos , Ratones , Ratones Noqueados , Polimorfismo de Nucleótido Simple , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The exocyst is a highly conserved eight-subunit protein complex (EXOC1-8) involved in the targeting and docking of exocytic vesicles translocating from the trans-Golgi network to various sites in renal cells. EXOC5 is a central exocyst component because it connects EXOC6, bound to the vesicles exiting the trans-Golgi network via the small GTPase RAB8, to the rest of the exocyst complex at the plasma membrane. In the kidney, the exocyst complex is involved in primary ciliognesis, cystogenesis, and tubulogenesis. The exocyst, and its regulators, have also been found in urinary extracellular vesicles, and may be centrally involved in urocrine signaling and repair following acute kidney injury. The exocyst is centrally involved in the development of other organs, including the eye, ear, and heart. The exocyst is regulated by many different small GTPases of the RHO, RAL, RAB, and ARF families. The small GTPases, and their guanine nucleotide exchange factors and GTPase-activating proteins, likely give the exocyst specificity of function. The recent development of a floxed Exoc5 mouse line will aid researchers in studying the role of the exocyst in multiple cells and organ types by allowing for tissue-specific knockout, in conjunction with Cre-driver mouse lines.
RESUMEN
Mitral valve prolapse (MVP) affects 1 in 40 people and is the most common indication for mitral valve surgery. MVP can cause arrhythmias, heart failure, and sudden cardiac death, and to date, the causes of this disease are poorly understood. We now demonstrate that defects in primary cilia genes and their regulated pathways can cause MVP in familial and sporadic nonsyndromic MVP cases. Our expression studies and genetic ablation experiments confirmed a role for primary cilia in regulating ECM deposition during cardiac development. Loss of primary cilia during development resulted in progressive myxomatous degeneration and profound mitral valve pathology in the adult setting. Analysis of a large family with inherited, autosomal dominant nonsyndromic MVP identified a deleterious missense mutation in a cilia gene, DZIP1 A mouse model harboring this variant confirmed the pathogenicity of this mutation and revealed impaired ciliogenesis during development, which progressed to adult myxomatous valve disease and functional MVP. Relevance of primary cilia in common forms of MVP was tested using pathway enrichment in a large population of patients with MVP and controls from previously generated genome-wide association studies (GWAS), which confirmed the involvement of primary cilia genes in MVP. Together, our studies establish a developmental basis for MVP through altered cilia-dependent regulation of ECM and suggest that defects in primary cilia genes can be causative to disease phenotype in some patients with MVP.
Asunto(s)
Cilios/patología , Prolapso de la Válvula Mitral/etiología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Secuencia de Bases , Matriz Extracelular/metabolismo , Femenino , Estudio de Asociación del Genoma Completo , Válvulas Cardíacas/diagnóstico por imagen , Válvulas Cardíacas/crecimiento & desarrollo , Humanos , Masculino , Ratones Noqueados , Prolapso de la Válvula Mitral/diagnóstico por imagen , Prolapso de la Válvula Mitral/genética , Morfogénesis , Linaje , Factores de Tiempo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Although the slit diaphragm proteins in podocytes are uniquely organized to maintain glomerular filtration assembly and function, little is known about the underlying mechanisms that participate in trafficking these proteins to the correct location for development and homeostasis. Identifying these mechanisms will likely provide novel targets for therapeutic intervention to preserve podocyte function following glomerular injury. Analysis of structural variation in cases of human nephrotic syndrome identified rare heterozygous deletions of EXOC4 in two patients. This suggested that disruption of the highly-conserved eight-protein exocyst trafficking complex could have a role in podocyte dysfunction. Indeed, mRNA profiling of injured podocytes identified significant exocyst down-regulation. To test the hypothesis that the exocyst is centrally involved in podocyte development/function, we generated homozygous podocyte-specific Exoc5 (a central exocyst component that interacts with Exoc4) knockout mice that showed massive proteinuria and died within 4 weeks of birth. Histological and ultrastructural analysis of these mice showed severe glomerular defects with increased fibrosis, proteinaceous casts, effaced podocytes, and loss of the slit diaphragm. Immunofluorescence analysis revealed that Neph1 and Nephrin, major slit diaphragm constituents, were mislocalized and/or lost. mRNA profiling of Exoc5 knockdown podocytes showed that vesicular trafficking was the most affected cellular event. Mapping of signaling pathways and Western blot analysis revealed significant up-regulation of the mitogen-activated protein kinase and transforming growth factor-ß pathways in Exoc5 knockdown podocytes and in the glomeruli of podocyte-specific Exoc5 KO mice. Based on these data, we propose that exocyst-based mechanisms regulate Neph1 and Nephrin signaling and trafficking, and thus podocyte development and function.
Asunto(s)
Eliminación de Gen , Glomérulos Renales/patología , Síndrome Nefrótico/patología , Podocitos/patología , Proteínas de Transporte Vesicular/fisiología , Animales , Apoptosis , Movimiento Celular , Exocitosis , Humanos , Glomérulos Renales/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Síndrome Nefrótico/genética , Fosforilación , Podocitos/metabolismo , Transporte de Proteínas , Proteinuria/etiología , Proteinuria/patología , Transducción de SeñalRESUMEN
Transforming growth factor-ß (TGF-ß) is known to play a critical role in the pathogenesis of many progressive podocyte diseases. However, the molecular mechanisms regulating TGF-ß signaling in podocytes remain unclear. Using a podocyte-specific myosin (Myo)1c knockout, we demonstrate whether Myo1c is critical for TGF-ß-signaling in podocyte disease pathogenesis. Specifically, podocyte-specific Myo1c knockout mice were resistant to fibrotic injury induced by Adriamycin or nephrotoxic serum. Further, loss of Myo1c also protected from injury in the TGF-ß-dependent unilateral ureteral obstruction mouse model of renal interstitial fibrosis. Mechanistic analyses showed that loss of Myo1c significantly blunted TGF-ß signaling through downregulation of canonical and non-canonical TGF-ß pathways. Interestingly, nuclear rather than the cytoplasmic Myo1c was found to play a central role in controlling TGF-ß signaling through transcriptional regulation. Differential expression analysis of nuclear Myo1c-associated gene promoters showed that nuclear Myo1c targeted the TGF-ß responsive gene growth differentiation factor (GDF)-15 and directly bound to the GDF-15 promoter. Importantly, GDF15 was found to be involved in podocyte pathogenesis, where GDF15 was upregulated in glomeruli of patients with focal segmental glomerulosclerosis. Thus, Myo1c-mediated regulation of TGF-ß-responsive genes is central to the pathogenesis of podocyte injury. Hence, inhibiting this process may have clinical application in treating podocytopathies.