RESUMO
Swelling of the brain or spinal cord (CNS edema) affects millions of people every year. All potential pharmacological interventions have failed in clinical trials, meaning that symptom management is the only treatment option. The water channel protein aquaporin-4 (AQP4) is expressed in astrocytes and mediates water flux across the blood-brain and blood-spinal cord barriers. Here we show that AQP4 cell-surface abundance increases in response to hypoxia-induced cell swelling in a calmodulin-dependent manner. Calmodulin directly binds the AQP4 carboxyl terminus, causing a specific conformational change and driving AQP4 cell-surface localization. Inhibition of calmodulin in a rat spinal cord injury model with the licensed drug trifluoperazine inhibited AQP4 localization to the blood-spinal cord barrier, ablated CNS edema, and led to accelerated functional recovery compared with untreated animals. We propose that targeting the mechanism of calmodulin-mediated cell-surface localization of AQP4 is a viable strategy for development of CNS edema therapies.
Assuntos
Aquaporina 4/metabolismo , Edema/metabolismo , Edema/terapia , Animais , Aquaporina 4/fisiologia , Astrócitos/metabolismo , Encéfalo/metabolismo , Edema Encefálico/metabolismo , Calmodulina/metabolismo , Sistema Nervoso Central/metabolismo , Edema/fisiopatologia , Masculino , Ratos , Ratos Sprague-Dawley , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/metabolismo , Trifluoperazina/farmacologiaRESUMO
Cancer invasion and metastasis are known to be potentiated by the expression of aquaporins (AQPs). Likewise, the expression levels of AQPs have been shown to be prognostic for survival in patients and have a role in tumor growth, edema, angiogenesis, and tumor cell migration. Thus, AQPs are key players in cancer biology and potential targets for drug development. Here, we present the single-particle cryo-EM structure of human AQP7 at 3.2-Å resolution in complex with the specific inhibitor compound Z433927330. The structure in combination with MD simulations shows that the inhibitor binds to the endofacial side of AQP7. In addition, cancer cells treated with Z433927330 show reduced proliferation. The data presented here serve as a framework for the development of AQP inhibitors.
Assuntos
Aquaporinas , Neoplasias , Humanos , Aquaporinas/metabolismo , Aquaporina 1/metabolismoRESUMO
The function of astrocytes in response to gut microbiota-derived signals has an important role in the pathophysiological processes of central nervous system (CNS) diseases. However, the specific effects of microbiota-derived metabolites on astrocyte activation have not been elucidated yet. Experimental autoimmune encephalomyelitis (EAE) was induced in female C57BL/6 mice as a classical MS model. The alterations of gut microbiota and the levels of short-chain fatty acids (SCFAs) were assessed after EAE induction. We observed that EAE mice exhibit low levels of Allobaculum, Clostridium_IV, Clostridium_XlVb, Lactobacillus genera, and microbial-derived SCFAs metabolites. SCFAs supplementation suppressed astrocyte activation by increasing the level of tryptophan (Trp)-derived AhR ligands that activating the AhR. The beneficial effects of SCFAs supplementation on the clinical scores, histopathological alterations, and the blood brain barrier (BBB)-glymphatic function were abolished by intracisterna magna injection of AAV-GFAP-shAhR. Moreover, SCFAs supplementation suppressed the loss of AQP4 polarity within astrocytes in an AhR-dependent manner. Together, SCFAs potentially suppresses astrocyte activation by amplifying Trp-AhR-AQP4 signaling in EAE mice. Our study demonstrates that SCFAs supplementation may serve as a viable therapy for inflammatory disorders of the CNS.
Assuntos
Aquaporina 4 , Astrócitos , Encefalomielite Autoimune Experimental , Ácidos Graxos Voláteis , Camundongos Endogâmicos C57BL , Receptores de Hidrocarboneto Arílico , Transdução de Sinais , Triptofano , Animais , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Astrócitos/metabolismo , Astrócitos/efeitos dos fármacos , Ácidos Graxos Voláteis/farmacologia , Ácidos Graxos Voláteis/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Camundongos , Triptofano/metabolismo , Triptofano/farmacologia , Feminino , Transdução de Sinais/efeitos dos fármacos , Aquaporina 4/metabolismo , Aquaporina 4/genética , Microbioma Gastrointestinal/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacosRESUMO
Protein kinase A (PKA) directly phosphorylates aquaporin-2 (AQP2) water channels in renal collecting ducts to reabsorb water from urine for the maintenance of systemic water homeostasis. More than 50 functionally distinct PKA-anchoring proteins (AKAPs) respectively create compartmentalized PKA signaling to determine the substrate specificity of PKA. Identification of an AKAP responsible for AQP2 phosphorylation is an essential step toward elucidating the molecular mechanisms of urinary concentration. PKA activation by several compounds is a novel screening strategy to uncover PKA substrates whose phosphorylation levels were nearly perfectly correlated with that of AQP2. The leading candidate in this assay proved to be an AKAP termed lipopolysaccharide-responsive and beige-like anchor protein (LRBA). We found that LRBA colocalized with AQP2 in vivo, and Lrba knockout mice displayed a polyuric phenotype with severely impaired AQP2 phosphorylation. Most of the PKA substrates other than AQP2 were adequately phosphorylated by PKA in the absence of LRBA, demonstrating that LRBA-anchored PKA preferentially phosphorylated AQP2 in renal collecting ducts. Furthermore, the LRBA-PKA interaction, rather than other AKAP-PKA interactions, was robustly dissociated by PKA activation. AKAP-PKA interaction inhibitors have attracted attention for their ability to directly phosphorylate AQP2. Therefore, the LRBA-PKA interaction is a promising drug target for the development of anti-aquaretics.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Aquaporina 2 , Água Corporal , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Aquaporina 2/genética , Aquaporina 2/metabolismo , Água Corporal/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Homeostase , Camundongos , FosforilaçãoRESUMO
The malignant brain cancer glioblastoma (GBM) contains groups of highly invasive cells that drive tumor progression as well as recurrence after surgery and chemotherapy. The molecular mechanisms that enable these GBM cells to exit the primary mass and disperse throughout the brain remain largely unknown. Here we report using human tumor specimens and primary spheroids from male and female patients that glial cell adhesion molecule (GlialCAM), which has normal roles in brain astrocytes and is mutated in the developmental brain disorder megalencephalic leukoencephalopathy with subcortical cysts (MLC), is differentially expressed in subpopulations of GBM cells. High levels of GlialCAM promote cell-cell adhesion and a proliferative GBM cell state in the tumor core. In contrast, GBM cells with low levels of GlialCAM display diminished proliferation and enhanced invasion into the surrounding brain parenchyma. RNAi-mediated inhibition of GlialCAM expression leads to activation of proinvasive extracellular matrix adhesion and signaling pathways. Profiling GlialCAM-regulated genes combined with cross-referencing to single-cell transcriptomic datasets validates functional links among GlialCAM, Mlc1, and aquaporin-4 in the invasive cell state. Collectively, these results reveal an important adhesion and signaling axis comprised of GlialCAM and associated proteins including Mlc1 and aquaporin-4 that is critical for control of GBM cell proliferation and invasion status in the brain cancer microenvironment.SIGNIFICANCE STATEMENT Glioblastoma (GBM) contains heterogeneous populations of cells that coordinately drive proliferation and invasion. We have discovered that glial cell adhesion molecule (GlialCAM)/hepatocyte cell adhesion molecule (HepaCAM) is highly expressed in proliferative GBM cells within the tumor core. In contrast, GBM cells with low levels of GlialCAM robustly invade into surrounding brain tissue along blood vessels and white matter. Quantitative RNA sequencing identifies various GlialCAM-regulated genes with functions in cell-cell adhesion and signaling. These data reveal that GlialCAM and associated signaling partners, including Mlc1 and aquaporin-4, are key factors that determine proliferative and invasive cell states in GBM.
Assuntos
Aquaporinas , Glioblastoma , Feminino , Humanos , Masculino , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Proteínas de Membrana/metabolismo , Microambiente Tumoral , Proliferação de Células , Invasividade NeoplásicaRESUMO
Acute pyelonephritis (APN) is most frequently caused by uropathogenic Escherichia coli (UPEC), which ascends from the bladder to the kidneys during a urinary tract infection. Patients with APN have been reported to have reduced renal concentration capacity under challenged conditions, polyuria, and increased aquaporin-2 (AQP2) excretion in the urine. We have recently shown increased AQP2 accumulation in the plasma membrane in cell cultures exposed to E. coli lysates and in the apical plasma membrane of inner medullary collecting ducts in a 5-day APN mouse model. This study aimed to investigate if AQP2 expression in host cells increases UPEC infection efficiency and to identify specific bacterial components that mediate AQP2 plasma membrane insertion. As the transepithelial water permeability in the collecting duct is codetermined by AQP3 and AQP4, we also investigated whether AQP3 and AQP4 localization is altered in the APN mouse model. We show that AQP2 expression does not increase UPEC infection efficiency and that AQP2 was targeted to the plasma membrane in AQP2-expressing cells in response to the two pathogen-associated molecular patterns (PAMPs), lipopolysaccharide and peptidoglycan. In contrast to AQP2, the subcellular localizations of AQP1, AQP3, and AQP4 were unaffected both in lysate-incubated cell cultures and in the APN mouse model. Our finding demonstrated that cellular exposure to lipopolysaccharide and peptidoglycan can trigger the insertion of AQP2 in the plasma membrane revealing a new regulatory pathway for AQP2 plasma membrane translocation, which may potentially be exploited in intervention strategies.NEW & NOTEWORTHY Acute pyelonephritis (APN) is associated with reduced renal concentration capacity and increased aquaporin-2 (AQP2) excretion. Uropathogenic Escherichia coli (UPEC) mediates changes in the subcellular localization of AQP2 and we show that in vitro, these changes could be elicited by two pathogen-associated molecular patterns (PAMPs), namely, lipopolysaccharide and peptidoglycan. UPEC infection was unaltered by AQP2 expression and the other renal AQPs (AQP1, AQP3, and AQP4) were unaltered in APN.
Assuntos
Aquaporina 2 , Aquaporina 3 , Pielonefrite , Escherichia coli Uropatogênica , Pielonefrite/metabolismo , Pielonefrite/microbiologia , Pielonefrite/patologia , Animais , Aquaporina 2/metabolismo , Camundongos , Escherichia coli Uropatogênica/metabolismo , Aquaporina 3/metabolismo , Aquaporina 3/genética , Doença Aguda , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Lipopolissacarídeos/toxicidade , Lipopolissacarídeos/farmacologia , Membrana Celular/metabolismo , Humanos , Aquaporina 4/metabolismo , Aquaporina 4/genética , Peptidoglicano/metabolismo , Rim/metabolismo , Rim/patologia , Camundongos Endogâmicos C57BL , Modelos Animais de DoençasRESUMO
The shuttling of renal collecting duct aquaporin-2 (AQP2) between intracellular vesicles and the apical plasma membrane is paramount for regulation of renal water reabsorption. The binding of the circulating antidiuretic hormone arginine vasopressin (AVP) to the basolateral AVP receptor increases intracellular cAMP, which ultimately leads to AQP2 plasma membrane accumulation via a dual effect on AQP2 vesicle fusion with the apical plasma membrane and reduced AQP2 endocytosis. This AQP2 plasma membrane accumulation increases water reabsorption and consequently urine concentration. Conventional fluorescent microscopy provides a lateral resolution of â¼250 nm, which is insufficient to resolve the AQP2-containing endosomes/vesicles. Therefore, detailed information regarding the AQP2 vesicular population is still lacking. Newly established 4.5x Expansion Microscopy (ExM) can increase resolution to 60-70 nm. Using 4.5x ExM, we detected AQP2 vesicles/endosomes as small as 79 nm considering an average expansion factor of 4.3 for endosomes. Using different markers of the endosomal system provided detailed information of the cellular AQP2 itinerary upon changes in endogenous cAMP levels. Before cAMP elevation, AQP2 colocalized with early and recycling, but not late endosomes. Forskolin-induced cAMP increase was characterized by AQP2 insertion into the plasma membrane and AQP2 withdrawal from large perinuclear endosomes as well as some localization to lysosomal compartments. Forskolin washout promoted AQP2 endocytosis where AQP2 localized to not only early and recycling endosomes but also late endosomes and lysosomes indicating increased AQP2 degradation. Thus, our results show that 4.5 ExM is an attractive approach to obtain detailed information regarding AQP2 shuttling.NEW & NOTEWORTHY Renal aquaporin-2 (AQP2) imaged by expansion microscopy provides unprecedented 3-D information regarding the AQP2 itinerary in response to changes in cellular cAMP.
Assuntos
Aquaporina 2 , Túbulos Renais Coletores , Aquaporina 2/metabolismo , Microscopia , Colforsina/farmacologia , Rim/metabolismo , Membrana Celular/metabolismo , Água/metabolismo , Túbulos Renais Coletores/metabolismoRESUMO
High concentrations of urinary calcium counteract vasopressin action via the activation of the Calcium-Sensing Receptor (CaSR) expressed in the luminal membrane of the collecting duct cells, which impairs the trafficking of aquaporin-2 (AQP2). In line with these findings, we provide evidence that, with respect to wild-type mice, CaSR knock-in (KI) mice mimicking autosomal dominant hypocalcaemia, display a significant decrease in the total content of AQP2 associated with significantly higher levels of AQP2 phosphorylation at Ser261, a phosphorylation site involved in AQP2 degradation. Interestingly, KI mice also had significantly higher levels of phosphorylated p38MAPK, a downstream effector of CaSR and known to phosphorylate AQP2 at Ser261. Moreover, ATF1 phosphorylated at Ser63, a transcription factor downstream of p38MAPK, was significantly higher in KI. In addition, KI mice had significantly higher levels of AQP2-targeting miRNA137 consistent with a post-transcriptional downregulation of AQP2. In vivo treatment of KI mice with the calcilytic JTT-305, a CaSR antagonist, increased AQP2 expression and reduced AQP2-targeting miRNA137 levels in KI mice. Together, these results provide direct evidence for a critical role of CaSR in impairing both short-term vasopressin response by increasing AQP2-pS261, as well as AQP2 abundance, via the p38MAPK-ATF1-miR137 pathway. KEY POINTS: Calcium-Sensing Receptor (CaSR) activating mutations are the main cause of autosomal dominant hypocalcaemia (ADH) characterized by inappropriate renal calcium excretion leading to hypocalcaemia and hypercalciuria. Current treatments of ADH patients with parathyroid hormone, although improving hypocalcaemia, do not improve hypercalciuria or nephrocalcinosis. In vivo treatment with calcilytic JTT-305/MK-5442 ameliorates most of the ADH phenotypes of the CaSR knock-in mice including hypercalciuria or nephrocalcinosis and reverses the downregulation of the vasopressin-sensitive aquaporin-2 (AQP2) expression, providing direct evidence for a critical role of CaSR in impairing vasopressin response. The beneficial effect of calcilytic in reducing the risk of renal calcification may occur in a parathyroid hormone-independent action through vasopressin-dependent inhibition of cAMP synthesis in the thick ascending limb and in the collecting duct. The amelioration of most of the abnormalities in calcium metabolism including hypercalciuria, renal calcification, and AQP2-mediated osmotic water reabsorption makes calcilytic a good candidate as a novel therapeutic agent for ADH.
Assuntos
Aquaporina 2 , Regulação para Baixo , Receptores de Detecção de Cálcio , Vasopressinas , Animais , Aquaporina 2/metabolismo , Aquaporina 2/genética , Receptores de Detecção de Cálcio/metabolismo , Receptores de Detecção de Cálcio/genética , Camundongos , Vasopressinas/metabolismo , Técnicas de Introdução de Genes , Rim/metabolismo , Rim/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Masculino , Transdução de Sinais , Fenótipo , Hipercalciúria/genética , Hipercalciúria/metabolismo , Hipercalciúria/tratamento farmacológico , Cálcio/metabolismo , Fosforilação , Hipocalcemia , Hipoparatireoidismo/congênitoRESUMO
The dietary approach to stop hypertension (DASH) diet combines the antihypertensive effect of a low sodium and high potassium diet. In particular, the potassium component of the diet acts as a switch in the distal convoluted tubule to reduce sodium reabsorption, similar to a diuretic but without the side effects. Previous trials to understand the mechanism of the DASH diet were based on animal models and did not characterize changes in human ion channel protein abundance. More recently, protein cargo of urinary extracellular vesicles (uEVs) has been shown to mirror tissue content and physiological changes within the kidney. We designed an inpatient open label nutritional study transitioning hypertensive volunteers from an American style diet to DASH diet to examine physiological changes in adults with stage 1 hypertension otherwise untreated (Sacks FM, Svetkey LP, Vollmer WM, Appel LJ, Bray GA, Harsha D, Obarzanek E, Conlin PR, Miller ER 3rd, Simons-Morton DG, Karanja N, Lin PH; DASH-Sodium Collaborative Research Group. N Engl J Med 344: 3-10, 2001). Urine samples from this study were used for proteomic characterization of a large range of pure uEVs (small to large) to reveal kidney epithelium changes in response to the DASH diet. These samples were collected from nine volunteers at three time points, and mass spectrometry identified 1,800 proteins from all 27 samples. We demonstrated an increase in total SLC12A3 [sodium-chloride cotransporter (NCC)] abundance and a decrease in aquaporin-2 (AQP2) in uEVs with this mass spectrometry analysis, immunoblotting revealed a significant increase in the proportion of activated (phosphorylated) NCC to total NCC and a decrease in AQP2 from day 5 to day 11. This data demonstrates that the human kidney's response to nutritional interventions may be captured noninvasively by uEV protein abundance changes. Future studies need to confirm these findings in a larger cohort and focus on which factor drove the changes in NCC and AQP2, to which degree NCC and AQP2 contributed to the antihypertensive effect and address if some uEVs function also as a waste pathway for functionally inactive proteins rather than mirroring protein changes.NEW & NOTEWORTHY Numerous studies link DASH diet to lower blood pressure, but its mechanism is unclear. Urinary extracellular vesicles (uEVs) offer noninvasive insights, potentially replacing tissue sampling. Transitioning to DASH diet alters kidney transporters in our stage 1 hypertension cohort: AQP2 decreases, NCC increases in uEVs. This aligns with increased urine volume, reduced sodium reabsorption, and blood pressure decline. Our data highlight uEV protein changes as diet markers, suggesting some uEVs may function as waste pathways. We analyzed larger EVs alongside small EVs, and NCC in immunoblots across its molecular weight range.
Assuntos
Aquaporina 2 , Vesículas Extracelulares , Humanos , Vesículas Extracelulares/metabolismo , Aquaporina 2/metabolismo , Aquaporina 2/urina , Masculino , Feminino , Pessoa de Meia-Idade , Abordagens Dietéticas para Conter a Hipertensão , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Simportadores de Cloreto de Sódio/metabolismo , Hipertensão/dietoterapia , Hipertensão/urina , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Adulto , Dieta Hipossódica , Pressão Sanguínea , Proteômica/métodos , Rim/metabolismoRESUMO
Sepsis-associated encephalopathy (SAE) is a significant cause of mortality in patients with sepsis. Despite extensive research, its exact cause remains unclear. Our previous research indicated a relationship between non-hepatic hyperammonemia (NHH) and SAE. This study aimed to investigate the relationship between NHH and SAE and the potential mechanisms causing cognitive impairment. In the in vivo experimental results, there were no significant abnormalities in the livers of mice with moderate cecal ligation and perforation (CLP); however, ammonia levels were elevated in the hippocampal tissue and serum. The ELISA study suggest that fecal microbiota transplantation in CLP mice can reduce ammonia levels. Reduction in ammonia levels improved cognitive dysfunction and neurological impairment in CLP mice through behavioral, neuroimaging, and molecular biology studies. Further studies have shown that ammonia enters the brain to regulate the expression of aquaporins-4 (AQP4) in astrocytes, which may be the mechanism underlying brain dysfunction in CLP mice. The results of the in vitro experiments showed that ammonia up-regulated AQP4 expression in astrocytes, resulting in astrocyte damage. The results of this study suggest that ammonia up-regulates astrocyte AQP4 expression through the gut-brain axis, which may be a potential mechanism for the occurrence of SAE.
Assuntos
Aquaporina 4 , Astrócitos , Eixo Encéfalo-Intestino , Hiperamonemia , Encefalopatia Associada a Sepse , Animais , Camundongos , Aquaporina 4/metabolismo , Aquaporina 4/genética , Aquaporina 4/biossíntese , Astrócitos/metabolismo , Hiperamonemia/metabolismo , Encefalopatia Associada a Sepse/metabolismo , Masculino , Eixo Encéfalo-Intestino/fisiologia , Camundongos Endogâmicos C57BL , Amônia/metabolismo , Amônia/sangue , Encéfalo/metabolismo , Transplante de Microbiota FecalRESUMO
The fluid transport of cerebrospinal fluid (CSF) and interstitial fluid in surrounding tissues plays an important role in the drainage pathway that facilitates waste clearance from the brain. This pathway is known as the glymphatic or perivascular system, and its functions are dependent on aquaporin-4 (AQP4). Recently, magnetization transfer indirect spin labeling (MISL) magnetic resonance imaging (MRI) has been proposed as a noninvasive and noncontrast-enhanced method for detecting water exchange between CSF and brain tissue. In this study, we first optimized the MISL sequence at preclinical 3 T MRI, and then studied the correlation of MISL in CSF with magnetization transfer (MT) in brain tissue, as well as the altered water exchange under AQP4 inhibition, using C57BL/6 mice. Results showed a strong correlation of MISL signal with MT signal. With the AQP4 inhibitor, we observed a significant decrease in MISL value (P < 0.05), suggesting that the hampered AQP4 activity led to decreased water exchange between CSF and brain tissue or the impairment of the glymphatic function. Overall, our findings demonstrate the potential application of MISL in assessing brain water exchange at 3 T MRI and its potential clinical translation.
Assuntos
Aquaporina 4 , Encéfalo , Líquido Cefalorraquidiano , Imageamento por Ressonância Magnética , Camundongos Endogâmicos C57BL , Marcadores de Spin , Animais , Aquaporina 4/metabolismo , Aquaporina 4/antagonistas & inibidores , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Camundongos , Líquido Cefalorraquidiano/metabolismo , Líquido Cefalorraquidiano/diagnóstico por imagem , Água/metabolismo , Masculino , Água Corporal/metabolismo , Niacinamida/análogos & derivados , TiadiazóisRESUMO
Transport of water is critical for maintaining the transparency of the avascular lens, and the lens is known to express at least five distinctly different water channels from the Aquaporin (AQP) family of proteins. In this study we report on the identification of a sixth lens AQP, AQP3 an aquaglyceroporin, which in addition to water also transports glycerol and H2O2. AQP3 was identified at the transcript level and protein levels using RT-PCR and Western blotting, respectively, in the mouse, rat, bovine and human lens, showing that its expression is conserved in the mammalian lens. Western blotting showed AQP3 in the lens exists as 25 kDa non-glycosylated and 37 kDa glycosylated monomeric forms in all lens species. To identify the regions in the lens where AQP3 is expressed Western blotting was repeated using epithelial, outer cortical and inner cortical/core fractions isolated from the mouse lens. AQP3 was found in all lens regions, with the highest signal of non-glycosylated AQP3 being found in the epithelium. While in the inner cortex/core region AQP3 signal was not only lower but was predominately from the glycosylated form of AQP3. Immunolabelling of lens sections with AQP3 antibodies confirmed that AQP3 is found in all regions of the adult mouse, and also revealed that the subcellular distribution of AQP3 changes as a function of fiber cell differentiation. In epithelial and peripheral fiber cells of the outer cortex AQP3 labelling was predominately associated with membrane vesicles in the cytoplasm, but in the deeper regions of the lens AQP3 labelling was associated with the plasma membranes of fiber cells located in the inner cortex and core of the lens. To determine how this adult pattern of AQP3 subcellular distribution was established, immunolabelling for AQP3 was performed on embryonic and postnatal lenses. AQP3 expression was first detected on embryonic day (E) 11 in the membranes of primary fiber cells that have started to elongate and fill the lumen of the lens vesicle, while later at E16 the AQP3 labelling in the primary fiber cells had shifted to a predominately cytoplasmic location. In the following postnatal (P) stages of lens growth at P3 and P6, AQP3 labelling remained cytoplasmic across all regions of the lens and it was not until P15 when the pattern of localisation of AQP3 changed to an adult distribution with cytoplasmic labelling detected in the outer cortex and membrane localisation detected in the inner cortex and core of the lens. Comparison of the AQP3 labelling pattern to those obtained previously for AQP0 and AQP5 showed that the subcellular distribution was more similar to AQP5 than AQP0, but there were still significant differences that suggest AQP3 may have unique roles in the maintenance of lens transparency.
Assuntos
Aquaporina 3 , Cristalino , Animais , Bovinos , Humanos , Camundongos , Ratos , Aquagliceroporinas/metabolismo , Aquaporina 3/genética , Aquaporina 3/metabolismo , Peróxido de Hidrogênio/metabolismo , Cristalino/metabolismo , Mamíferos , Água/metabolismoRESUMO
AQP5 plays a crucial role in maintaining corneal transparency and the barrier function of the cornea. Here, we found that in the corneas of Aqp5-/- mice at older than 6 months, loss of AQP5 significantly increased corneal neovascularization, inflammatory cell infiltration, and corneal haze. The results of immunofluorescence staining showed that upregulation of K1, K10, and K14, and downregulation of K12 and Pax6 were detected in Aqp5-/- cornea and primary corneal epithelial cells. Loss of AQP5 aggravated wound-induced corneal neovascularization, inflammation, and haze. mRNA sequencing, western blotting, and qRT-PCR showed that Wnt2 and Wnt6 were significantly decreased in Aqp5-/- corneas and primary corneal epithelial cells, accompanied by decreased aggregation in the cytoplasm and nucleus of ß-catenin. IIIC3 significantly suppressed corneal neovascularization, inflammation, haze, and maintained corneal transparent epithelial in Aqp5-/- corneas. We also found that pre-stimulated Aqp5-/- primary corneal epithelial cells with IIIC3 caused the decreased expression of K1, K10, and K14, the increased expression of K12, Pax6, and increased aggregation in the cytoplasm and nucleus of ß-catenin. These findings revealed that AQP5 may regulate corneal epithelial homeostasis and function through the Wnt/ß-catenin signaling pathway. Together, we uncovered a possible role of AQP5 in determining corneal epithelial cell fate and providing a potential therapeutic target for corneal epithelial dysfunction.
Assuntos
Neovascularização da Córnea , Via de Sinalização Wnt , Camundongos , Animais , Aquaporina 5/genética , Neovascularização da Córnea/metabolismo , beta Catenina/metabolismo , Córnea/metabolismo , Células Epiteliais/metabolismo , Inflamação/metabolismoRESUMO
Although anti-TNF antibodies are extensively used to treat Crohn's disease (CD), a significant proportion of patients, up to 40%, exhibit an inadequate response to this therapy. Our objective was to identify potential targets that could improve the effectiveness of anti-TNF therapy in CD. Through the integration and analysis of transcriptomic data from various CD databases, we found that the expression of AQP9 was significantly increased in anti-TNF therapy-resistant specimens. The response to anti-TNF therapy in the CD mouse model was significantly enhanced by specifically inhibiting AQP9. Further experiments found that the blockade of AQP9, which is dominantly expressed in macrophages, decreased inflamed macrophage functions and cytokine expression. Mechanistic studies revealed that AQP9 transported glycerol into macrophages, where it was metabolized to LPA, which was further metabolized to LPA, resulting in the activation of the LPAR2 receptor and downstream hippo pathway, finally promoting the expression of cytokines, especially IL23 and IL1ßâ¡ Taken together, the expansion of AQP9+ macrophages is associated with resistance to anti-TNF therapy in Crohn's disease. These findings indicated that AQP9 could be a potential target for enhancing anti-TNF therapy in Crohn's disease.
Assuntos
Aquaporinas , Doença de Crohn , Via de Sinalização Hippo , Lisofosfolipídeos , Macrófagos , Animais , Humanos , Masculino , Camundongos , Aquaporinas/metabolismo , Aquaporinas/genética , Aquaporinas/antagonistas & inibidores , Doença de Crohn/tratamento farmacológico , Doença de Crohn/metabolismo , Citocinas/metabolismo , Via de Sinalização Hippo/efeitos dos fármacos , Lisofosfolipídeos/metabolismo , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Inibidores do Fator de Necrose Tumoral/uso terapêutico , Inibidores do Fator de Necrose Tumoral/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The global incidence of cardiac diseases is increasing, imposing a substantial socioeconomic burden on healthcare systems. The pathogenesis of cardiovascular disease is complex and not fully understood, and the physiological function of the heart is inextricably linked to well-regulated cardiac muscle movement. Myosin light chain kinase (MLCK) is essential for myocardial contraction and diastole, cardiac electrophysiological homeostasis, vasoconstriction of vascular nerves and blood pressure regulation. In this sense, MLCK appears to be an attractive therapeutic target for cardiac diseases. MLCK participates in myocardial cell movement and migration through diverse pathways, including regulation of calcium homeostasis, activation of myosin light chain phosphorylation, and stimulation of vascular smooth muscle cell contraction or relaxation. Recently, phosphorylation of myosin light chains has been shown to be closely associated with the activation of myocardial exercise signaling, and MLCK mediates systolic and diastolic functions of the heart through the interaction of myosin thick filaments and actin thin filaments. It works by upholding the integrity of the cytoskeleton, modifying the conformation of the myosin head, and modulating innervation. MLCK governs vasoconstriction and diastolic function and is associated with the activation of adrenergic and sympathetic nervous systems, extracellular transport, endothelial permeability, and the regulation of nitric oxide and angiotensin II. Additionally, MLCK plays a crucial role in the process of cardiac aging. Multiple natural products/phytochemicals and chemical compounds, such as quercetin, cyclosporin, and ML-7 hydrochloride, have been shown to regulate cardiomyocyte MLCK. The MLCK-modifying capacity of these compounds should be considered in designing novel therapeutic agents. This review summarizes the mechanism of action of MLCK in the cardiovascular system and the therapeutic potential of reported chemical compounds in cardiac diseases by modifying MLCK processes.
Assuntos
Quinase de Cadeia Leve de Miosina , Transdução de Sinais , Humanos , Quinase de Cadeia Leve de Miosina/metabolismo , Animais , Transdução de Sinais/efeitos dos fármacos , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/fisiopatologia , Doenças Cardiovasculares/enzimologia , Fármacos Cardiovasculares/uso terapêutico , Fármacos Cardiovasculares/farmacologiaRESUMO
Brain edema is a critical complication arising from stroke and traumatic brain injury (TBI) with an important impact on patient recovery and can lead to long-term consequences. Therapeutic options to reduce edema progression are limited with variable patient outcomes. Aquaporin 4 (AQP4) is a water channel that allows bidirectional water diffusion across the astrocyte membrane and participates in the distinct phases of cerebral edema. The absence or inhibition of this channel has been demonstrated to ameliorate edema and brain damage. The endocannabinoid system (ECS) is a neuromodulator system with a wide expression in the brain and its activation has shown neuroprotective properties in diverse models of neuronal damage. This review describes and discusses the major features of ECS and AQP4 and their role during brain damage, observing that ECS stimulation reduces edema and injury size in diverse models of brain damage, however, the relationship between AQP4 expression and dynamics and ECS activation remains unclear. The research on these topics holds promising therapeutic implications for the treatment of brain edema following stroke and TBI.
Assuntos
Aquaporina 4 , Edema Encefálico , Lesões Encefálicas , Endocanabinoides , Endocanabinoides/metabolismo , Aquaporina 4/metabolismo , Humanos , Animais , Edema Encefálico/metabolismo , Edema Encefálico/etiologia , Lesões Encefálicas/metabolismo , Lesões Encefálicas Traumáticas/metabolismoRESUMO
AIMS: To discuss the role of autocrine/paracrine signaling of urothelial arginine vasopressin (AVP) on mammalian bladder capacities and micturition thresholds, impact of distension on water/urea reabsorption from the bladder, review of the literature to better characterize the central/peripheral effects of AVP, desmopressin (dAVP) toxicity, and urine biomarkers of nocturia. METHODS: This review summarizes discussions during an International Consultation on Incontinence-Research Society 2024 think tank with respect to the role of urothelial AVP in aged individuals with nocturnal polyuria, impact of solute and water reabsorption by the bladder on uninterrupted sleep, central effects of AVP, pharmacological basis of dAVP toxicity, and biomarkers in nocturia/lower urinary tract dysfunction (LUTD) with neurological diseases. RESULTS: Consensus recognized AVP function and pathways in the central nervous system (CNS), pre-proAVP localized using immunohistochemistry in bladder sections from adult/aged noncancerous human punch biopsies and rodent bladder sections is likely to accelerate the systemic uptake of water and urea from the bladder of anesthetized mice instilled with 3H-water and 14C-urea. Mechanisms for charged and uncharged solutes and water transport across the bladder, mechanism of dAVP toxicity, and utility of urine biomarkers in those with neurological diseases/nocturia were determined from literature reviews. CONCLUSION: Pre-proAVP is present in human/rodent bladders and may be involved in water reabsorption from bladder that prevents the sensation of fullness for uninterrupted sleep in healthy adults. The mechanism of action of AVP in the CNS was discussed, as was electrolyte/water transport across the bladder, the basis for dAVP toxicity, and feasibility of urine biomarkers to identify nocturia/LUTD with neurological diseases.
RESUMO
For adult anuran amphibians, the kidney and urinary bladder play important osmoregulatory roles through water reabsorption. In the present study, we have examined ontogenetic expression of aquaporins, i.e., AQP2, AQPamU (AQP6ub, AQPa2U), and AQP3, in these organs using the Japanese tree frog, Dryophytes japonicus. Immunohistochemistry using the metamorphosing larvae at stages 40-43 localized AQP2 protein to the collecting ducts in the dorsal zone of the mesonephric kidney. At prometamorphic stages 40 and 41, labelling of AQP2 protein was observed in the apical/ subapical regions of the collecting duct cells. At climax stages 42 and 43, labels for AQP2 and AQP3 became observed in the apical/subapical regions and basolateral membrane of the collecting duct cells, respectively, as seen in the adults. As for the urinary bladder, immuno-positive labels for AQPamU were localized to the apical/subapical regions of granular cells in the mucosal epithelium at stages 40-43. On the other hand, AQP3 immunoreactivity was hardly observed in the urinary bladder at stage 40, and weakly appeared in many granular cells at stage 41. Thereafter, labels for AQP3 became evident along the basolateral membrane of granular cells at stages 42 and 43, together with AQPamU in the apical/subapical regions. These results suggest that the kidney and urinary bladder might be capable of water reabsorption, via AQP2, AQPamU, and AQP3, at stage 42, contributing to the acclimation of the tree frogs to terrestrial environments.
Assuntos
Aquaporina 2 , Bexiga Urinária , Animais , Japão , Anuros , Rim , ÁguaRESUMO
Neuromyelitis optica (NMO) is an autoimmune inflammatory disease of the central nervous system (CNS) characterized by transverse myelitis and optic neuritis. The pathogenic serum IgG antibody against the aquaporin-4 (AQP4) on astrocytes triggers the activation of the complement cascade, causing astrocyte injury, followed by oligodendrocyte injury, demyelination, and neuronal loss. Complement C3 is positioned as a central player that relays upstream initiation signals to activate downstream effectors, potentially stimulating and amplifying host immune and inflammatory responses. However, whether targeting the inhibition of C3 signaling could ameliorate tissue injury, locomotor defects, and visual impairments in NMO remains to be investigated. In this study, using the targeted C3 inhibitor CR2-Crry led to a significant decrease in complement deposition and demyelination in both slice cultures and focal intracerebral injection models. Moreover, the treatment downregulated the expression of inflammatory cytokines and improved motor dysfunction in a systemic NMO mouse model. Similarly, employing serotype 2/9 adeno-associated virus (AAV2/9) to induce permanent expression of CR2-Crry resulted in a reduction in visual dysfunction by attenuating NMO-like lesions. Our findings reveal the therapeutic value of inhibiting the complement C3 signaling pathway in NMO.
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Complemento C3 , Neuromielite Óptica , Animais , Camundongos , Complemento C3/genética , Complemento C3/metabolismo , Neuromielite Óptica/patologia , Aquaporina 4/metabolismo , Transtornos da Visão/complicações , Transtornos da Visão/patologia , Astrócitos/metabolismo , Transdução de Sinais , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Immune checkpoint inhibitors (ICIs) are a pharmacological group increasingly used in Oncology and Hematology. These treatments can lead to autoimmune complications, with neurological conditions, especially central nervous system (CNS) involvement, being rare. We describe a case of seropositive neuromyelitis optica in a patient with locally advanced lung adenocarcinoma treated with Atezolizumab.