Aquaporins (AQPs) are water channels that mediate a variety of biological processes. However, their role in the immune system is poorly understood. We recently reported that AQP4 is expressed by naïve and memory T cells and that AQP4 blockade with a small molecule inhibitor prolongs murine heart allograft survival at least partially through diminishing T cell activation, proliferation and trafficking. The goal of this study was to determine how AQP4 function impacts T cells in the absence of antigen stimulation. AQP4 inhibition transiently reduced the number of circulating CD4+ and CD8+ T cells in naïve non-transplanted mice in the absence of systemic T cell depletion. Adoptive transfer studies demonstrated T cell intrinsic effect of AQP4 inhibition. AQP4 blockade altered T cell gene and protein expression of chemokine receptors S1PR1 and CCR7, and their master regulator KLF-2, and reduced chemotaxis toward S1P and CCL21. Consistent with the in vitro data, in vivo AQP4 inhibition reduced T lymphocyte numbers in the lymph nodes with simultaneous accumulation in the liver. Our findings indicate that blocking AQP4 reversibly alters T lymphocyte trafficking pattern. This information can be explored for the treatment of undesirable immune responses in transplant recipients or in patients with autoimmune diseases.

Aquaporin-4 (AQP4) is a predominant water channel expressed in astrocytes in the mammalian brain. AQP4 is crucial for the regulation of homeostatic water movement across the blood-brain barrier (BBB). Although the molecular mechanisms regulating AQP4 levels in the cerebral cortex under pathological conditions have been intensively investigated, those under normal physiological conditions are not fully understood. Here we demonstrate that AQP4 is selectively expressed in astrocytes in the mouse cerebral cortex during development. BMP signaling was preferentially activated in AQP4-positive astrocytes. Furthermore, activation of BMP signaling by in utero electroporation markedly increased AQP4 levels in the cerebral cortex, and inhibition of BMP signaling strongly suppressed them. These results indicate that BMP signaling alters AQP4 levels in the mouse cerebral cortex during development.

Aquaporin-4 (AQP4) has been implicated in post-traumatic syringomyelia (PTS), a disease characterised by the formation of fluid-filled cysts in the spinal cord. This study investigated the expression of AQP4 around a mature cyst (syrinx) and the effect of pharmacomodulation of AQP4 on syrinx size. PTS was induced in male Sprague-Dawley rats by computerized spinal cord impact and subarachnoid kaolin injection. Immunofluorescence of AQP4 was carried out on mature syrinx tissue 12 weeks post-surgery. Increased AQP4 expression corresponded to larger, multiloculated cysts (R2 = 0.94), yet no localized changes to AQP4 expression in perivascular regions or the glia limitans were present. In a separate cohort of animals, at 6 weeks post-surgery, an AQP4 agonist (AqF026), antagonist (AqB050), or vehicle was administered daily over 4 days, with MRIs performed before and after the completion of treatment. Histological analysis was performed at 12 weeks post-surgery. Syrinx volume and length were not altered with AQP4 modulation. The correlation between increased AQP4 expression with syrinx area suggests that AQP4 or the glia expressing AQP4 are recruited to regulate water movement. Given this, further investigation should examine AQP4 modulation with dose regimens at earlier time-points after PTS induction, as these may alter the course of syrinx development.

The water channel aquaporin 4 (AQP4) regulates the flux of water across the cell membrane, maintaining cellular homeostasis. Since AQP4 is enriched in the sarcolemma of skeletal muscle, a functional defect in AQP4 may cause skeletal muscle dysfunction. To investigate a novel mechanism underlying skeletal muscle atrophy, we examined AQP4 expression and its regulation in muscle using the rotator cuff tear (RCT) model. Human and mouse AQP4 expression was significantly decreased in atrophied muscle resulting from RCT. The size and the number of myotubes were reduced following AQP4 knockdown. Atrogin 1-mediated ubiquitination of AQP4 was verified with an ubiquitination assay after immunoprecipitation of AQP4 with an anti-AQP4 antibody. In this study, we identified high mobility group box 1 (HMGB1) as a potent upstream regulator of atrogin 1 expression. Atrogin 1 expression was increased by recombinant mouse HMGB1 protein, and the HMGB1-induced atrogin 1 expression was mediated via NF-κB signaling. Our study suggests that loss of AQP4 appears to be involved in myocyte shrinkage after RCT, and its degradation is mediated by atrogin 1-dependent ubiquitination. HMGB1, in its function as a signaling molecule upstream of the ubiquitin ligase atrogin 1, was found to be a novel regulator of muscle atrophy.

Proinflammatory cytokines, such as (IL: interleukin) IL-6 and IL-17A, and complement fixation are critical in the immunopathogenesis of neuromyelitis optica spectrum disorders (NMOSD). Blocking the IL-6 receptor or the C5 complement pathway reduces relapse risk. However, the role of interleukin (IL)-6 and complement in aquaporin-4 (AQP4) autoimmunity remains unclear. To investigate the role of the anti-AQP4 immunoglobulin (AQP4-IgG)/AQP4 immunocomplex on the induction and profile of ex vivo cytokine and surface marker expression in peripheral blood mononuclear cells (PBMC) culture. Isolated PBMCs obtained from 18 patients with AQP4-IgG-seropositive-NMOSD (8 treatment-naive, 10 rituximab-treated) or ten healthy controls were cultured with AQP4-immunocomplex with or without complement. Changes in PBMC surface markers and cytokine expression were profiled using flow cytometry and ELISA. PBMCs derived from treatment-naive NMOSD patients stimulated with a complex mixture of serum complement proteins produced significant elevations of IL-17A and IL-6. Rituximab-treated patients also exhibited higher IL-6 but not IL-17A release. IL-6 and IL-17A elevations are not observed without complement. Co-stimulation of PBMCs with AQP4-IgG/AQP4 immunocomplex and complement prompts a Th17-biased response consistent with the inflammatory paradigm observed in NMOSD. A possible inflammation model is proposed via antigen-specific autoreactive peripheral blood cells, including NK/NKT cells.

We aimed to compare longitudinal brain atrophy in patients with neuromyelitis optica spectrum disorder (NMOSD) with healthy controls (HCs). The atrophy rate in patients with anti-aquaporin-4 antibody-positive NMOSD (AQP4 + NMOSD) was compared with age-sex-matched HCs recruited from the Japanese Alzheimer's Disease Neuroimaging Initiative study and another study performed at Chiba University. Twenty-nine patients with AQP4 + NMOSD and 29 HCs were enrolled in the study. The time between magnetic resonance imaging (MRI) scans was longer in the AQP4 + NMOSD group compared with the HCs (median; 3.2 vs. 2.9 years, P = 0.009). The annualized normalized white matter volume (NWV) atrophy rate was higher in the AQP4 + NMOSD group compared with the HCs (median; 0.37 vs. - 0.14, P = 0.018). The maximum spinal cord lesion length negatively correlated with NWV at baseline MRI in patients with AQP4 + NMOSD (Spearman's rho =  - 0.41, P = 0.027). The annualized NWV atrophy rate negatively correlated with the time between initiation of persistent prednisolone usage and baseline MRI in patients with AQP4 + NMOSD (Spearman's rho =  - 0.43, P = 0.019). Patients with AQP4 + NMOSD had a greater annualized NWV atrophy rate than HCs. Suppressing disease activity may prevent brain atrophy in patients with AQP4 + NMOSD.

White blood cell (WBC) count profiles in anti-aquaporin-4 antibody-positive neuromyelitis optica spectrum disorder (AQP4-NMOSD) and anti-myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) are still unknown. This study evaluated the total WBC count, differential WBC counts, monocyte-to-lymphocyte ratio (MLR), and neutrophil-to-lymphocyte ratio (NLR) in patients with these diseases within three months from an attack before acute treatment or relapse prevention and compared the profiles with those in matched volunteers or in multiple sclerosis (MS) patients. AQP4-NMOSD patients (n = 13) had a higher neutrophil count (p = 0.0247), monocyte count (p = 0.0359), MLR (p = 0.0004), and NLR (p = 0.0037) and lower eosinophil (p = 0.0111) and basophil (p = 0.0283) counts than those of AQP4-NMOSD-matched volunteers (n = 65). Moreover, patients with MOGAD (n = 26) had a higher overall WBC count (p = 0.0001), neutrophil count (p < 0.0001), monocyte count (p = 0.0191), MLR (p = 0.0320), and NLR (p = 0.0002) than those of MOGAD-matched volunteers (n = 130). The three demyelinating diseases showed similar levels of the total and differential WBC counts; however, MOGAD and MS showed different structures in the hierarchical clustering and distributions on a two-dimensional canonical plot using differential WBC counts from the other three groups. WBC count profiles were similar in patients with MOGAD and MS but differed from profiles in matched volunteers or patients with AQP4-NMOSD.

We performed multi-b and multi-diffusion-time diffusion-weighted magnetic resonance imaging on aquaporin-4-expressing (AQ) and -non-expressing (noAQ) cells, and demonstrated a clear difference between the signals from the two cell types. The data were interpreted using a two-compartment (intra and extracellular spaces) model including inter-compartmental exchange. It was also assumed that restricted diffusion of water molecules inside the cells leads to the intracellular diffusion coefficient being inversely proportional to the diffusion-time. Estimates of the water-exchange-times obtained with this model are compared to those measured using an independent optical imaging technique (coherent anti-Stokes Raman scattering imaging, CARS). For both techniques it was found that the exchange-time estimated for the noAQ cells was significantly longer than that for the AQ cells.

Aquaporin incorporated biomimetic membranes are anticipated to offer unprecedented desalination capabilities. However, the lack of accurate methods to quantify the reconstituted aquaporin presents a huge hurdle in investigating aquaporin performance and optimizing membrane fabrication. Herein, we present three quantification methods to determine the Aquaporin-Z reconstituted into E. coli lipid vesicles: 1) nanogold labeling with transmission electron microscopy (TEM) visualization, 2) nickel labeling with inductively coupled plasma-mass spectrometry (ICP-MS) and 3) gel electrophoresis. The TEM method serves as a quick way to determine if aquaporin has been reconstituted, but is not quantitative. The numerical results from quantitative methods, ICP-MS and gel electrophoresis, correlate closely, showing that 60 ± 20% vs 66 ± 4% of Aquaporin-Z added is successfully reconstituted into vesicles respectively. These methods allow more accurate determination of Aquaporin-Z reconstituted and loss during reconstitution, with relatively commonly available equipment and without complex sample handling, or lengthy data analysis. These would allow them to be widely applicable to scientific studies of protein function in the biomimetic environment and engineering studies on biomimetic membrane fabrication.

Aquaporin TIP2;1 is a protein channel permeable to both water and ammonia. The structural origin of ammonia selectivity remains obscure, but experiments have revealed that a double mutation renders it impermeable to ammonia without affecting water permeability. Here, we aim to reproduce and explain these observations by performing an extensive mutational study using microsecond long molecular dynamics simulations, applying the two popular force fields CHARMM36 and Amber ff99SB-ILDN. We calculate permeabilities and free energies along the channel axis for ammonia and water. For one force field, the permeability of the double mutant decreases by a factor of 2.5 for water and 4 for ammonia, increasing water selectivity by a factor of 1.6. We attribute this effect to decreased entropy of water in the pore, due to the observed increase in pore-water interactions and narrower pore. Additionally, we observe spontaneous opening and closing of the pore on the cytosolic side, which suggests a gating mechanism for the pore. Our results show that sampling methods and simulation times are sufficient to delineate even subtle effects of mutations on structure and function and to capture important long-timescale events, but also underline the importance of improving models further.

Prolonged hypokalemia induces a decrease of urinary concentrating ability via down-regulation of aquaporin 2 (AQP2); however, the precise mechanisms remain unknown. To investigate the role of autophagy in the degradation of AQP2, we generated the principal cell-specific Atg7 deletion (Atg7Δpc) mice. In hypokalemic Atg7-floxed (Atg7f/f) mice, huge irregular shaped LC3-positive autophagic vacuoles accumulated mainly in inner medullary collecting duct (IMCD) cells. Total- and pS261-AQP2 were redistributed from apical and subapical domains into these vacuoles, which were not co-localized with RAB9. However, in the IMCD cells of hypokalemic Atg7Δpc mice, these canonical autophagic vacuoles were markedly reduced, whereas numerous small regular shaped LC3-negative/RAB9-positive non-canonical autophagic vacuoles were observed along with diffusely distributed total- and pS261-AQP2 in the cytoplasm. The immunoreactivity of pS256-AQP2 in the apical membrane of IMCD cells was markedly decreased, and no redistribution was observed in both hypokalemic Atg7f/f and Atg7Δpc mice. These findings suggest that AQP2 down regulation in hypokalemia was induced by reduced phosphorylation of AQP2, resulting in a reduction of apical plasma labeling of pS256-AQP2 and degradation of total- and pS261-AQP2 via an LC3/ATG7-dependent canonical autophagy pathway.

Hypokalemia (low serum potassium level) is a common electrolyte imbalance that can cause a defect in urinary concentrating ability, i.e., nephrogenic diabetes insipidus (NDI), but the molecular mechanism is unknown. We employed proteomic analysis of inner medullary collecting ducts (IMCD) from rats fed with a potassium-free diet for 1 day. IMCD protein quantification was performed by mass spectrometry using a label-free methodology. A total of 131 proteins, including the water channel AQP2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the down-regulated proteins were associated with the biological processes of generation of precursor metabolites and energy, actin cytoskeleton organization, and cell-cell adhesion. Targeted LC-MS/MS and immunoblotting studies further confirmed the down regulation of 18 selected proteins. Electron microscopy showed autophagosomes/autophagolysosomes in the IMCD cells of rats deprived of potassium for only 1 day. An increased number of autophagosomes was also confirmed by immunofluorescence, demonstrating co-localization of LC3 and Lamp1 with AQP2 and several other down-regulated proteins in IMCD cells. AQP2 was also detected in autophagosomes in IMCD cells of potassium-deprived rats by immunogold electron microscopy. Thus, enhanced autophagic degradation of proteins, most notably including AQP2, is an early event in hypokalemia-induced NDI.

The central region of the intervertebral disc (IVD) is rich in proteoglycans, leading to a hyperosmotic environment, which fluctuates with daily loading. The cells of the nucleus pulposus (NP cells) have adapted to this environment via the function of tonicity enhancer binding protein (TonEBP), and NP cells have been shown to express several water channels known as aquaporins (AQP). We have previously shown that AQP1 and 5 decrease during IVD degeneration. Here, the regulation of AQP1 and 5 by hyperosmotic conditions and the role of TonEBP in this regulation was investigated. AQP1 and 5 gene expression was upregulated by hyperosmotic conditions mimicking the osmolality of the healthy IVD, which was abrogated by TonEBP knockdown. Furthermore, AQP1 and 5 immunopositivity was significantly reduced in TonEBPΔ/Δ E17.5 mice when compared with wildtype controls, indicating in vivo expression of AQP1 and 5 is controlled at least in part by TonEBP. This hyperosmotic regulation of AQP1 and 5 could help to explain the decreased AQP1 and 5 expression during degeneration, when the osmolality of the NP decreases. Together this data suggests that TonEBP-regulated osmo-adaptation may be disrupted during IVD degeneration when the expression of both AQPs is reduced.

Vasopressin (AVP) increases water permeability in the renal collecting duct through the regulation of aquaporin-2 (AQP2) trafficking. Several disorders, including hypertension and inappropriate antidiuretic hormone secretion (SIADH), are associated with abnormalities in water homeostasis. It has been shown that certain phytocompounds are beneficial to human health. Here, the effects of the Olive Leaf Extract (OLE) have been evaluated using in vitro and in vivo models. Confocal studies showed that OLE prevents the vasopressin induced AQP2 translocation to the plasma membrane in MCD4 cells and rat kidneys. Incubation with OLE decreases the AVP-dependent increase of the osmotic water permeability coefficient (Pf). To elucidate the possible effectors of OLE, intracellular calcium was evaluated. OLE increases the intracellular calcium through the activation of the Calcium Sensing Receptor (CaSR). NPS2143, a selective CaSR inhibitor, abolished the inhibitory effect of OLE on AVP-dependent water permeability. In vivo experiments revealed that treatment with OLE increases the expression of the CaSR mRNA and decreases AQP2 mRNA paralleled by an increase of the AQP2-targeting miRNA-137. Together, these findings suggest that OLE antagonizes vasopressin action through stimulation of the CaSR indicating that this extract may be beneficial to attenuate disorders characterized by abnormal CaSR signaling and affecting renal water reabsorption.

Altered aquaporin 5 (AQP5) expression in immune cells impacts on key mechanisms of inflammation and is associated with sepsis survival. Since epigenetic regulation via DNA methylation might contribute to a differential AQP5 expression in sepsis, we tested the hypotheses that DNA methylation of the AQP5 promotor (1) influences AQP5 expression, (2) is associated with the 30-day survival of septic patients, and (3) alters the nuclear transcription factor NF-κB binding. AQP5 mRNA expression was quantified by real-time PCR in whole blood samples of 135 septic patients. In silico computer analysis of the AQP5 promoter (nt-567 to nt-975) revealed seven putative inflammatory transcription factor binding sites and methylation of these sites was analyzed. Electrophoretic mobility shift assays were performed to assess the binding of nuclear NF-κB to the AQP5 promoter region nt-937. After adjustment for multiple testing, a greater methylation rate was found at cytosine site nt-937 in the AQP5 promoter linked to NF-κB binding in non-survivors compared to survivors (p = 0.002, padj = 0.014). This was associated with greater AQP5 mRNA expression in non-survivors (p = 0.037). Greater (≥16%) promoter methylation at nt-937 was also associated with an independently increased risk of death within 30 days (HR: 3.31; 95% CI: 1.54-6.23; p = 0.002). We detected a functionally important AQP5 promoter cytosine site (nt-937) linked to the binding of the inflammatorily acting nuclear transcription factor NF-κB, with increased methylation in sepsis non-survivors. Thus, nt-937 APQ5 promoter methylation, presumably related to NF-κB binding, is prognostically relevant in sepsis and demonstrates that epigenetic changes impact on sepsis outcome.

The inability to over-express Aquaporin 6 (AQP6) in the plasma membrane of heterologous cells has hampered efforts to further characterize the function of this aquaglyceroporin membrane protein at atomic detail using crystallographic approaches. Using an Aquaporin 3-tGFP Reporter (AGR) system we have identified a region within loop C of AQP6 that is responsible for severely hampering plasma membrane expression. Serine substitution corroborated that amino acids present within AQP6194-213 of AQP6 loop C contribute to intracellular endoplasmic reticulum (ER) retention. This intracellular retention signal may preclude proper plasma membrane trafficking and severely curtail expression of AQP6 in heterologous expression systems.

Patients with stress-induced exhaustion disorder (SED) demonstrate cognitive dysfunction similar to patients with minor traumatic brain injury (TBI). We have previously detected elevated concentrations of astrocyte-derived extracellular vesicles (EVs) in patients with TBI. As such, we hypothesized that astrocyte-derived EVs could be higher in patients with SED than in patients with major depressive disorder (MDD) and healthy controls. Patients with SED (n = 31), MDD (n = 31), and healthy matched controls (n = 61) were included. Astrocyte-derived EVs (previously known as microparticles) were measured in plasma with flow cytometry and labeled against glial fibrillary acidic protein (GFAP) and aquaporin 4 (AQP4). In addition, platelet EVs and their CD40 ligand expression were measured. Patients with SED had significantly higher concentrations of AQP4 and GFAP-positive EVs and EVs co-expressing AQP4/GFAP than patients with MDD and healthy controls. Patients with MDD had significantly higher concentrations of GFAP-positive EVs and EVs co-expressing AQP4/GFAP than healthy controls. Platelet EVs did not differ between groups. CD40 ligand expression was significantly higher in patients with SED and MDD than in controls. In conclusion, the present study suggests that patients with SED, and to some extent, patients with MDD, have increased leakage of astrocyte-derived EVs through the blood-brain barrier.

Being able to directly obtain micron-sized cell blebs, giant plasma membrane vesicles (GPMVs), with native membrane proteins and deposit them on a planar support to form supported plasma membranes could allow the membrane proteins to be studied by various surface analytical tools in native-like bilayer environments. However, GPMVs do not easily rupture on conventional supports because of their high protein and cholesterol contents. Here, we demonstrate the possibility of using compression generated by the air-water interface to efficiently rupture GPMVs to form micron-sized supported membranes with native plasma membrane proteins. We demonstrated that not only lipid but also a native transmembrane protein in HeLa cells, Aquaporin 3 (AQP3), is mobile in the supported membrane platform. This convenient method for generating micron-sized supported membrane patches with mobile native transmembrane proteins could not only facilitate the study of membrane proteins by surface analytical tools, but could also enable us to use native membrane proteins for bio-sensing applications.

Carnitine/organic cation transporter 1 (OCTN1) is the only known uptake transporter for ergothioneine which is a food-derived strong antioxidant amino acid that is absorbed by OCTN1. We previously reported the roles of OCTN1/ergothioneine in the progression of kidney fibrosis in ischemic kidney disease. In this study, we evaluated the roles of OCTN1 in the progression of diabetic kidney disease. A diabetic kidney disease model was induced in octn1 knockout and wild-type mice by streptozotocin (STZ). Oxidative stress, represented by urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG), were higher in the octn1 knockout mice. Azan- and Sirius red-positive areas increased significantly in the octn1 knockout mice. Gene expression was evaluated by cluster analysis, and shown to be different in the octn1 knockout mice compared with the wild-type mice. In a pathway analysis, the pathway associated with the cytoskeleton and cell adhesion increased. In accordance with interstitial fibrosis in octn1 knockout mice, gene expression of moesin in the injured kidney, known as an associated protein of cytoskeleton and cell membranes, was doubled 28 weeks after STZ injection. In addition, the moesin protein was expressed in a part of α-SMA-positive renal tubular epithelial cells. These findings were confirmed by cultured murine proximal tubular epithelial cells: The expression of moesin was induced under oxidative stress with hydrogen peroxide. These data indicate that OCTN1 would play some roles in progression of interstitial fibrosis under oxidative stress via moesin expression in diabetic kidney disease.

We examined the effect of total and afferent renal denervation (RDN) on hypertension and the renin-angiotensin system (RAS) in a rodent model of juvenile-onset polycystic kidney disease (PKD). Lewis Polycystic Kidney (LPK) and control rats received total, afferent or sham RDN by periaxonal application of phenol, capsaicin or normal saline, respectively, and were monitored for 4-weeks. Afferent RDN did not affect systolic blood pressure (SBP) determined by radiotelemetry in either strain (n = 19) while total RDN significantly reduced SBP in Lewis rats 4-weeks post-denervation (total vs. sham, 122 ± 1 vs. 130 ± 2 mmHg, P = 0.002, n = 25). Plasma and kidney renin content determined by radioimmunoassay were significantly lower in LPK vs. Lewis (plasma: 278.2 ± 6.7 vs. 376.5 ± 11.9 ng Ang I/ml/h; kidney: 260.1 ± 6.3 vs. 753.2 ± 37.9 ng Ang I/mg/h, P < 0.001, n = 26). These parameters were not affected by RDN. Intrarenal mRNA expression levels of renin, angiotensinogen, angiotensin-converting enzyme (ACE)2, and angiotensin II receptor type 1a were significantly lower, whereas ACE1 expression was significantly higher in the LPK vs. Lewis (all P < 0.05, n = 26). This pattern of intrarenal RAS expression was not changed by RDN. In conclusion, RDN does not affect hypertension or the RAS in the LPK model and indicates RDN might not be a suitable antihypertensive strategy for individuals with juvenile-onset PKD.