[In silico analysis of molecular mimicry between human aquaporin 3, Aspergillus fumigatus aquaporin and aquaporins from allergic sources]. / Análisis en silico del mimetismo molecular entre acuaporina 3 humana y Aspergillus fumigatus: implicaciones para el potencial de reactividad cruzada.

OBJECTIVE: Conduct an in-silico assessment of potential molecular mimicry between human aquaporins, A. fumigatus, and diverse allergenic sources. METHODS: Amino acid sequences of human AQP3 and A. fumigatus aquaporin were compared through multiple alignments with 25 aquaporins from diverse allergenic sources. Phylogenetic analysis and homology-based modeling were executed, and the ElliPro server predicted conserved antigenic regions on 3D structures. RESULTS: Global identity among studied aquaporins was 32.6%, with a specific conserved local region at 71.4%. Five monophyletic clades (A-E) were formed, and Group B displayed the highest identity (95%), including 6 mammalian aquaporins, notably AQP3. A. fumigatus aquaporin exhibited the highest identity with Malassezia sympodialis (35%). Three linear and three discontinuous epitopes were identified in both human and A. fumigatus aquaporins. The Root Mean Square Deviation (RMSD) from overlapping aquaporin structures was 1.006. CONCLUSION: Identification of potential linear and conformational epitopes on human AQP3 suggests likely molecular mimicry with A. fumigatus aquaporins. High identity in a specific antigenic region indicates potential autoreactivity and a probable antigenic site involved in cross-reactivity. Validation through in vitro and in vivo studies is essential for further understanding and confirmation.

OBJETIVO: Realizar una evaluación in silico del posible mimetismo molecular entre las acuaporinas humanas, A. fumigatus y diversas fuentes alergénicas. MÉTODOS: Se compararon secuencias de aminoácidos de AQP3 humana y acuaporina de A. fumigatus mediante alineamientos múltiples con 25 acuaporinas de diversas fuentes alergénicas. Se ejecutaron análisis filogenéticos y modelos basados en homología, y el servidor ElliPro predijo regiones antigénicas preservadas en estructuras 3D. RESULTADOS: La identidad global entre las acuaporinas estudiadas fue del 32.6%, con una región local específica preservada en el 71.4%. Se formaron cinco clados monofiléticos (A-E), y el grupo B mostró la identidad más alta (95%), incluidas 6 acuaporinas de mamíferos, en particular AQP3. A. fumigatus aquaporin exhibió la mayor identidad con Malassezia sympodialis (35%). Se identificaron tres epítopos lineales y tres discontinuos en acuaporinas tanto humanas como de A. fumigatus. La desviación cuadrática media (RMSD) de las estructuras de acuaporinas superpuestas fue de 1,006. CONCLUSIÓN: La identificación de posibles epítopos lineales y conformacionales en AQP3 humano sugiere un probable mimetismo molecular con acuaporinas de A. fumigatus. La identidad alta en una región antigénica específica indica autorreactividad potencial y un sitio antigénico probable implicado en la reactividad cruzada. La validación mediante estudios in vitro e in vivo es desicivo para una mayor comprensión y confirmación.

Intact caveolae are required for proper extravillous trophoblast migration and differentiation.

Caveolae constitute membrane domains critical for the organization and synchronization of different signaling molecules related to numerous cell processes such as cell migration, invasion, and differentiation. Caveolin-1 (Cav-1) is the main integral membrane protein of these domains. Recently, it was found that a normal expression of aquaporin-3 (AQP3) is required for extravillous trophoblast (EVT) cell migration. Our aim was to investigate the role of caveolae in the migration, invasion, and endovascular differentiation of human EVT cells during placentation and its interaction with AQP3. EVT cells (Swan 71 cell line) were cultured in complete Dulbecco's modified Eagle's medium-nutrient mixture F12 and treated with 5 mM methyl-ß-cyclodextrin (MßCD) to disrupt caveolae. We found that after MßCD treatment, Cav-1 protein was undetectable. In this condition, the ability of the cells to migrate was significantly decreased compared with the control cells, while no differences were observed in the number of invading cells and the metalloproteinases activity between control and MßCD-treated cells. Surprisingly, the disruption of caveolae significantly enhanced EVT endovascular differentiation. On the contrary, the silencing of AQP3, negatively affected tube-like formation. The theoretical analysis of the primary sequence of AQP3 protein revealed a putative Cav-1-binding site. In addition, immunoprecipitation and double immunofluorescence assays showed that AQP3 colocalized with Cav-1. These results showed that during placentation an intact caveola in EVT cells may be necessary for AQP3 and Cav-1 interaction and any perturbations might result in serious pregnancy disorders.

Effect of aquaporin 3 knockdown by RNA interference on antrum formation in sheep secondary follicles cultured in vitro.

SummaryThe objectives were to develop an effective protocol for transfection of ovine secondary follicles and to assess the effect of attenuating aquaporin 3 (AQP3) using a small interfering RNA (siRNA-AQP3) on antrum formation and follicular growth in vitro. Various combinations of Lipofectamine® volumes (0.5, 0.75 or 1.0 µl), fluorescent oligonucleotide (BLOCK-iT ™) concentrations (3.18, 27.12 or 36.16 nM) and exposure times (12, 14, 16, 18 or 20 h) were tested. The BLOCK-iT™ was replaced by siRNA-AQP3 in the transfection complex. Ovine secondary follicles were isolated and cultured in vitro for 6 days using standard protocols. Follicles were transfected on day 0 or 3 or on both days (0 and 3) and then cultured for an additional 3 or 6 days. As revealed by the fluorescence signal, the Lipofectamine®/BLOCK-iT™ complex (0.75 µl + 27.12 nM by 12 h of incubation) crossed the basement membrane and granulosa cell and reached the oocytes. In general, the rate of intact follicles was higher and the rate of antrum formation was lower in transfected follicles compared with control follicles. In conclusion, ovine secondary follicles can be successfully transfected during in vitro culture, and siRNA-mediated attenuation of AQP3 gene reduced antrum formation of secondary follicles.

The blocking of aquaporin-3 (AQP3) impairs extravillous trophoblast cell migration.

Several aquaporins (AQPs) are expressed in extravillous (EVT) and villous trophoblast cells. Among them, AQP3 is the most abundant AQP expressed in chorionic villi samples from first trimester, followed by AQP1 and AQP9. Although AQP3 expression persists in term placentas, it is significantly decreased in placentas from preeclamptic pregnancies. AQP3 is involved in the migration of different cell types, however its role in human placenta is still unknown. Here, we evaluated the role of AQP3 in the migration of EVT cells during early gestation. Our results showed that Swan 71 cells expressed AQP1, AQP3 and AQP9 but only the blocking of AQP3 by CuSO4 or the silencing of its expression by siRNA significantly attenuates EVT cell migration. Our work provides evidence that AQP3 is required for EVT cell migration and suggests that an altered expression of placental AQP3 may produce failures in placentation such as in preeclampsia.

Age-related changes in renal AQP3 and AQP4 expression in Sprague Dawley rats.

Aquaporin (AQP) 3 and AQP4 are important in urine concentrating mechanisms and in other physiological functions such as brain water balance, cell migration, cell proliferation, fat metabolism, and epidermal hydration. The results of studies investigating AQP3 and AQP4 expression in the kidneys are inconsistent, and systematic research is rare. This study aimed to obtain a better understanding of the changes in renal AQP3 and AQP4 mRNA expression that take place with age. The expression of AQP3 and AQP4 mRNA, during prenatal and postnatal development, and during aging, was investigated in kidneys from Sprague-Dawley rats. The pattern of AQP3 expression was similar to that of AQP4 expression during development, and both were detected at gestational day 19 in the rat kidney where they maintained a stable level to postnatal day 14. Subsequently, a significant increase in expression was observed from day 21 to day 35, with peak expression occurring at day 35. No significant change in AQP3 or AQP4 mRNA expression was observed after day 35, apart from AQP4, which increased at day 540. Moreover, the expression of both AQP3 and AQP4 on day 850 was higher than on day -2, and lower than on days 28 and 35. The expression of AQP3 and AQP4 was similar on days 1, 7, 14, and 21. These findings indicate that mRNA expression of AQP3 and AQP4 varies with age, which should be considered when treating kidney disease in pediatric and elderly patients.

Ménière's Disease: Molecular Analysis of Aquaporins 2, 3 and Potassium Channel KCNE1 Genes in Brazilian Patients.

OBJECTIVES: Ménière's disease (MD) is a complex disease of unknown etiology characterized by a symptomatic tetrad of vertigo, hearing loss, tinnitus, and aural fullness. In addition to factors related to homeostasis of the inner ear, genetic factors have been implicated in its pathophysiology, including genes related to the transport of water and ionic composition maintenance of the endolymph, such as the aquaporin genes AQP2 and AQP3, and the potassium channel gene KCNE1. The aim of this study was to identify polymorphisms of these genes and determine their association with clinical characteristics of patients with MD. DESIGN: A case-control genetic association study was carried out, including 30 patients with definite Ménière's disease and 30 healthy controls. The coding regions of the target genes were amplified from blood samples by polymerase chain reaction (PCR), followed by direct sequencing. The associations of polymorphisms with clinical characteristics were analyzed with logistic regression. RESULTS: Five polymorphisms were identified: rs426496 in AQP2; rs591810 in AQP3; and rs1805127, rs1805128, and rs17173510 in KCNE1. After adjustment, rs426496 was significantly associated with tinnitus during the initial crisis and with altered electronystagmography, and rs1805127 was significantly associated with nephropathy. CONCLUSIONS: The genetic variant rs426496 in AQP2; rs591810 in AQP3 and rs1805127, rs1805128, and rs17173510, in KCNE1 were found in patients with Ménière's disease. The polymorphism rs426496, in AQP2, is associated with tinnitus at the onset of Ménière's disease and altered electronystagmography. In addition, rs1805127, in KCNE1, is associated with the presence of nephropathy.

Effect of retinoic acid on aquaporin 3 expression in keratinocytes.

To explore the possible mechanism of the third-generation retinoic acid drugs (isotretinoin, acitretin, adapalene) in inducing skin and mucosa dryness and rhagades; specifically, mechanism by which these drugs influence keratinocyte cell culture models in vitro (HaCaT) and aquaporin channel (AQP3) protein expression was investigated. Isotretinoin, acitretin, and adapalene were applied to human keratinocyte HaCaT cells. Immunohistochemistry, reverse transcriptase polymerase chain reaction, and western blotting were used to detect their effects on AQP3 expression in HaCaT cells at different concentrations (0.000, 0.001, 0.010, 0.060, and 0.100 mg/mL) or different at times (0, 6, 12, 24, and 48 h). At 0.010 mg/mL, maximal AQP3 expression was observed in HaCaT cells; this was significantly higher than the expressions at the other concentrations (P < 0.05). After treatment with isotretinoin, acitretin, or adapalene at 0.010 mg/mL for 12 h, the expression of AQP3 was the highest in the isotretinoin group, followed by the acitretin group, with the lowest expression in the adapalene group. However, the differences were not statistically significant (P > 0.05). Retinoic acid can increase AQP3 expression in HaCaT cells, with significant effects observed with 0.010 mg/mL isotretinoin treatment for 12 h. The side effects, namely skin and mucosa dryness caused by retinoic acid might be related to its effects on AQP3 expression.

Effect of crotamine, a cell-penetrating peptide, on blastocyst production and gene expression of in vitro fertilized bovine embryos.

The present study investigated the effects of crotamine, a cell-penetrating peptide from rattlesnake venom, at different exposure times and concentrations, on both developmental competence and gene expression (ATP1A1, AQP3, GLUT1 and GLUT3) of in vitro fertilized (IVF) bovine embryos. In Experiment 1, presumptive zygotes were exposed to 0.1 µM crotamine for 6, 12 or 24 h and control groups (vehicle and IVF) were included. In Experiment 2, presumptive zygotes were exposed to 0 (vehicle), 0.1, 1 and 10 µM crotamine for 24 h. Additionally, to visualize crotamine uptake, embryos were exposed to rhodamine B-labelled crotamine and subjected to confocal microscopy. In Experiment 1, no difference (P > 0.05) was observed among different exposure times and control groups for cleavage and blastocyst rates and total cells number per blastocyst. Within each exposure time, mRNA levels were similar (P > 0.05) in embryos cultured with or without crotamine. In Experiment 2, concentrations as high as 10 µM crotamine did not affect (P > 0.05) the blastocyst rate. Crotamine at 0.1 and 10 µM did not alter mRNA levels when compared with the control (P > 0.05). Remarkably, only 1 µM crotamine decreased both ATP1A1 and AQP3 expression levels relative to the control group (P < 0.05). Also, it was possible to visualize the intracellular localization of crotamine. These results indicate that crotamine can translocate intact IVF bovine embryos and its application in the culture medium is possible at concentrations from 0.1-10 µM for 6-24 h.

Evidence of Positive Selection of Aquaporins Genes from Pontoporia blainvillei during the Evolutionary Process of Cetaceans.

BACKGROUND: Marine mammals are well adapted to their hyperosmotic environment. Several morphological and physiological adaptations for water conservation and salt excretion are known to be present in cetaceans, being responsible for regulating salt balance. However, most previous studies have focused on the unique renal physiology of marine mammals, but the molecular bases of these mechanisms remain poorly explored. Many genes have been identified to be involved in osmotic regulation, including the aquaporins. Considering that aquaporin genes were potentially subject to strong selective pressure, the aim of this study was to analyze the molecular evolution of seven aquaporin genes (AQP1, AQP2, AQP3, AQP4, AQP6, AQP7, and AQP9) comparing the lineages of cetaceans and terrestrial mammals. RESULTS: Our results demonstrated strong positive selection in cetacean-specific lineages acting only in the gene for AQP2 (amino acids 23, 83, 107,179, 180, 181, 182), whereas no selection was observed in terrestrial mammalian lineages. We also analyzed the changes in the 3D structure of the aquaporin 2 protein. Signs of strong positive selection in AQP2 sites 179, 180, 181, and 182 were unexpectedly identified only in the baiji lineage, which was the only river dolphin examined in this study. Positive selection in aquaporins AQP1 (45), AQP4 (74), AQP7 (342, 343, 356) was detected in cetaceans and artiodactyls, suggesting that these events are not related to maintaining water and electrolyte homeostasis in seawater. CONCLUSIONS: Our results suggest that the AQP2 gene might reflect different selective pressures in maintaining water balance in cetaceans, contributing to the passage from the terrestrial environment to the aquatic. Further studies are necessary, especially those including other freshwater dolphins, who exhibit osmoregulatory mechanisms different from those of marine cetaceans for the same essential task of maintaining serum electrolyte balance.

Expression of aquaporins 1 and 3 in degenerative tissue of the lumbar intervertebral disc.

Lumbar intervertebral disc degeneration is induced by multiple factors, but few studies have examined the effects of aquaporins on this process. We compared the expression levels of aquaporins 1 and 3 in normal and degenerative lumbar intervertebral discs. Fifteen normal and 15 degenerative lumbar intervertebral disc tissues were excised from lumbar burst fracture patients during orthopedic operations at the Dali College subsidiary hospital. Tissues were stained with hematoxylin-eosin and the expression levels of aquaporins 1 and 3 were measured by immunohistochemistry. Hematoxylin-eosin-staining results illustrated that the structures of the intervertebral disc tissues from the control group were clear, with distinct collagen fiber shapes and slight edema but without mucoid degeneration. Structures of the intervertebral disc tissues from the disease group were obscure and disordered with hyperplastic collagen fibers and tissues of severe inflammatory edemas with necrosis mucoid degeneration. Immunohistochemistry results demonstrated that the average absorbances of aquaporins 1 and 3 in the disease group were significantly lower than those in the control group (P < 0.01), suggesting that the reduction of aquaporins 1 and 3 may be a factor resulting in lumbar intervertebral disc degeneration.

Expression and localization of Aquaporin 3 (AQP3) in folliculogenesis of ewes.

The mRNA expression and localization of Aquaporin 3 (AQP3) were investigated in the ovarian follicles of ewes at different stages of development (primordial, primary, secondary, small, and large antral). The gene expression was quantified by qPCR, while the protein identification and localization were determined by Western blot and immunohistochemistry, respectively. Analysis revealed that AQP3 mRNA was detected only in the antral follicles, whereas the protein expression was detected in the oocyte and granulosa cells in all stages of follicular development. The latter observation suggests that the presence of AQP3 in follicles of all categories, especially in the antral follicles, provides novel insights on the mechanisms that regulate the flow of water between cells during the formation of antral follicles in sheep.

Osmotic challenge and expression of aquaporin 3 and Na/K ATPase genes in bovine embryos produced in vitro.

The aim of this study was to evaluate the effect of culture media and stage of development in the osmotic ability of in vitro-fertilized bovine embryos and the expression of aquaporin 3 (Aqp3) and Na/K ATPase isoform 1 (ATPAse1) genes in embryos (i) with different ability to undergo rehydration and (ii) following vitrification. In experiment 1, in vitro fertilized presumptive zygotes were co-cultured in SOFaac or modified CR2aa medium and embryos at blastocyst and expanded blastocyst stages at day 7 post-insemination were exposed to NaCl hypertonic medium (900 mOsm) for 5 min following 120 min of culture in isotonic medium in order to evaluate dehydration and rehydration, respectively. No difference (P>0.05) on blastocyst rate was found between CR2aa and SOFaac medium but embryos co-cultured in SOFaac medium underwent greater (P<0.05) dehydration. Embryos at expanded blastocyst stage underwent greater dehydration but slower rehydration than embryos at blastocysts stage (P<0.05). In the experiment 2, the amount of Aqp3 and ATPase1 transcripts were quantified in blastocysts with high or low rehydration after exposure to hypertonic medium. No difference (P>0.05) on relative amount of transcripts was found in either genes. In the experiment 3, expanded blastocysts produced in a co-culture system were vitrified, warmed and then cultured for 72 h for analysis of embryo survival and amount of Aqp3 and ATPase1 transcripts. Lower (P<0.05) embryo survival rate was found for vitrified-warmed embryos (57.9%) than for their fresh counterparts (84.6%). There was no difference on expression of ATPase1 gene but lower (P<0.01) amount of Aqp3 transcripts was found in the vitrified-warmed embryos. In conclusion, embryo ability to undergo shrinkage and swelling is influenced by medium used in a co-culture system and by embryo stage. Rehydrating ability of embryos after exposure to NaCl hypertonic medium is not associated with variations on expression of Aqp3 and ATPase1 genes, but the vitrification can alter gene expression of in vitro-fertilized bovine embryos produced in a co-culture system.

Expression of differential genes involved in the maintenance of water balance in human skin by Piptadenia colubrina extract.

BACKGROUND: Hydration and integrity of the stratum corneum (SC) is an important determinant of skin appearance, metabolism, mechanical properties, and barrier function. The presence of aquaglyceroporins and envelope proteins are crucial to provide greater corneocyte cohesion to keep water and other moisturizers in the skin. AIMS: In this study, we evaluated the ability of Piptadenia colubrina, a plant native of South American rain forests, in the expression of genes involved in skin capacitance and SC integrity. METHODS: The expression of genes for aquaporin-3 (AQP3), loricrin, involucrin (INV), and filaggrin (FLG) was measured by real-time PCR, using an in vitro model of human keratinocytes incubated with concentrations of 2.5, 5, 10, and 20 mg/mL of a hydroglycolic extract of P. colubrina (HEPC). The amount of AQP3 protein was also tested by immunohistochemistry in human skin explants. Clinical trials were conducted to evaluate the effects of a gel-cream containing HEPC on the glycerol index and skin capacitance. RESULTS: Hydroglycolic extract of P. colubrina increased both the expression and immunoreactivity of AQP3 in cultured keratinocytes and human skin explants. The gene induction to envelope proteins FLG and INV was also observed after cell incubation with HEPC. Skin capacitance was significantly improved in human volunteers under treatment with HEPC-containing cream. CONCLUSIONS: The extract of P. colubrina promotes cellular hydration and induces gene expression of envelope proteins providing greater corneocyte cohesion to keep water and other moisturizers in the skin and an appropriate epidermal adhesion. The in vitro findings were clinically confirmed and encourage the clinical use of this compound in skin care products.