Molecular basis for human aquaporin inhibition.

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.

A novel interaction between aquaporin 1 and caspase-3 in pulmonary arterial smooth muscle cells.

Pulmonary hypertension (PH) is a condition in which remodeling of the pulmonary vasculature leads to hypertrophy of the muscular vascular wall and extension of muscle into nonmuscular arteries. These pathological changes are predominantly due to the abnormal proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), enhanced cellular functions that have been linked to increases in the cell membrane protein aquaporin 1 (AQP1). However, the mechanisms underlying the increased AQP1 abundance have not been fully elucidated. Here we present data that establishes a novel interaction between AQP1 and the proteolytic enzyme caspase-3. In silico analysis of the AQP1 protein reveals two caspase-3 cleavage sites on its C-terminal tail, proximal to known ubiquitin sites. Using biotin proximity ligase techniques, we establish that AQP1 and caspase-3 interact in both human embryonic kidney (HEK) 293A cells and rat PASMCs. Furthermore, we demonstrate that AQP1 levels increase and decrease with enhanced caspase-3 activity and inhibition, respectively. Ultimately, further work characterizing this interaction could provide the foundation for novel PH therapeutics.NEW & NOTEWORTHY Pulmonary arterial smooth muscle cells (PASMCs) are integral to pulmonary vascular remodeling, a characteristic of pulmonary arterial hypertension (PAH). PASMCs isolated from robust animal models of disease demonstrate enhanced proliferation and migration, pathological functions associated with increased abundance of the membrane protein aquaporin 1 (AQP1). We present evidence of a novel interaction between the proteolytic enzyme caspase-3 and AQP1, which may control AQP1 abundance. These data suggest a potential new target for novel PAH therapies.

AQP1 Promoter Variant, Water Transport, and Outcomes in Peritoneal Dialysis.

BACKGROUND: Variability in ultrafiltration influences prescriptions and outcomes in patients with kidney failure who are treated with peritoneal dialysis. Variants in AQP1, the gene that encodes the archetypal water channel aquaporin-1, may contribute to that variability. METHODS: We gathered clinical and genetic data from 1851 patients treated with peritoneal dialysis in seven cohorts to determine whether AQP1 variants were associated with peritoneal ultrafiltration and with a risk of the composite of death or technique failure (i.e., transfer to hemodialysis). We performed studies in cells, mouse models, and samples obtained from humans to characterize an AQP1 variant and investigate mitigation strategies. RESULTS: The common AQP1 promoter variant rs2075574 was associated with peritoneal ultrafiltration. Carriers of the TT genotype at rs2075574 (10 to 16% of patients) had a lower mean (±SD) net ultrafiltration level than carriers of the CC genotype (35 to 47% of patients), both in the discovery phase (506±237 ml vs. 626±283 ml, P = 0.007) and in the validation phase (368±603 ml vs. 563±641 ml, P = 0.003). After a mean follow-up of 944 days, 139 of 898 patients (15%) had died and 280 (31%) had been transferred to hemodialysis. TT carriers had a higher risk of the composite of death or technique failure than CC carriers (adjusted hazard ratio, 1.70; 95% confidence interval [CI], 1.24 to 2.33; P = 0.001), as well as a higher risk of death from any cause (24% vs. 15%, P = 0.03). In mechanistic studies, the rs2075574 risk variant was associated with decreases in AQP1 promoter activity, aquaporin-1 expression, and glucose-driven osmotic water transport. The use of a colloid osmotic agent mitigated the effects of the risk variant. CONCLUSIONS: A common variant in AQP1 was associated with decreased ultrafiltration and an increased risk of death or technique failure among patients treated with peritoneal dialysis. (Funded by the Swiss National Science Foundation and others.).

A Dual-Gene Reporter-Amplifier Architecture for Enhancing the Sensitivity of Molecular MRI by Water Exchange.

The development of genetic reporters for magnetic resonance imaging (MRI) is essential for investigating biological functions in vivo. However, current MRI reporters have low sensitivity, making it challenging to create significant contrast against the tissue background, especially when only a small fraction of cells express the reporter. To overcome this limitation, we developed an approach for amplifying the sensitivity of molecular MRI by combining a chemogenetic contrast mechanism with a biophysical approach to increase water diffusion through the co-expression of a dual-gene construct comprising an organic anion transporting polypeptide, Oatp1b3, and a water channel, Aqp1. We first show that the expression of Aqp1 amplifies MRI contrast in cultured cells engineered to express Oatp1b3. We demonstrate that the contrast amplification is caused by Aqp1-driven increase in water exchange, which provides the gadolinium ions internalized by Oatp1b3-expressing cells with access to a larger water pool compared with exchange-limited conditions. We further show that our methodology allows cells to be detected using approximately 10-fold lower concentrations of gadolinium than that in the Aqp1-free scenario. Finally, we show that our approach enables the imaging of mixed-cell cultures containing a low fraction of Oatp1b3-labeled cells that are undetectable on the basis of Oatp1b3 expression alone.

Hypo-osmolality activates Wnt/ß-catenin mediated by AQP1 in nucleus pulposus cells degeneration.

The goals of this study were to investigate whether Wnt/ß-catenin signaling plays a role in hypo-osmolality-related degeneration of nucleus pulposus (NP) cells, and if so, to define the mechanism underlying AQP1 in this effect. Human NP cells were cultured under hypo-osmotic (300/350/400 mOsm) and iso-osmotic (450 mOsm) conditions. The cell viability, AQP1, the expression of Wnt/ß-catenin signaling, collagen II/I, and MMP3/9 were evaluated. To determine the effects of the Wnt/ß-catenin signaling, we used the inhibitor and the activator of Wnt during the hypo-osmotic culture of NP cells. We also examined whether the silencing and overexpressing of the AQP1 gene would affect the Wnt/ß-catenin expression in NP cells. Hypo-osmolality caused NP cell degeneration and activated the Wnt/ß-catenin signaling but suppressed the AQP1 level. Inhibiting the Wnt/ß-catenin signaling alleviated the hypo-osmolality-induced NP cell degeneration. On the contrary, activating Wnt/ß-catenin aggravated the NP cell degeneration under hypo-osmotic conditions, which did not affect AQP1 expression. AQP1-overexpressed NP cells exhibited decreased Wnt/ß-catenin signaling and alleviated cell degeneration under the hypo-osmotic condition. Besides, AQP1 silencing accelerated NP cell degeneration and activated Wnt/ß-catenin expression compared with untreated control. Hypo-osmolality promotes NP cell degeneration via activating Wnt/ß-catenin signaling, which is suppressed by AQP1 expression. The upregulation of AQP1 suppressed the Wnt/ß-catenin signaling and alleviated the hypo-osmolality induced by the NP cell degeneration.

Critical role of Aquaporin-1 and telocytes in infantile hemangioma response to propranolol beta blockade.

Propranolol, a nonselective ß-adrenergic receptor (ADRB) antagonist, is the first-line therapy for severe infantile hemangiomas (IH). Since the incidental discovery of propranolol efficacy in IH, preclinical and clinical investigations have shown evidence of adjuvant propranolol response in some malignant tumors. However, the mechanism for propranolol antitumor effect is still largely unknown, owing to the absence of a tumor model responsive to propranolol at nontoxic concentrations. Immunodeficient mice engrafted with different human tumor cell lines were treated with anti-VEGF bevacizumab to create a model sensitive to propranolol. Proteomics analysis was used to reveal propranolol-mediated protein alteration correlating with tumor growth inhibition, and Aquaporin-1 (AQP1), a water channel modulated in tumor cell migration and invasion, was identified. IH tissues and cells were then functionally investigated. Our functional protein association networks analysis and knockdown of ADRB2 and AQP1 indicated that propranolol treatment and AQP1 down-regulation trigger the same pathway, suggesting that AQP1 is a major driver of beta-blocker antitumor response. Examining AQP1 in human hemangioma samples, we found it exclusively in a perivascular layer, so far unrecognized in IH, made of telocytes (TCs). Functional in vitro studies showed that AQP1-positive TCs play a critical role in IH response to propranolol and that modulation of AQP1 in IH-TC by propranolol or shAQP1 decreases capillary-like tube formation in a Matrigel-based angiogenesis assay. We conclude that IH sensitivity to propranolol may rely, at least in part, on a cross talk between lesional vascular cells and stromal TCs.

Anethole mitigates H2 O2 -induced inflammation in HIG-82 synoviocytes by suppressing the aquaporin 1 expression and activating the protein kinase A pathway.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease affecting approximately 1% of the global population, with a higher prevalence in women than in men. Chronic inflammation and oxidative stress play pivotal roles in the pathogenesis of RA. Anethole, a prominent compound derived from fennel (Foeniculum vulgare), possesses a spectrum of therapeutic properties, including anti-arthritic, anti-inflammatory, antioxidant, and tumor-suppressive effects. However, its specific impact on RA remains underexplored. This study sought to uncover the potential therapeutic value of anethole in treating RA by employing an H2 O2 -induced inflammation model with HIG-82 synovial cells. Our results demonstrated that exposure to H2 O2 induced the inflammation and apoptosis in these cells. Remarkably, anethole treatment effectively countered these inflammatory and apoptotic processes triggered by H2 O2 . Moreover, we identified the aquaporin 1 (AQP1) and protein kinase A (PKA) pathway as critical regulators of inflammation and apoptosis. H2 O2 stimulation led to an increase in the AQP1 expression and a decrease in p-PKA-C, contributing to cartilage degradation. Conversely, anethole not only downregulated the AQP1 expression but also activated the PKA pathway, effectively suppressing cell inflammation and apoptosis. Furthermore, anethole also inhibited the enzymes responsible for cartilage degradation. In summary, our findings highlight the potential of anethole as a therapeutic agent for mitigating H2 O2 -induced inflammation and apoptosis in synovial cells, offering promising prospects for future RA treatments.

Effects of Modulating BMP9, BMPR2, and AQP1 on BMP Signaling in Human Pulmonary Microvascular Endothelial Cells.

Pulmonary arterial hypertension (PAH) is a chronic disease characterized by a progressive increase in mean pulmonary arterial pressure. Mutations in the BMPR2 and AQP1 genes have been described in familial PAH. The bone morphogenetic proteins BMP9 and BMP10 bind with high affinity to BMPR2. Administration of BMP9 has been proposed as a potential therapeutic strategy against PAH, although recent conflicting evidence dispute the effect of such a practice. Considering the involvement of the above molecules in PAH onset, progression, and therapeutic value, we examined the effects of modulation of BMP9, BMPR2, and AQP1 on BMP9, BMP10, BMPR2, AQP1, and TGFB1 expression in human pulmonary microvascular endothelial cells in vitro. Our results demonstrated that silencing the BMPR2 gene resulted in increased expression of its two main ligands, namely BMP9 and BMP10. Exogenous administration of BMP9 caused the return of BMP10 to basal levels, while it restored the decreased AQP1 protein levels and the decreased TGFB1 mRNA and protein expression levels caused by BMPR2 silencing. Moreover, AQP1 gene silencing also resulted in increased expression of BMP9 and BMP10. Our results might possibly imply that the effect of exogenously administered BMP9 on molecules participating in the BMP signaling pathway could depend on the expression levels of BMPR2. Taken together, these results may provide insight into the highly complex interactions of the BMP signaling pathway.

Aquaporin 2 in Cerebral Edema: Potential Prognostic Marker in Craniocerebral Injuries.

Despite continuous medical advancements, traumatic brain injury (TBI) remains a leading cause of death and disability worldwide. Consequently, there is a pursuit for biomarkers that allow non-invasive monitoring of patients after cranial trauma, potentially improving clinical management and reducing complications and mortality. Aquaporins (AQPs), which are crucial for transmembrane water transport, may be significant in this context. This study included 48 patients, with 27 having acute (aSDH) and 21 having chronic subdural hematoma (cSDH). Blood plasma samples were collected from the participants at three intervals: the first sample before surgery, the second at 15 h, and the third at 30 h post-surgery. Plasma concentrations of AQP1, AQP2, AQP4, and AQP9 were determined using the sandwich ELISA technique. CT scans were performed on all patients pre- and post-surgery. Correlations between variables were examined using Spearman's nonparametric rank correlation coefficient. A strong correlation was found between aquaporin 2 levels and the volume of chronic subdural hematoma and midline shift. However, no significant link was found between aquaporin levels (AQP1, AQP2, AQP4, and AQP9) before and after surgery for acute subdural hematoma, nor for AQP1, AQP4, and AQP9 after surgery for chronic subdural hematoma. In the chronic SDH group, AQP2 plasma concentration negatively correlated with the midline shift measured before surgery (Spearman's ρ -0.54; p = 0.017) and positively with hematoma volume change between baseline and 30 h post-surgery (Spearman's ρ 0.627; p = 0.007). No statistically significant correlation was found between aquaporin plasma levels and hematoma volume for AQP1, AQP2, AQP4, and AQP9 in patients with acute SDH. There is a correlation between chronic subdural hematoma volume, measured radiologically, and serum AQP2 concentration, highlighting aquaporins' potential as clinical biomarkers.

Leptin Promotes Vasculogenic Mimicry in Breast Cancer Cells by Regulating Aquaporin-1.

Leptin is an obesity-related hormone that plays an important role in breast cancer progression. Vasculogenic mimicry (VM) refers to the formation of vascular channels lined by tumor cells. This study aimed to investigate the relationship between leptin and VM in human breast cancer cells. VM was measured by a 3D culture assay. Signal transducers and activators of transcription 3 (STAT3) signaling, aquaporin-1 (AQP1), and the expression of VM-related proteins, including vascular endothelial cadherin (VE-cadherin), twist, matrix metalloproteinase-2 (MMP-2), and laminin subunit 5 gamma-2 (LAMC2), were examined by Western blot. AQP1 mRNA was analyzed by a reverse transcriptase-polymerase chain reaction (RT-PCR). Leptin increased VM and upregulated phospho-STAT3, VE-cadherin, twist, MMP-2, and LAMC2. These effects were inhibited by the leptin receptor-blocking peptide, Ob-R BP, and the STAT3 inhibitor, AG490. A positive correlation between leptin and AQP1 mRNA was observed and was confirmed by RT-PCR. Leptin upregulated AQP1 expression, which was blocked by Ob-R BP and AG490. AQP1 overexpression increased VM and the expression of VM-related proteins. AQP1 silencing inhibited leptin-induced VM and the expression of VM-related proteins. Thus, these results showed that leptin facilitates VM in breast cancer cells via the Ob-R/STAT3 pathway and that AQP1 is a key mediator in leptin-induced VM.

Tanshinone IIA changed the amniotic fluid volume and regulated expression of AQP1 and AQP3 in amniotic epithelium cells: a promising drug treating abnormal amniotic fluid volume.

BACKGROUND: Many studies have confirmed the association of aquaporins (AQPs) with abnormal amniotic fluid volume (AFV). In our previous experiments, we found that Tanshinone IIA was able to regulate the expression of AQP1 and AQP3. However, the exact mechanism by which Tanshinone IIA regulates AQPs protein expression and its effect on AFV remains unclear. The purpose of this study was to investigate the effects of Tanshinone IIA on AFV and the possible molecular mechanism of regulation of AQP1 and AQP3. METHODS: The expression of AQPs protein in the amniotic membranes was compared between pregnant women with normal pregnancy and those with isolated oligohydramnios. The AQP1 knockout (AQP1-KO) mice and wild-type (WT) mice were treated with saline or Tanshinone IIA (10 mg/kg) at 13.5GD and 16.5GD. Human amniotic epithelium cells (hAECs) from pregnant women with normal AFV and isolated oligohydramnios were incubated with 35 µmmol/L Tanshinone IIA or 25 mmol/L LiCl [inhibitor of glycogen synthetic kinase 3ß (GSK-3ß)]. The protein expressions of AQPs, GSK-3ß, phospho-GSK-3ß (Ser9) in fetal membranes of mice and human amniotic epithelium cells were detected by western blotting. RESULTS: The expression of AQP1 protein in the amniotic membrane of isolated oligohydramnios was increased compared with normal pregnancy. The AFV in AQP1-KO mice is higher than that in WT mice. In wild-type mice, AFV in Tanshinone IIA group was significantly higher than that in control group, and AQP1 protein expression was significantly lower than that in control group, but in AQP1 knockout mice, Tanshinone IIA reduced amniotic fluid volume and AQP3 protein expression at 16.5GD. Tanshinone IIA reduced AQP1, AQP3 and p-GSK-3ß (Ser9) protein expression in normal hAECs, and this effect was inhibited by LiCl. In hAECs with oligohydramnios, the down-regulation of AQP1 and up-regulation of AQP3 by Tanshinone IIA was independent of GSK-3ß signaling pathway. CONCLUSIONS: Tanshinone IIA may increase AFV in normal pregnancy by downregulating AQP1 protein expression in the fetal membranes, which may be associated with p-GSK-3ß signaling pathway. But a larger AFV in AQP1-KO mice was significantly attenuated by Tanshinone IIA, which may be related to AQP3. Tanshinone IIA is a promising drug for the treatment of amniotic fluid abnormality.

Aquaporin water channels affect the response of conventional anticancer therapies of 3D grown breast cancer cells.

Aquaporin (AQP) water channels facilitate water transport across cellular membranes and are essential in regulation of body water balance. Moreover, several AQPs are overexpressed or ectopically expressed in breast cancer. Interestingly, several in vitro studies have suggested that AQPs can affect the response to conventional anticancer chemotherapies. Therefore, we took a systematic approach to test how AQP1, AQP3 and AQP5, which are often over-/ectopically expressed in breast cancer, affect total viability of 3-dimensional (3D) breast cancer cell spheroids when treated with the conventional anticancer chemotherapies Cisplatin, 5-Fluorouracil (5-FU) and Doxorubicin, a Combination of the three drugs as well as the Combination plus the Ras inhibitor Salirasib. Total viability of spheroids overexpressing AQP1 were decreased by all treatments except for 5-FU, which increased total viability by 20% compared to DMSO treated controls. All treatments reduced viability of spheroids overexpressing AQP3. In contrast, only Doxorubicin, Combination and Combination + Salirasib reduced total viability of spheroids overexpressing AQP5. Thus, this study supports a significant role of AQPs in the response to conventional chemotherapies. Evaluating the role of individual proteins that contribute to resistance to chemotherapies is essential in advancing personalized medicine in breast carcinomas.

Neural crest cells bulldoze through the microenvironment using Aquaporin 1 to stabilize filopodia.

Neural crest migration requires cells to move through an environment filled with dense extracellular matrix and mesoderm to reach targets throughout the vertebrate embryo. Here, we use high-resolution microscopy, computational modeling, and in vitro and in vivo cell invasion assays to investigate the function of Aquaporin 1 (AQP-1) signaling. We find that migrating lead cranial neural crest cells express AQP-1 mRNA and protein, implicating a biological role for water channel protein function during invasion. Differential AQP-1 levels affect neural crest cell speed and direction, as well as the length and stability of cell filopodia. Furthermore, AQP-1 enhances matrix metalloprotease activity and colocalizes with phosphorylated focal adhesion kinases. Colocalization of AQP-1 with EphB guidance receptors in the same migrating neural crest cells has novel implications for the concept of guided bulldozing by lead cells during migration.

Function and evolution of the aquaporin IsAQP1 in the Lyme disease vector Ixodes scapularis.

Ticks are important vectors of pathogenic viruses, bacteria, and protozoans to humans, wildlife, and domestic animals. Due to their life cycles, ticks face significant challenges related to water homeostasis. When blood-feeding, they must excrete water and ions, but when off-host (for stretches lasting several months), they must conserve water to avoid desiccation. Aquaporins (AQPs), a family of membrane-bound water channels, are key players in osmoregulation in many animals but remain poorly characterized in ticks. Here, we bioinformatically identified AQP-like genes from the deer tick Ixodes scapularis and used phylogenetic approaches to map the evolution of the aquaporin gene family in arthropods. Most arachnid AQP-like sequences (including those of I. scapularis) formed a monophyletic group clustered within aquaglycerolporins (GLPs) from bacteria to vertebrates. This gene family is absent from insects, revealing divergent evolutionary paths for AQPs in different hematophagous arthropods. Next, we sequenced the full-length cDNA of I. scapularis aquaporin 1 (IsAQP1) and expressed it heterologously in Xenopus oocytes to functionally characterize its permeability to water and solutes. Additionally, we examined IsAQP1 expression across different life stages and adult female organs. We found IsAQP1 is an efficient water channel with high expression in salivary glands prior to feeding, suggesting it plays a role in osmoregulation before or during blood feeding. Its functional properties are unique: unlike most GLPs, IsAQP1 has low glycerol permeability, and unlike most AQPs, it is insensitive to mercury. Together, our results suggest IsAQP1 plays an important role in tick water balance physiology and that it may hold promise as a target of novel vector control efforts.

Fasting influences aquaporin expression, water transport and adipocyte metabolism in the peritoneal membrane.

BACKGROUND: The water channels aquaporin-1 (AQP1) and AQP7 are abundantly expressed in the peritoneal membrane. While AQP1 facilitates water transport during peritoneal dialysis (PD), the role of AQP7, which mediates glycerol transport during fasting, remains unknown. METHODS: We investigated the distribution of AQP7 and AQP1 and used a mouse model of PD to investigate the role of AQP7 in the peritoneal membrane at baseline and after fasting. RESULTS: Single nucleus RNA-sequencing revealed that AQP7 was mostly detected in mature adipocytes, whereas AQP1 was essentially expressed in endothelial cells. Fasting induced significant decreases in whole body fat, plasma glucose, insulin and triglycerides, as well as higher plasma glycerol and corticosterone levels in mice, paralleled by major decreases in adipocyte size and levels of fatty acid synthase and leptin, and increased levels of hormone-sensitive lipase mRNAs in the peritoneum. Mechanistically, fasting upregulated the expression of AQP1 and AQP7 in the peritoneum, with increased ultrafiltration but no change in small solute transport. Studies based on Aqp1 and Aqp7 knockout mice and RU-486 inhibition demonstrated that the glucocorticoid induction of AQP1 mediates the increase in ultrafiltration whereas AQP7 regulates the size of adipocytes in the peritoneum. CONCLUSIONS: Fasting induces a coordinated regulation of lipolytic and lipogenic factors and aqua(glycero)porins in the peritoneum, driving structural and functional changes. These data yield novel information on the specific roles of aquaporins in the peritoneal membrane and indicate that fasting improves fluid removal in a mouse model of PD.

Mifepristone increases AQP1 mRNA expression, angiogenesis, and cell permeability through the ERK MAPK pathway.

AIMS: The purpose of this study was to investigate the mechanism of mifepristone serves as an anti-implantation contraceptive drug on aquaporins 1 (AQP1) expression. METHODS: Human umbilical vein endothelial cells (HUVECs) were used to detect the effects of different concentrations of mifepristone (0, 0.065, 0.2, and 1 µmol/L) on the activity of angiogenesis and AQP1 expression. The expression of AQP1 was tested by the real-time PCR. The angiogenesis and penetration function of HUVECs was investigated by Matrigel lumen formation and trans-well assay, respectively. RESULTS: The expression of AQP1, angiogenesis and cell permeability were significantly higher than control groups in HUVECs treatment with mifepristone at 1 µmol/L for 12 h. Estrogen and progesterone decreased the up-regulation of AQP1 and cell permeability, not angiogenesis, induced by mifepristone. Mifepristone increased protein levels of p-ERK, not p-p38 or p-JNK, and pre-treatment with ERK MAPK-specific inhibitor significantly inhibited the up-regulation of AQP1 mRNA expression, angiogenesis and cell permeability induced by mifepristone. si-AQP1 significantly reduced the up-regulation of angiogenesis, cell permeability and p-ERK/ERK ratio expression induced by mifepristone treatment. Overexpression of AQP1 enhanced the increase of expression ratio of p-ERK/ERK induced by mifepristone. CONCLUSIONS: Low-dose mifepristone increased cell permeability, angiogenesis and AQP1 expression, which was involved in MAPK pathways. This provides new insights into the molecular mechanism of mifepristone serves as an anti-implantation contraceptive drug.

Actin cytoskeleton aggregation involves the water channel protein aquaporin 1-mediated human chondrocyte degeneration.

Water metabolism and actin cytoskeleton remoulding act as essential characters in the process of osteoarthritis (OA). However, the relation between water channel protein aquaporin 1 (AQP1) and actin filament during chondrocytes (CHs) degeneration is not evident. Therefore, the present study aimed to evaluate the role of actin remoulding in the AQP1 mediated CHs degeneration. Primary CHs were collected from human hip cartilage and were degenerated from long-time monolayer culture or IL-1ß stimulation. Besides, the CHs were transfected with AQP1­specific siRNA or vectors to mediate the AQP1 gene expression. The potent inhibitor of actin polymerization Cytochalasin D was also supplemented during culture. RT-PCR was performed to determine the relative gene expression. AQP1 and F-actin fluorescence staining were performed to determine the AQP1 and F-actin organization. Moreover, the cell area and viability were also analyzed. AQP1 and F-actin organization were both increased during seven days' CHs culture or three days' IL-1ß stimulation. Silencing of AQP1 prevented the cell area spreading and degenerated phenotype of CHs with suppression of F-actin aggregation in both natural or IL-1ß-caused inflammatory-related degeneration. Besides, upregulating the AQP1 in the CHs via gene editing promoted the cell area spreading, and F-actin accumulation, and accelerated the CHs degeneration, which can be alleviated by Cytochalasin D treatment. These findings suggested that AQP1-mediated human CHs degeneration is related to F-actin aggregation.

Blood-nerve barrier enhances chronic postsurgical pain via the HIF-1α/ aquaporin-1 signaling axis.

BACKGROUND: Blood nerve barrier (BNB) participates in the development of neuropathic pain. AQP1 is involved in peripheral pain perception and is negatively correlated with HIF-1α phenotype, which regulates endothelial permeability. However, the role of HIF-1α-AQP1-mediated BNB dysfunction in Chronic Postsurgical Pain (CPSP) has not been reported. METHODS: Male Sprague-Dawley rats were randomized into 5 groups: (i) Naive group; (ii) Sham group; (iii) SMIR group: skin/muscle incision and retraction for one hour. Behavioral tests were performed for the three groups, BNB vascular permeability and western blotting were conducted to determine HIF-1α and AQP1 protein expression. (iv) The SMIR + HIF-1α inhibitor group; (v) SMIR + DMSO group. Rats in the two groups were administered with HIF-1α inhibitor (2ME2) or DMSO intraperitoneally on the third day post-SMIR surgery followed by performance of behavioral tests, BNB permeability assessment, and determination of HIF-1α, AQP1 and NF200 protein levels. RESULTS: The permeability of BNB was significantly increased and the expression of AQP1 was downregulated on the 3rd and 7th days post-operation. AQP1 is mainly located in neurons and NF200, CGRP-positive nerve fibers. HIF-1α was highly expressed on the third day post-operation. HIF-1α inhibitor reversed the decrease in AQP1 expression and increase in NF200 expression, barrier permeability and hyperalgesia induced by SMIR on the 3rd day post-surgery. CONCLUSIONS: Early dysfunction of BNB mediated by HIF-1α/AQP1 activated by SMIR may be an important mechanism to promote acute postoperative painful transformation of CPSP. Preadaptive protection of endothelial cells around nerve substructures may be an important countermeasure to inhibit CPSP transformation. Early impairment of BNB function mediated by HIF-1α/AQP1 activated by SMIR may be an important mechanism for promoting acute postoperative pain transformation of CPSP.

Aquaporin Inhibitors.

Aquaporins (AQP) working as membrane channels facilitated water transport, play vital roles in various physiological progress including cell migration, energy metabolism, inflammation, etc. They are quite important drug targets, but elusive for discovery due to their undruggable properties. In this chapter, we summarized most fluently used methods for screening AQP inhibitors, including cell swelling assay, cell shrinking assay, and stopped-flow assay. And three classes of AQP inhibitors have been discussed, including metal-related inhibitors, quaternary ammonium salts, and small molecule inhibitors which further divided into four parts, sulfanilamide analogies, TGN-020, antiepileptic drugs, and others. It has been suggested that although they showed inhibition effects on AQP1, AQP3, AQP4, AQP7, or AQP9 in some researches, none of them could be asserted as AQP inhibitors to some extent. Discovering AQP inhibitors is a big challenge, but if successful, it will be a great contribution for human health.

Non-Aquaporin Water Channels.

Water transport through membrane is so intricate that there are still some debates. AQPs are entirely accepted to allow water transmembrane movement depending on osmotic gradient. Cotransporters and uniporters, however, are also concerned in water homeostasis. UT-B has a single-channel water permeability that is similar to AQP1. CFTR was initially thought as a water channel but now not believed to transport water directly. By cotransporters, such as KCC4, NKCC1, SGLT1, GAT1, EAAT1, and MCT1, water is transported by water osmosis coupling with substrates, which explains how water is transported across the isolated small intestine. This chapter provides information about water transport mediated by other membrane proteins except AQPs.