Aquaporin-4 Protein Expression Research Articles

The Role of Aquaporin 4 in Lacrimal Gland Ductal Fluid Secretion in Mice.

Earlier reports highlighted the predominant presence of aquaporin 4 (AQP4) in the duct cells of rabbit lacrimal glands (LGs). Whereas significant alterations in AQP4 mRNA levels have been observed in experimental dry eye and during pregnancy, the impact of AQP4 in LG ductal fluid production remains unclear. In our recent work, the role of AQP4 in LG ductal fluid secretion was investigated utilizing wild type (WT) and AQP4 knock out (KO) mice. Tear production was assessed in both WT and KO animals. Immunostaining was used to identify AQP4 protein. Duct segments were harvested from LGs of WT and KO mice. Fluid secretion and filtration permeability (Pf) were quantified using video-microscopy. Ductal tear production, elicited by a cell-permeable cAMP analogue (8-bromo cAMP), carbachol, vasoactive intestinal peptide (VIP), and phenylephrine (PHE), were assessed in both WT and KO ducts. A higher expression of AQP4 protein was noted in the duct cells from WT mice when compared to acinar cells. Pf did not show notable alterations between WT and AQP4 KO ducts. Carbachol elicited comparable secretory responses in ducts from both WT and KO animals. However, 8-bromo cAMP, VIP, and PHE stimulation resulted in decreased secretion in ducts from AQP4 KO LGs. Our findings underscore the functional relevance of AQP4 in the fluid production of mouse LG ducts. AQP4 seems to play different roles in fluid secretions elicited by different secretagogues. Specifically, cAMP-mediated, and adrenergic agonist-related secretions were reduced in AQP4 KO ducts.

Preventive effect of kiwi berry (Actinidia arguta) on loperamide-induced constipation

Constipation is a common intestinal disease. Kiwi berries can effectively prevent constipation. However, studies have yet to be done to determine how kiwi berries prevent constipation. For 2 weeks, mice in this study were continually orally gavaged with kiwi berry, loperamide, or a combination of the two. This study found that the kiwi group's feces had more water than the constipated mice. In addition, kiwi berries can speed up gastrointestinal transit (GI), shorten the time it takes to pass the first dark stool, and dramatically enhance body weight gain. In the interstitial cells of Cajal (ICC) cells and colon tissues, alterations in the protein expression of vasoactive intestinal peptide (VIP), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and aquaporin-3 (AQP3) were found. At 3, 6, and 12 h of ICC cells and mouse colon, the kiwi group’s VIP, cAMP, PKA, and AQP3 protein expression levels were lower than those of the constipated mice. The kiwi berry can decrease the Firmicutes to Bacteroidetes ratio and boost the diversity and quantity of gut microbiota. By influencing the gut microbiota and VIP-cAMP-PKA-AQP3 signaling pathway, kiwi berries prevent constipation.

Alterations in aquaporin 4 expression in the chicken ovary following tamoxifen treatment

Previously, we demonstrated the follicular size- and a layer of the follicular wall-dependend expression of aquaporin 4 (AQP4) in the chicken ovary. In this study, we aimed to examine the mRNA and protein expression of aquaporin 4 (AQP4) in the chicken ovary during a pause in laying induced by a tamoxifen (TMX, oestrogen receptor modulator) treatment. Ovarian white and yellowish follicles and the granulosa and theca layers of the largest yellow preovulatory follicles were harvested from control and TMX-treated (daily until the cessation of egg-laying) hens. It was found that the TMX treatment lowered the AQP4 transcript abundance in the white follicles, but increased it in the theca layer of the F3-F1 follicles. Moreover, the TMX caused a decrease in AQP4 protein abundance in the theca layer of the F2 follicle. We propose that AQP4 plays a role in the ovarian follicle development and that there is a relationship between oestradiol action and the AQP4 gene and protein expression in chicken ovarian follicles.

TNF inhibits AQP2 expression via a miR137-dependent pathway.

As miR-137 is a regulator of aquaporin (AQP)2 expression and tumor necrosis factor (TNF) inhibits the expression of several extrarenal AQPs, we tested the hypothesis that TNF inhibits AQP2 in the kidney via a miR-137-dependent mechanism. AQP2 mRNA and protein expression decreased ∼70% and 53%, respectively, in primary renal inner medullary collecting duct (IMCD) cells transfected with a miRNA mimic of mmu-miR-137, suggesting that miR-137 directly targets AQP2 mRNA in these cells. Exposure of IMCD cells for 2 h to 400 mosmol/kgH2O medium increased mmu-miR-137 mRNA expression about twofold, conditions that also increased TNF production approximately fourfold. To determine if the increase in mmu-miR-137 mRNA expression was related to the concomitant increase in TNF, IMCD cells were transfected with a lentivirus construct to silence TNF. This construct decreased mmu-miR-137 mRNA expression by ∼63%, suggesting that TNF upregulates the expression of miR-137. Levels of miR-137 also increased approximately twofold in IMCD tubules isolated from male mice given 1% NaCl in the drinking water for 3 days. Intrarenal lentivirus silencing of TNF increased AQP2 mRNA levels and protein expression concomitant with a decrease in miR-137 levels in tubules isolated from mice given NaCl. The changes in AQP2 expression levels affected the diluting ability of the kidney, which was assessed by measuring urine osmolality and urine volume, as the decrease in these parameters after renal silencing of TNF was prevented on intrarenal administration of miR-137. The study reveals a novel TNF function via a miR-137-dependent mechanism that regulates AQP2 expression and function.NEW & NOTEWORTHY An emerging intratubular tumor necrosis factor system, functioning during normotensive noninflammatory conditions, acts as a breaking mechanism that attenuates both the increases in Na+-K+-2Cl- cotransporter and aquaporin-2 induced by arginine vasopressin, thereby contributing to the regulation of electrolyte balance and blood pressure. A greater appreciation for the role of cytokines as mediators of immunophysiological responses may help reveal the relationship between the immune system and other physiological systems.

Mannitol inhibits the proliferation of neural stem cell by a p38 mitogen-activated protein kinase-dependent signaling pathway

PurposeMannitol is one of the first-line drugs for reducing cerebral edema through increasing the extracellular osmotic pressure. However, long-term administration of mannitol in the treatment of cerebral edema triggers damage to neurons and astrocytes. Given that neural stem cell (NSC) is a subpopulation of main regenerative cells in the central nervous system after injury, the effect of mannitol on NSC is still elusive. The present study aims to elucidate the role of mannitol in NSC proliferation. MethodsC57 mice were derived from the animal house of Zunyi Medical University. A total of 15 pregnant mice were employed for the purpose of isolating NSCs in this investigation. Initially, mouse primary NSCs were isolated from the embryonic cortex of mice and subsequently identified through immunofluorescence staining. In order to investigate the impact of mannitol on NSC proliferation, both cell counting kit-8 assays and neurospheres formation assays were conducted. The in vitro effects of mannitol were examined at various doses and time points. In order to elucidate the role of Aquaporin 4 (AQP4) in the suppressive effect of mannitol on NSC proliferation, various assays including reverse transcription polymerase chain reaction, western blotting, and immunocytochemistry were conducted on control and mannitol-treated groups. Additionally, the phosphorylated p38 (p-p38) was examined to explore the potential mechanism underlying the inhibitory effect of mannitol on NSC proliferation. Finally, to further confirm the involvement of the p38 mitogen-activated protein kinase-dependent (MAPK) signaling pathway in the observed inhibition of NSC proliferation by mannitol, SB203580 was employed. All data were analyzed using SPSS 20.0 software (SPSS, Inc., Chicago, IL). The statistical analysis among multiple comparisons was performed using one-way analysis of variance (ANOVA), followed by Turkey's post hoc test in case of the data following a normal distribution using a Shapiro-Wilk normality test. Comparisons between 2 groups were determined using Student's t-test, if the data exhibited a normal distribution using a Shapiro-Wilk normality test. Meanwhile, data were shown as median and interquartile range and analyzed using the Mann-Whitney U test, if the data failed the normality test. A p < 0.05 was considered as significant difference. ResultsPrimary NSC were isolated from the mice, and the characteristics were identified using immunostaining analysis. Thereafter, the results indicated that mannitol held the capability of inhibiting NSC proliferation in a dose-dependent and time-dependent manner using cell counting kit-8, neurospheres formation, and immunostaining of Nestin and Ki67 assays. During the process of mannitol suppressing NSC proliferation, the expression of AQP4 mRNA and protein was downregulated, while the gene expression of p-p38 was elevated by reverse transcription polymerase chain reaction, immunostaining, and western blotting assays. Subsequently, the administration of SB203580, one of the p38 MAPK signaling pathway inhibitors, partially abrogated this inhibitory effect resulting from mannitol, supporting the fact that the p38 MAPK signaling pathway participated in curbing NSC proliferation induced by mannitol. ConclusionsMannitol inhibits NSC proliferation through downregulating AQP4, while upregulating the expression of p-p38 MAPK.

Hypomethylation of a CpG Site in the CpG Island of the Aquaporin 1 Gene May Be Involved in the Formation of Adult-Onset Non-communicating Hydrocele Testis.

Background Water channel aquaporin 1 (AQP1) protein expression is enhanced in the tunica vaginalis of patients with adult-onset non-communicating hydrocele testis and may contribute to the development of non-communicating hydrocele testis. We performed genetic and epigenetic analyses of the AQP1 gene in the tunica vaginalis of patients with adult-onset non-communicating hydrocele testis to elucidate the cause of enhanced AQP1 protein expression. Methodology The genotype was determined for Tag single-nucleotide polymorphisms (SNPs) representing the AQP1 gene and SNPs in the 5'-upstream region of the AQP1 gene. Then, by performing association analysis, the applicability of various genetic models was investigated for each SNP. Moreover, the methylation rate of CpG sites was examined for the CpG island related to the AQP1 gene. Results There was no significant association between each SNP and hydrocele testis for any of the genetic models. The average methylation rate of the 17 CpG sites evaluated was not significantly different between controls and hydrocele testis, but the methylation rate was lower in hydrocele testis than in controls at one CpG site. Conclusions There was a significant decrease in the methylation rate at one of the CpG sites in the CpG island associated with the AQP1 gene in the tunica vaginalis of patients with non-communicating hydrocele testis. This may increase AQP1 protein expression and contribute to the formation of hydrocele testis. SNPs related to the AQP1 gene were not associated with hydrocele testis.

Vitamin D attenuates DNCB-induced atopic dermatitis-like skin lesions by inhibiting immune response and restoring skin barrier function.

Atopic dermatitis (AD) is a common chronic inflammatory skin disease causing erythema and itching. The etiology of AD is complex and not yet clear. Vitamin D is a fat-soluble vitamin that promotes skin cell growth and differentiation and regulates immune function. This study aimed to explore the therapeutic effect of calcifediol, the active metabolite of vitamin D, on experimental AD and the possible mechanism of action. We found that the levels of vitamin D binding protein (VDBP) and vitamin D receptor (VDR) in biopsy skin samples from AD patients decreased compared with controls. We used 2,4-dinitrochlorobenzene (DNCB) to induce an AD mouse model on the ear and back of BALB/c mice. A total of five groups were used: the control group, the AD group, the AD+calcifediol group, the AD+dexamethasone group, and the calcifediol alone group. Under calcifediol treatment, mice exhibited reduced spinous layer thickening, reduced inflammatory cell infiltration, downregulated aquaporin 3 (AQP3) expression, and restored the barrier function of the skin. Simultaneous calcifediol treatment decreased STAT3 phosphorylation, inhibited inflammation and chemokine release, decreased AKT1 and mTOR phosphorylation, and suppressed epidermal cell proliferation and abnormal differentiation. In conclusion, our study demonstrated that calcifediol significantly protected mice against DNCB-induced AD. In a mouse model of AD, calcifediol may reduce inflammatory cell infiltration and chemokines by inhibiting the phosphorylation of STAT3 and may restore skin barrier function through the downregulation of AQP3 protein expression and inhibition of cell proliferation.

Loss of aquaporin-1 expression is associated with worse clinical outcomes in clear cell renal cell carcinoma: an immunohistochemical study.

Aquaporin (AQP) expression has been investigated in various malignant neoplasms, and the overexpression of AQP is related to poor prognosis in some malignancies. However, the expression of AQP protein in clear cell renal cell carcinoma (ccRCC) has not been extensively investigated by immunohistochemistry with large sample size. We evaluated the AQP expression in 827 ccRCC with immunohistochemical staining in tissue microarray blocks and classified the cases into two categories, high and low expression. High expression of aquaporin-1 (AQP1) was found in 320 cases (38.7%), but aquaporin-3 was not expressed in ccRCC. High AQP1 expression was significantly related to younger age, low TNM stage, low World Health Organization/International Society of Urologic Pathology nuclear grade, and absence of distant metastasis. Furthermore, high AQP1 expression was also significantly associated with longer overall survival (OS; p<.001) and progression-specific survival (PFS; p<.001) and was an independent predictor of OS and PFS in ccRCC. Our study revealed the prognostic significance of AQP1 protein expression in ccRCC. These findings could be applied to predict the prognosis of ccRCC.

Aquaporin 1 Facilitates Ferroptosis, M1 Polarization, Mitochondrial Dysfunction, and Autophagy Damage on Lipopolysaccharide-Induced Macrophage Through Down-Regulation of P53 Signaling Pathway.

This study was designed to investigate the role of aquaporin 1 (AQP1) in ferroptosis, macrophage polarization, mitochondrial dysfunction, and impaired autophagy of lipopolysaccharide (LPS)-stimulated RAW264.7 cells and explored the underlying mechanisms. Si-AQP1-mediated AQP1 silencing RAW264.7 cells was constructed. Si-P53-mediated P53 silencing or pcDNA-P53 overexpression RAW264.7 cells was constructed. Assays of ATP, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and Mitochondrial membrane potential (JC-1) staining were performed to evaluate mitochondrial biological function. Assays of flow cytometry, reactive oxygen species (ROS) staining, western blot (WB), RT-qPCR, malondialdehyde (MDA), glutathione (GSH), and total superoxide dismutase (SOD) were performed to detect cell ferroptosis, macrophage polarization, and impaired autophagy. The involvement of the P53 pathway was revealed by WB. The results showed that LPS (30 μg/mL) could induce ferroptosis, M1 polarization, mitochondrial dysfunction, and autophagy damage in RAW264.7 cells. Meanwhile, the expression of AQP1 was increased and the expression of P53 was decreased. In addition, Pifithrin-α (PIF; 15 μM), a P53 inhibitor, significantly aggravated ferroptosis, M1 polarization, mitochondrial dysfunction, and autophagy damage as well as up-regulation of AQP1 protein expression in LPS-induced RAW264.7 cells. Interestingly, this phenomenon was markedly alleviated by Kevetrin hydrochloride (70 μM), a P53 agonist. Mechanistically, silencing AQP1 significantly alleviated ferroptosis, M1 polarization, mitochondrial dysfunction, and autophagy damage by up-regulating the expression of P53 in LPS-stimulated RAW264.7 cells. Indeed, inhibition of P53 expression by PIF treatment dramatically reversed this effect on the basis of LPS+si-AQP1. Therefore, we concluded for the first time that AQP1 can promote ferroptosis, M1 polarization, mitochondrial dysfunction, and autophagy impairment by inhibiting the expression of P53 in LPS-stimulated RAW264.7 cells, and AQP1 or P53 may be considered as a crucial determiner that can regulate the biological behavior of RAW264.7 cells stimulated by LPS.

Klotho gene might antagonize ischemic injury in stroke rats by reducing the expression of AQP4 via P38MAPK pathway

ObjectivesThis study was aimed at exploring whether klotho improved neurologic function in rats with cerebral infarction by inhibiting P38 mitogen-activated protein kinase (MAPK) activation and thus down-regulating aquaporin 4 (AQP4). MethodsIn this study, we induced intracerebral Klotho overexpression in 6-week-old Sprague Dawley rats by injecting lentivirus carrying full-length rat Klotho cDNA into the lateral ventricle of the brain, followed by middle cerebral artery occlusion (MCAO) surgery after three days. Neurologic function was evaluated by neurological deficit scores. Infarct volume was assessed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. The expressions of Klotho, AQP4, and P38 MAPK were detected by Western blot and Immunofluorescence. Resultswhen rats were subjected to cerebral ischemia, their neurologic function was impaired, the protein expressions of klotho downregulated, the protein expressions of AQP4 and P38 MAPK increased, and the ratios of AQP4 and P-P38-positive area were significantly increased compared with the sham group rats. LV-KL-induced Klotho overexpression greatly improved neurobehavioral deficits and reduced infarct volume in MCAO rats. Klotho overexpression significantly reduced AQP4 and P38 MAPK pathway-related protein expression levels and the ratios of P-P38 and AQP4-positive area in MCAO rats. In addition, SB203580, a P38 MAPK signal pathway inhibitor, improved neurobehavioral deficits, reduced infarct volume, downregulated the expressions levels of AQP4 and P38 MAPK, and reduced the size of P-P38 and AQP4-positive area in MCAO rats. ConclusionKlotho could alleviate the infraction volume and neurological dysfunction in MCAO rats, and its mechanism may involve AQP4 expression downregulation by suppressing P38-MAPK activation.

Upregulation of ITGB6 in primary palmar hyperhidrosis.

The regulatory effect of integrin β6 (ITGB6) on sweat gland cells in primary palmar hyperhidrosis (PPH) remains unclear. This study investigated the involvement of ITGB6 in the pathogenesis of PPH. Sweat gland tissues were collected from PPH patients and healthy volunteers. The expression levels of ITGB6 in sweat gland tissues were detected with quantitative polymerase chain reaction (qPCR), western blot and immunohistochemical staining. Sweat gland cells were extracted from PPH patients, and identified with immunofluorescence staining of CEA and CK7. The expression of aquaporin 5 (AQP5) and Na-K-Cl cotransporter 1 (NKCC1) in primary sweat gland cells that overexpress ITGB6 were also detected. Through a series of bioinformatic methods, differentially expressed genes in sweat gland tissues were examined and validated via comparing PPH samples and controls. The key proteins and biological functions enriched in PPH were determined using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The ITGB6 was upregulated in sweat gland tissues of PPH patients compared to that of healthy volunteers. The CEA and CK7 were positively expressed in sweat gland cells extracted from PPH patients. The overexpression of ITGB6 upregulated AQP5 and NKCC1 protein expression in the sweat gland cells of PPH patients. A total of 562 differentially expressed mRNAs were identified using high-throughput sequencing (394 upregulated, 168 downregulated), which were mainly active in the chemokine and Wnt signaling pathways. After verification with qPCR and western blot, the overexpression of ITGB6 significantly upregulated CXCL3, CXCL5, CXCL10, and CXCL11, and downregulated Wnt2 mRNA and protein expression in sweat gland cells. The ITGB6 is upregulated in PPH patients. It may be involved in the pathogenesis of PPH by upregulating AQP5, NKCC1, CXCL3, CXCL5, CXCL10, and CXCL11, and downregulating Wnt2 expression in sweat glands.

Aquaporin 4 Mediates the Effect of Iron Overload on Hydrocephalus After Intraventricular Hemorrhage.

Iron overload plays an important role in hydrocephalus development following intraventricular hemorrhage (IVH). Aquaporin 4 (AQP4) participates in the balance of cerebrospinal fluid secretion and absorption. The current study investigated the role of AQP4 in the formation of hydrocephalus caused by iron overload after IVH. There were three parts to this study. First, Sprague-Dawley rats received an intraventricular injection of 100µl autologous blood or saline control. Second, rats had IVH and were treated with deferoxamine (DFX), an iron chelator, or vehicle. Third, rats had IVH and were treated with 2-(nicotinamide)-1,3,4-thiadiazole (TGN-020), a specific AQP4 inhibitor, or vehicle. Rats underwent T2-weighted and T2* gradient-echo magnetic resonance imaging to assess lateral ventricular volume and intraventricular iron deposition at 7, 14, and 28days after intraventricular injection and were then euthanized. Real-time quantitative polymerase chain reaction, western blot analysis, and immunofluorescence analyses were conducted on the rat brains to evaluate the expression of AQP4 at different time points. Hematoxylin and eosin-stained brain sections were obtained to assess the ventricular wall damage on day 28. Intraventricular injection of autologous blood caused a significant ventricular dilatation, iron deposition, and ventricular wall damage. There was increased AQP4 mRNA and protein expression in the periventricular tissue in IVH rats through day 7 to day 28. The DFX treatment group had a lower lateral ventricular volume and less intraventricular iron deposition and ventricular wall damage than the vehicle-treated group after IVH. The expression of AQP4 protein in periventricular tissue was also inhibited by DFX on days 14 and 28 after IVH. The use of TGN-020 attenuated hydrocephalus development after IVH and inhibited the expression of AQP4 protein in the periventricular tissue between day 14 and day 28 without a significant effect on intraventricular iron deposition or ventricular wall damage. AQP4 located in the periventricular area mediated the effect of iron overload on hydrocephalus after IVH.

TNF inhibits AQP2 expression via a miR137-dependent pathway

We previously showed that production of tumor necrosis factor-alpha (TNF) by the kidney, under normotensive, non-inflammatory conditions, inhibits expression of angiotensinogen in the proximal tubule and NKCC2 isoform A in the thick ascending limb of Henle’s loop via micro-RNA (miRNA)-dependent mechanisms. As miR-137 is a regulator of aquaporin-2 (AQP2) expression and TNF inhibits the expression of several extrarenal aquaporins, we tested the hypothesis that TNF inhibits AQP2 in the kidney via a miR-137-dependent mechanism. Target seed regions of mmu-miR-137 are conserved in the 3'-untranslated region (3’-UTR) of mouse AQP2 mRNA and luciferase assays showed that mmu-miR-137 directly targeted the 3’-UTR of mouse AQP2. Moreover, AQP2 expression was decreased by approximately 70% (P&lt;0.05) in primary renal inner medullary collecting duct (IMCD) cells transfected with a miRNA mimic of mmu-miR-137, indicating that miR-137 directly targets AQP2 mRNA in these cells. The decrease in AQP2 mRNA accumulation was accompanied by a 53% decrease (P&lt;0.05) in AQP2 protein expression when mmu-miR-137 was overexpressed in IMCD cells. Exposure of IMCD cells for 2 hr to 400 mosmol/kg H2O medium, made by increasing the osmolality by adding NaCl, increased mmu-miR-137 mRNA expression about 2-fold (P&lt;0.05) as determined by quantitative RT-PCR analysis. These conditions also increased TNF production approximately 2-fold (P&lt;0.05). To determine if the increase in mmu-miR-137 mRNA expression was related to the concomitant increase in TNF, cells were transfected with either a lentivirus construct to silence TNF or a control construct. The silencing construct, EGFP-shTNF-ex4, but not U6 control, decreased mmu-miR-137 mRNA expression in the IMCD cells exposed to 400 mosmol/kg H2O medium by approximately 63% (P&lt;0.05) indicating that TNF upregulates mmu-miR-137 mRNA expression in IMCD cells. The levels of miR-137 also increased approximately 2-fold (P&lt;0.05) in inner medulla isolated from age- and sex-matched male or female mice given 1% NaCl in the drinking water (HS) for 3 days. Six days after intrarenal injection of lentivirus EGFP-shTNF-ex4 to silence TNF, renal AQP2 mRNA levels increased approximately 3-fold (P&lt;0.05) in inner medullary tissue isolated from male or female mice given HS compared with their respective control groups. In addition, renal silencing of TNF in male or female mice given HS exhibited an increase in AQP2 protein expression the inner medulla compared with control group as determined by Western blot analysis. Similarly, EGFP-shTNF-ex4, but not U6, decreased miR-137 levels in the inner medulla in male or female mice given HS. Taken together, these data reveal a novel mechanism by which TNF mediates the inhibition of AQP2 via miR-137, indicating that miR-137 may be important for studying TNF-regulated effects involving the AQP2 pathway on renal water excretion and blood pressure regulation. NIH grants R01 HL133077 and HL153525 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Combination of Merocel sponge with Lipopolysaccharide to establish rat rhinosinusitis model

ObjectiveThe study aimed to investigate the feasibility of establishing rhinosinusitis model in rats combinated with Lipopolysaccharide (LPS) and merocel sponge. MethodsSD (Sprague Dawley) rats that underwent nasal obstruction using Merocel sponge packing, rats with LPS instillation alone, and rats with both nasal obstruction and LPS instillation were used to establish rat models of rhinosinusitis. After the models were established, the nasal symptoms of rats were recorded, the histopathological examination and Transmission Electron Microscopy (TME) of the sinus tissue were performed and the levels of Tumor Necrosis Factor-α (TNF-α), Interleukin-6 (IL-6) in the blood were also analyzed. The expressions of Aquaporin-5 (AQP5), Occludin, Toll-Like Receptor-4 (TLR4), Medullary differentiation factor 88 (MyD88) and phosphorylated (p)-p65 protein were detected by Western blot to evaluate the effect and mechanism of the experimental models. ResultsWe found that compared with the control group and LPS group, the sinusitis symptom scores in the Merocel sponge combined with LPS group were significantly increased; the respiratory epithelia of the maxillary sinus were degenerated, cilia were detached, and even inflammatory cell infiltration occurred; the levels of TNF-α and IL-6 were increased; the expression of AQP5 and Occludin protein was decreased; and the expressions of TLR4, MyD88, and p-p65 protein were increased. ConclusionFor the first time, we successfully established a rat rhinosinusitis model using Merocel sponge with LPS and explored the possible mechanism of LPS action.

Activating Blood Circulation, Anti-Inflammatory and Diuretic Effects of Leonurus japonicus Extract on a Rat Model of Trauma Blood Stasis and Its Phytochemical Profiling.

Leonurus japonicus Houtt. has been traditionally used to treat many ailments. This study evaluated the activating blood circulation, anti-inflammatory, and diuretic effects of L. japonicus extract (LJ) and identified its phytochemicals. In this work, the phytochemicals in LJ were identified using liquid chromatography mass spectrometry. Rats were randomly assigned to three groups (n=8): Control group was treated with saline, while the Model group (saline) and LJ group (426 mg/kg) had induced traumatic injury. All rats were treated with once by daily oral gavage for one week. The biochemical indices and protein expression were measured. Herein, 79 constituents were identified in LJ, which were effective in elevating body weight, food consumption, water intake, and urinary excretion volume, as well as in ameliorating traumatic muscle tissues in model rats. In addition, LJ prominently decreased the contents of plasma viscosity, platelet aggregation rate, thrombin time, prothrombin time, activated partial thromboplastin time, fibrinogen, thromboxane B2 (TXB2), TXB2/6-keto-prostaglandin F1α (6-keto-PGF1α), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitor 1 (PAI-1), PAI-1/tissue-type PA (t-PA), and PAI-1/u-PA, while significantly increasing antithrombin III, 6-keto-PGF1α, and t-PA contents. Furthermore, LJ notably inhibited tumor necrosis factor alpha, interleukin 6 (IL-6), IL-8, angiotensin II, antidiuretic hormone, aldosterone, aquaporin 1 (AQP1), AQP2, and AQP3 levels, and markedly elevating IL-10 and natriuretic peptide levels. Finally, LJ markedly reduced the protein expression of AQP1, AQP2, and AQP3 compared to the model group. Collectively, LJ possessed prominent activating blood circulation, anti-inflammatory, and diuretic effects, thus supporting the clinical application of L. japonicus.

Advanced Glycation End Products and Activation of Toll-like Receptor-2 and -4 Induced Changes in Aquaporin-3 Expression in Mouse Keratinocytes.

Prolonged inflammation and impaired re-epithelization are major contributing factors to chronic non-healing diabetic wounds; diabetes is also characterized by xerosis. Advanced glycation end products (AGEs), and the activation of toll-like receptors (TLRs), can trigger inflammatory responses. Aquaporin-3 (AQP3) plays essential roles in keratinocyte function and skin wound re-epithelialization/re-generation and hydration. Suberanilohydroxamic acid (SAHA), a histone deacetylase inhibitor, mimics the increased acetylation observed in diabetes. We investigated the effects of TLR2/TLR4 activators and AGEs on keratinocyte AQP3 expression in the presence and absence of SAHA. Primary mouse keratinocytes were treated with or without TLR2 agonist Pam3Cys-Ser-(Lys)4 (PAM), TLR4 agonist lipopolysaccharide (LPS), or AGEs, with or without SAHA. We found that (1) PAM and LPS significantly upregulated AQP3 protein basally (without SAHA) and PAM downregulated AQP3 protein with SAHA; and (2) AGEs (100 µg/mL) increased AQP3 protein expression basally and decreased AQP3 levels with SAHA. PAM and AGEs produced similar changes in AQP3 expression, suggesting a common pathway or potential crosstalk between TLR2 and AGEs signaling. Our findings suggest that TLR2 activation and AGEs may be beneficial for wound healing and skin hydration under normal conditions via AQP3 upregulation, but that these pathways are likely deleterious in diabetes chronically through decreased AQP3 expression.

Heterogeneity evaluation of multi-high b-value apparent diffusion coefficient on cerebral ischemia in MCAO rat.

To assess brain damage in a rat model of cerebral ischemia based on apparent diffusion coefficient (ADC) data obtained from multi-high b-values and evaluate the relationship between Aquaporin 4 (AQP4) expression and ADC. Thirty eight male Sprague-Dawley rats were randomized into two groups: (1) sham controls (n = 6) and (2) cerebral ischemia (successful model, n = 19). All rats underwent diffusion-weighted imaging (DWI) with both standard b-values and multi-high b-values (2,500-4,500 s/mm2) using a 3.0-T device. Standard ADC (ADCst) maps and multi-high b-value ADCs (ADCmh) were calculated, respectively. Aquaporin 4 expression was quantified using Western blot. Relative values of ADCst and ADCmh, AQP4 expression were compared between the sham group and the ischemia group. Correlations between ADC values and AQP4 expression were evaluated. At 0.5 h after suture insertion, the value of ADCmh on the lesion was obviously decreased, and there was no difference in lesion volume when compared with ADCst. After reperfusion, besides similar regions where ADCst values decreased, we also found additional large values on ADCmh within the cortex of the ipsilateral side or surrounding the lesion. The lesion evolution of the large value on ADCmh was quite different from other indicators. But the total ADCmh values were still significantly associated with ADCst. The AQP4 protein expression level was appreciably increased after middle cerebral artery occlusion (MCAO), but there was no correlation between AQP4 expression either with ADCmh or ADCst. We found the large values on ADCmh during the progression of cerebral infarction is varied, but there was no correlation between ADCmh values and AQP4 expression. ADCmh may indicate the heterogeneity of ischemia lesions, but the underlying pathological basis should be further explored.

Bumetanide Rescues Aquaporin-4 Depolarization via Suppressing β-Dystroglycan Cleavage and Provides Neuroprotection in Rat Retinal Ischemia-Reperfusion Injury

Aquaporin-4 (AQP4) regulates retinal water homeostasis and participates in retinal oedema pathophysiology. β-dystroglycan (β-DG) is responsible for AQP4 polarization and can be cleaved by matrix metalloproteinase-9 (MMP9). Retinal oedema induced by ischemia–reperfusion (I/R) injury is an early complication. Bumetanide (BU) has potential efficacy against cytotoxic oedema. Our study investigated the effects of β-DG cleavage on AQP4 and the roles of BU in a rat retinal I/R injury model. The model was induced by applying 110 mm Hg intraocular pressure to the anterior eye chamber. BU and U0126 (a selective ERK inhibitor) were intraperitoneally administered 15 and 30 min, respectively, before I/R induction. Rhodamine isothiocyanate extravasation detection, quantitative real-time PCR, transmission electron microscopy, hematoxylin-eosin staining, immunofluorescence staining, western blotting, and TUNEL staining were performed. AQP4 lost its polarization in the retinal perivascular domain as a result of β-DG cleavage. BU rescued AQP4 depolarization, suppressed AQP4 protein expression, attenuated retinal cytotoxic oedema, and downregulated β-DG and AQP4 mRNA expression. BU suppressed glial responses and mitochondria-mediated apoptotic protein expression, including that of Caspase-3 and Cyto C, raised the Bcl-2/Bax ratio, and lowered the number of apoptotic cells in the retina. Both BU and U0126 downregulated p-ERK and MMP9 expression. Thus, BU treatment suppressed β-DG cleavage, recovered AQP4 polarization partially via inhibiting ERK/MMP9 signaling pathway, and possess potential neuroprotective efficacy in the rat retinal ischemia–reperfusion injury model.

The Interplay between Aquaporin-1 and the Hypoxia-Inducible Factor 1α in a Lipopolysaccharide-Induced Lung Injury Model in Human Pulmonary Microvascular Endothelial Cells.

Aquaporin-1 (AQP1), a water channel, and the hypoxia-inducible factor 1α (HIF1A) are implicated in acute lung injury responses, modulating among others pulmonary vascular leakage. We hypothesized that the AQP1 and HIF1A systems interact, affecting mRNA, protein levels and function of AQP1 in human pulmonary microvascular endothelial cells (HPMECs) exposed to lipopolysaccharide (LPS). Moreover, the role of AQP1 in apoptosis and wound healing progression was examined. Both AQP1 mRNA and protein expression levels were higher in HPMECs exposed to LPS compared to untreated HPMECs. However, in the LPS-exposed HIF1A-silenced cells, the mRNA and protein expression levels of AQP1 remained unaltered. In the permeability experiments, a statistically significant volume increase was observed at the 360 s time-point in the LPS-exposed HPMECs, while LPS-exposed HIF1A-silenced HPMECs did not exhibit cell swelling, implying a dysfunctional AQP1. AQP1 did not seem to affect cell apoptosis yet could interfere with endothelial migration and/or proliferation. Based on our results, it seems that HIF1A silencing negatively affects AQP1 mRNA and protein expression, as well as AQP1 function, in the setting of lung injury.

AQP4-A25Q Point Mutation in Mice Depolymerizes Orthogonal Arrays of Particles and Decreases Polarized Expression of AQP4 Protein in Astrocytic Endfeet at the Blood-Brain Barrier.

Aquaporin-4 (AQP4) is characterized by the formation of orthogonal arrays of particles (OAPs) comprising its M1 and M23 isoforms in the plasma membrane. However, the biological importance of OAP formation is obscure. Here, we developed an OAP depolymerization male mouse model by transgenic knock-in of an AQP4-A25Q mutation. Analyses of the mutant brain tissue using blue native polyacrylamide gel electrophoresis, super-resolution imaging, and immunogold electron microscopy revealed remarkably reduced OAP structures and glial endfeet localization of the AQP4-A25Q mutant protein without effects on its overall mRNA and protein expression. AQP4A25Q/A25Q mice showed better survival and neurologic deficit scores when cerebral edema was induced by water intoxication or middle cerebral artery occlusion/reperfusion. The brain water content and swelling of pericapillary astrocytic endfeet processes in AQP4A25Q/A25Q mice were significantly reduced, functionally supporting decreased AQP4 protein expression at the blood-brain barrier. The infarct volume and neuronal damage were also reduced in AQP4A25Q/A25Q mice in the middle cerebral artery occlusion/reperfusion model. Astrocyte activation in the brain was alleviated in AQP4A25Q/A25Q mice, which may be associated with decreased cell swelling. We conclude that the OAP structure of AQP4 plays a key role in its polarized expression in astrocytic endfeet processes at the blood-brain barrier. Therefore, our study provided new insights into intervention of cerebral cellular edema caused by stroke and traumatic brain injury through regulating AQP4 OAP formation.SIGNIFICANCE STATEMENT Aquaporin-4 (AQP4) is characterized by orthogonal arrays of particles (OAPs) comprising the M1 and M23 isoforms in the membrane. Here, an OAP depolymerization male mouse model induced by AQP4-A25Q mutation was first established, and the functions of OAP depolymerization in cerebral edema have been studied. The results revealed that AQP4 lost its OAP structure without affecting AQP4 mRNA and protein levels in AQP4-A25Q mice. AQP4-A25Q mutation mice has neuroprotective effects on cerebral edema induced by water intoxication and middle cerebral artery occlusion/reperfusion through relieving the activation of astrocytes and suppressed microglia-mediated neuroinflammation. We concluded that the OAP structure of AQP4 plays a key role in its polarized expression in astrocytic endfeet processes at the blood-brain barrier. Therefore, our study provided new insights into intervention of cerebral cellular edema caused by stroke and traumatic brain injury through regulating AQP4 OAP formation.