Reticulated Retinoic Acid Synthesis is Implicated in the Pathogenesis of Dry Eye in Aqp5 Deficiency Mice.

Purpose: Abnormalities in aquaporins are implicated in the pathological progression of dry eye syndrome. Retinoic acid (RA) regulates cellular proliferation, differentiation, and apoptosis in the cornea, thereby being associated with dry eye disease (DED). The objective of this study is to explore the underlying mechanisms responsible for RA metabolic abnormalities in corneas lacking aquaporin 5 (AQP5). Methods: Dry eye (DE) models were induced via subcutaneous scopolamine hydrobromide. Aqp5 knockout (Aqp5-/-) mice and DE mice were utilized to assess corneal epithelial alterations. Tear secretion, goblet cell counts, and corneal punctate defects were evaluated. The impact of Aqp5 on RA-related enzymes and receptors was investigated using pharmacological RA or SR (A JunB inhibitor), a transcription factor JunB inhibitor, treatment in mouse corneal epithelial cells (CECs), or human corneal epithelial cells (HCECs). The HCECs and NaCl-treated HCECs underwent quantitative real-time PCR (qRT-PCR), immunofluorescent, Western blot, and TUNEL assays. The regulation of transcription factor JunB on Aldh1a1 was explored via ChIP-PCR. Results: Aqp5 and Aldh1a1 were reduced in both CECs of DE mice and NaCl-induced HCECs. Aqp5-/- mice exhibited DE phenotype and reduced Aldh1a1. RA treatment reduced apoptosis, promoted proliferation, and improved the DE phenotype in Aqp5-/- mice. JunB enrichment in the Aldh1a1 promoter was identified by ChIP-PCR. SR significantly increased Aldh1a1 expression, Ki67, and ΔNp63-positive cells, and decreased TUNEL-positive cells in CECs and HCECs. Conclusions: Our findings demonstrated the downregulation of Aqp5 expression and aberrant RA metabolism in DE conditions. Knockout of Aqp5 resulted in reduced production of RA through activation of JunB, subsequently leading to the manifestation of DE symptoms.

McH-lpr/lpr-RA1 mice: A novel spontaneous mouse model of autoimmune sialadenitis.

Many studies of the autoimmune disease Sjögren's syndrome have been performed using spontaneous mouse models. In the present study, we describe the characteristics of McH/lpr-RA1 mice and propose their use as a novel murine model of autoimmune sialadenitis. The McH/lpr-RA1 mouse is a recombinant congenic strain derived from generation F54 or more of MRL-Faslpr x (MRL- Faslpr x C3H- Faslpr) F1. We show for the first time that this mouse spontaneously develops autoimmune sialadenitis and vasculitis in submandibular gland tissues. Sialadenitis was accompanied by extensive inflammatory cell infiltration and tissue destruction. Immunohistochemical studies revealed that the salivary gland lesions strongly expressed four sialadenitis-related molecules: SSA and SSB (autoantigens of Sjögren's syndrome), gp91phox (an accelerator of reactive oxygen species production) and single strand DNA (a marker of apoptotic cells). In contrast, expression of aquaporin-5 (AQP5), which stimulates salivary secretion was weak or negligible. Statistical correlation analyses indicated that the apoptosis of salivary gland cells provoked by oxidative stress contributed to the severe sialadenitis and reduced expression of AQP5. Our study has demonstrated that McH/lpr-RA1 mice spontaneously develop the pathognomonic features of autoimmune sialadenitis and thus could be used as a new animal model of Sjögren's syndrome.

Different Prognostic Implications of Aquaporin-1 and Aquaporin-5 Expression among Different Histological Types of Ovarian Carcinoma.

The aquaporins (AQPs) are a family of transmembrane water channel proteins that are distributed in various human tissues. Recent studies have suggested that AQP expression correlates with various aspects of cancer biology that determine the aggressiveness of different cancers. Ovarian carcinoma is one of the most lethal gynecological cancers. Some studies have suggested that AQPs are expressed in ovarian carcinoma, and are associated with cancer cell growth and migration. In this study, we immunohistochemically evaluated the expression of AQP1, 3, 5, and 9 in a total of 300 ovarian carcinomas using tissue microarrays. In our analyses of correlations between aquaporin expression and overall survival, high AQP5 expression was significantly associated with poorer prognosis (P = 0.029). For AQP1, the low expression group trended towards poorer prognosis than the high expression group, but the difference was not statistically significant. When ovarian carcinomas were divided by histological types, high AQP5 expression correlated with poorer prognosis in serous carcinoma (P = 0.015), and low AQP1 expression correlated with poorer prognosis in clear cell carcinomas (P = 0.0055). By contrast, high AQP1 expression correlated with poorer prognosis in mucinous carcinoma (P = 0.0001) and endometrioid carcinoma (P = 0.021). Our studies suggest that AQPs can be useful prognostic markers in ovarian carcinoma, but their correlation with prognosis depends on the histological type of ovarian carcinoma.

Effect of low radiation dose on the expression and location of aquaporins in rat submandibular gland.

Head and neck cancers are common in several regions of the world and the treatment usually includes radiotherapy. This treatment can generate adverse effects to the salivary flow, with a relationship between the dose and the damage caused. Salivary gland cells are highly permeable to water and therefore, they express aquaporins (AQPs). This study analyzed changes in the expression and location of these proteins and identified morphological changes induced by low radiation in rat submandibular gland. Female rats were divided into control and irradiated groups. Immunohistochemistry analysis allowed confirming the presence of AQP1 in the blood vessel endothelium. Intense and steady labelling granules were also observed in the cytoplasm of submandibular gland ductal cells. In addition, there was AQP5 positive labelling in ductal cells delimiting the lumen of intercalated duct, in the cytoplasm and membrane of acinar cells. Finally, the decrease of AQP labelling in irradiated animal glands validated their radiosensitivity. Thus, the decrease in AQP1 protein levels in the endothelium and AQP5 in gland ductal cells of irradiated animals may have hindered the removal of water from the lumen of ductal cells, inducing a delay in water absorption and triggering a slight lumen increase.

P2X7R: independent modulation of aquaporin 5 expression in CdCl2-injured alveolar epithelial cells.

The expression of aquaporin 5 in alveolar epithelial type I cells under conditions of cadmium-induced injury has not yet been discovered. We investigated the effect of the P2X7R agonist BzATP under this condition, since P2X7R is involved in altered regulation of aquaporin 5 in pulmonary fibrosis. CdCl2/TGF-ß1 treatment of lung epithelial MLE-12 cells was leading to increasing P2X7R, and aquaporin 5 protein levels. The aquaporin 5 expression was P2X7R-independent in MLE-12 cells under cadmium, as was shown in blocking experiments with oxATP. Further, the expression of both proteins increased after 24 h CdCl2/TGF-ß1 treatment of precision-cut lung slices, but decreased after 72 h. Using immunohistochemistry, the activation of the P2X7R with the agonist BzATP modulated the aquaporin 5 immunoreactivity in the alveolar epithelium of precision-cut lung slices from wild-type but not from P2X7R knockout mice. Similarly, aquaporin 5 protein was reduced in BzATP-treated immortal lung epithelial E10 cells. Surprisingly, untreated alveolar epithelial type II cells of P2X7R knockouts exhibited a pronounced apical immunoreactivity in addition to the remaining alveolar epithelial type I cells. BzATP exposure did not alter this distribution pattern, but increased the number of apoptotic alveolar epithelial type II cells in wild-type lung slices.

Heat shock exerts anticancer effects on liver cancer via autophagic degradation of aquaporin 5.

Previous studies described that the expression of aquaporin 5 (AQP5) was altered in tumors of various organs. AQP5 is attracting attention as a new cancer therapeutic target. In the present study, heat shock-induced changes in AQP5 expression were evaluated by immunofluorescent staining (IF) and western blotting (WB) of liver cancer cells. AQP5 knockdown experiments or a heat shock treatment were conducted, and their effects on cell volume, proliferation, cell cycle, the activity of apoptosis and migration/invasion were compared. Cycloheximide (CHX) chase experiments and double IF of AQP5 and light chain 3B (LC3B) were performed to investigate the mechanisms underlying changes in AQP5 expression. The results showed that IF and WB revealed decrease in AQP5 expression on cellular membranes and in the cytoplasm of heated cells. AQP5 knockdown and heat shock similarly decreased cell volume, suppressed migration/invasion and proliferation, and induced early apoptosis and partial G0/G1 arrest. CHX chase experiments revealed that heat shock accelerated the degradation of AQP5, which was rescued under CHX and the autophagy inhibitor, bafilomycin A1 (BafA1). Double IF showed the co-localization of AQP5 and LC3B on BafA1-treated heated cells. In conclusion, we demonstrated that heat shock decreased AQP5 on cellular membranes and in the cytoplasm by activating autophagic degradation, and heat shock and AQP5 knockdown exerted similar anticancer effects, suggesting that heat shock exerts anticancer effects via the autophagic degradation of AQP5.

Vasoactive Intestinal Peptide Protects Salivary Glands against Structural Injury and Secretory Dysfunction via IL-17A and AQP5 Regulation in a Model of Sjögren Syndrome.

OBJECTIVE: Sjögren syndrome (SS) is an autoimmune disease involving exocrine glands. Currently, drugs that can improve both abnormal immunity and exocrine gland function are needed. The study aimed to investigate the effect and mechanism of vasoactive intestinal peptide (VIP) on the immune response and exocrine gland function in SS. METHODS: We investigated the effects of VIP on the immune response and secretory function of submandibular glands using NOD mice, and analyzed the expression of IL-17A and AQP5 (aquaporin 5). The submandibular gland cells from healthy 8-day-old Sprague-Dawley rats were used to observe the influence of VIP on AQP5 expression. RESULTS: Our study shows that treatment with VIP in an SS mouse model could not only reduce the immune injury to exocrine glands but also improve the secretory function of these glands. Furthermore, VIP was shown to improve the abnormal immune status by downregulating IL-17A expression in the exocrine glands. It also enhanced the secretory function of exocrine glands by upregulating AQP5 expression. CONCLUSIONS: Using a model of SS, we found that VIP could not only modulate the immune response but also affect exocrine gland function, and that these therapeutic effects were associated with IL-17A and AQP5 regulation.

AQP5 Variants Affect Tumoral Expression of AQP5 and Survival in Patients with Early Breast Cancer.

BACKGROUND: Our previous study showed the association of AQP5 upregulation with cancer proliferation and migration in breast cancer cell lines and with unfavorable prognosis in patients with early breast cancer (EBC). In the current study, we analyzed the association of AQP5 variants or their haplotypes with AQP5 expression and their prognostic impact for survival in patients with EBC. METHODS: Three AQP5 polymorphisms (rs74091166, rs3736309, and rs1964676) were selected based on the SNP database and genotyped using the Sequenom MassARRAY in 374 out of 447 patients with EBC in whom AQP5 expression had been investigated in our previous study. RESULTS: The allele frequencies of the selected variants in the current study were similar to those from Asian data previously reported. In a univariate analysis, both rs74091166 and rs1964676 were statistically associated with survival as a dominant model of minor allele. Moreover, a multivariate survival analysis revealed that the CC genotype of rs1964676 is an independent prognostic marker of survival in EBC patients, regardless of stage, tumor subtype, and adjuvant treatment [hazard ratio = 0.399, 0.384, and 0.205; p = 0.021, 0.027, and 0.016 for disease-free survival (DFS), distant DFS, and disease-specific survival, respectively]. In particular, the CT/TT genotype of rs1964676 showed an association with strong expression of AQP5 (58.6 vs. 26.0%; p = 0.001), without any associations with clinical or pathological characteristics including tumor subtype, stage, or histologic grade. CONCLUSION: The current study suggests AQP5 rs1964676 as a new potential prognostic marker in patients with EBC involved in AQP5 expression.

Increased aquaporin 1 and 5 membrane expression in the lens epithelium of cataract patients.

In this work we have analyzed the expression levels of the main aquaporins (AQPs) expressed in human lens epithelial cells (HLECs) using 112 samples from patients treated with cataract surgery and 36 samples from individuals treated with refractive surgery, with transparent lenses as controls. Aquaporin-1 (AQP1) is the main AQP, representing 64.1% of total AQPs in HLECs, with aquaporin-5 (AQP5) representing 35.9% in controls. A similar proportion of each AQP in cataract was found. Although no differences were found at the mRNA level compared to controls, a significant 1.65-fold increase (p=0.001) in AQP1protein expression was observed in HLECs from cataract patients, with the highest differences being found for nuclear cataracts (2.1-fold increase; p<0.001). A similar trend was found for AQP5 (1.47-fold increase), although the difference was not significant (p=0.161). Moreover we have shown increased membrane AQP5 protein expression in HLECs of patients with cataracts. No association of AQP1 or AQP5 expression levels with age or sex was observed in either group. Our results suggest regulation of AQP1 and AQP5 at the post-translational level and support previous observations on the implication of AQP1 and 5 in maintenance of lens transparency in animal models. Our results likely reflect a compensatory response of the crystalline lens to delay cataract formation by increasing the water removal rate.

Emodin ameliorates acute lung injury induced by severe acute pancreatitis through the up-regulated expressions of AQP1 and AQP5 in lung.

The present study investigates the ameliorating effects of emodin on acute lung injury (ALI) induced by severe acute pancreatitis (SAP). An ALI rat model was constructed by sodium ursodeoxycholate and they were divided into four groups: SHAM, ALI, emodin and dexamethasone (DEX) (n=24 per group). Blood samples and lung tissues were collected 6, 12 and 24 hours after the induction of SAP-associated ALI. Lung wet/dry ratio, blood gases, serum amylase and tumor necrosis factor-α (TNF-α) were measured at each time point. The expressions of AQP1 and AQP5 in lung tissue were detected by immunohistochemical staining, western blotting and real-time PCR. As the results show, there were no statistical differences in the levels of serum amylase, lung wet/dry ratio, blood gases indexes, serum TNF-α and pathological changes between emodin and DEX groups. However, significant differences were observed when compared with the ALI group. AQP1 and AQP5 expressions were significantly increased and lung oedemas were alleviated with the treatment of emodin and DEX. The expressions of AQP1 and AQP5 were significantly decreased in SAP-associated ALI rats. Emodin up-regulated the expression of AQP1 and AQP5, it could reduce pulmonary oedema and ameliorate SAP-induced ALI. Regulations on AQP1 and AQP5 expression had a great value in clinical application.

Pistacia integerrima ameliorates airway inflammation by attenuation of TNF-α, IL-4, and IL-5 expression levels, and pulmonary edema by elevation of AQP1 and AQP5 expression levels in mouse model of ovalbumin-induced allergic asthma.

BACKGROUND: Natural products are considered as an essential source for the search of new drugs. Pistacia integerrima galls (PI) have been used for the treatment of asthma and cough in traditional system of medicine. AIM/HYPOTHESIS: Current study investigates the immunomodulatory and anti-inflammatory activities of P. integerrima in mouse model of ovalbumin-induced allergic asthma. METHODS: Mice were intraperitoneally sensitized and subsequently challenged intranasally with ovalbumin to induce allergic asthma. Experimental group mice were treated with methanol extract of P. integerrima extract (200mg/kg b. w.) and Methylprednisolone (MP) (15mg/kg b. w.) for 07 consecutive days, alongside intranasal challenge. Lung tissues were stained with Hematoxyline and Eosin (H & E), and Periodic Acid-Schiff (PAS) stains for histopathological evaluation. Lung wet/dry weight ratio was measured as an index of lung tissue edema. Albumin was injected in the right ear 24h before sacrificing the mice and difference of weight was taken as a degree of delayed type hypersensitivity (DTH). mRNA expression levels of TNF-α, IL-4, IL-5, Aquaporin-1 (AQP1), and AQP5 were evaluated using reverse transcription polymerase chain reaction (RT-PCR) followed by gel electrophoresis. RESULTS: The data showed both PI extract and MP significantly alleviated DTH and nearly normalized total leukocyte count and differential leukocyte count in both blood and BALF. We found significantly suppressed goblet cell hyperplasia and inflammatory cell infiltration after treatment with both PI extract and MP. Expression levels of TNF-α, IL-4, and IL-5 were also found significantly reduced after treatment with both PI extract and MP, which might have resulted in the amelioration of airway inflammation. Current study displayed that both PI extract and MP significantly decreased lung wet/dry ratio, suggesting reduction in pulmonary edema. RT-PCR analysis showed significant increase in AQP1 and AQP5 expression levels after treatment with both PI extract and MP, which might have caused the alleviation of pulmonary edema. CONCLUSION: Our study displays that P. integerrima possesses significant anti-asthmatic activity which may be attributed to reduction in TNF-α, IL-4, and IL-5 expression levels, and increase in AQP1 and AQP5 expression levels.

Up-regulation of Store-operated Ca2+ Entry and Nuclear Factor of Activated T Cells Promote the Acinar Phenotype of the Primary Human Salivary Gland Cells.

The signaling pathways involved in the generation and maintenance of exocrine gland acinar cells have not yet been established. Primary human salivary gland epithelial cells, derived from salivary gland biopsies, acquired an acinar-like phenotype when the [Ca(2+)] in the serum-free medium (keratinocyte growth medium, KGM) was increased from 0.05 mm (KGM-L) to 1.2 mm (KGM-H). Here we examined the mechanism underlying this Ca(2+)-dependent generation of the acinar cell phenotype. Compared with cells in KGM-L, those in KGM-H display enhancement of Orai1, STIM1, STIM2, and nuclear factor of activated T cells 1 (NFAT1) expression together with an increase in store-operated Ca(2+) entry (SOCE), SOCE-dependent nuclear translocation of pGFP-NFAT1, and NFAT-dependent but not NFκB-dependent gene expression. Importantly, AQP5, an acinar-specific protein critical for function, is up-regulated in KGM-H via SOCE/NFAT-dependent gene expression. We identified critical NFAT binding motifs in the AQP5 promoter that are involved in Ca(2+)-dependent up-regulation of AQP5. These important findings reveal that the Ca(2+)-induced switch of salivary epithelial cells to an acinar-like phenotype involves remodeling of SOCE and NFAT signaling, which together control the expression of proteins critically relevant for acinar cell function. Our data provide a novel strategy for generating and maintaining acinar cells in culture.

Aquaporin 5 expression is frequent in prostate cancer and shows a dichotomous correlation with tumor phenotype and PSA recurrence.

Aquaporin 5 (AQP5) is an androgen-regulated member of a family of small hydrophobic integral transmembrane water channel proteins regulating cellular water homeostasis and growth signaling. To evaluate its clinical impact and relationship with key genomic alterations in prostate cancer, AQP5 expression was analyzed by immunohistochemistry on a tissue microarray containing 12427 prostate cancers. The analysis revealed weak to moderate immunostaining in normal prostate epithelium. In prostate cancers AQP5 staining levels were more variable and also included completely negative and highly overexpressing cases. Negative, weak, moderate, and strong AQP5 staining was found in 25.0%, 32.5%, 32.5%, and 10.0% of 10239 interpretable tumors. Comparison of AQP5 expression levels with tumor characteristics showed a dichotomous pattern with both high and low staining levels being linked to unfavorable tumor phenotype. AQP5 was negative in 28%, 23%, 24%, and 35% of tumors with Gleason score ≤3 + 3, 3 + 4, 4 + 3 and ≥4 + 4, while the rate of strongly positive cases continuously increased from 7.0% over 10.0% and 12.0% to 13.0% in cancers with Gleason score ≤3 + 3, 3 + 4, 4 + 3 and ≥4 + 4. AQP5 expression was also related to ERG positivity and phosphatase and tensin homolog (PTEN) deletion (P < .0001 each). Strong AQP5 positivity was seen in 15.5% of ERG-positive and 5.8% of ERG-negative cancers (P < .0001) as well as in 14.7% of cancers with PTEN deletion and 9.4% of cancers without PTEN deletion. Remarkably, both negativity and strong positivity of AQP5 were linked to unfavorable disease outcome. This was however only seen in subgroups defined by TMPRSS2-ERG fusion and/or PTEN deletion. In summary, AQP5 can be both overexpressed and lost in subgroups of prostate cancers. Both alterations are linked to unfavorable outcome in molecularly defined cancer subgroups. It is hypothesized that this dichotomous role of AQP5 is due to two highly different mechanisms as to how the protein can influence cancer cells, that is, hydraulic motility regulation and Ras/MAPK pathway activation.

AQP1, AQP5, Bcl-2 and p16 in pharyngeal squamous cell carcinoma.

OBJECTIVE: This study aimed to link expression patterns of AQP1, AQP5, Bcl-2 and p16 to clinicopathological characteristics of oro-hypopharyngeal squamous cell carcinomas. METHODS: Immunohistochemical expression of AQP1, AQP5, Bcl-2 and p16 was investigated in 107 consecutive oro-hypopharyngeal squamous cell carcinoma cases. Molecular interrelationship and correlations with clinicopathological parameters and survival were computed. RESULTS: AQP1 was expressed exclusively by a subgroup of basaloid-like squamous cell carcinomas. AQP5 was detected in 25.2 per cent of the samples, showing significant association with the absence of p16 and Bcl-2 (p = 0.018; p = 0.010). In multivariate analysis, overexpression of p16 was significantly correlated with favourable overall survival (p = 0.014). CONCLUSION: AQP5 defined a subset of patients with Bcl-2-negative and p16-negative tumours with a poor clinical outcome. AQP1 was found to be a marker of a subgroup of aggressive basaloid-like squamous cell carcinomas. These findings suggest that AQP1 and AQP5 are interesting candidates for further studies on risk group classification and personalised treatment of oro-hypopharyngeal squamous cell carcinomas.

Aquaporin 1 and 5 expression decreases during human intervertebral disc degeneration: Novel HIF-1-mediated regulation of aquaporins in NP cells.

Objectives of this study were to investigate whether AQP1 and AQP5 expression is altered during intervertebral disc degeneration and if hypoxia and HIF-1 regulate their expression in NP cells. AQP expression was measured in human tissues from different degenerative grades; regulation by hypoxia and HIF-1 was studied using promoter analysis and gain- and loss-of-function experiments. We show that both AQPs are expressed in the disc and that mRNA and protein levels decline with human disease severity. Bioinformatic analyses of AQP promoters showed multiple evolutionarily conserved HREs. Surprisingly, hypoxia failed to induce promoter activity or expression of either AQP. While genomic chromatin immunoprecipitation showed limited binding of HIF-1α to conserved HREs, their mutation did not suppress promoter activities. Stable HIF-1α suppression significantly decreased mRNA and protein levels of both AQPs, but HIF-1α failed to induce AQP levels following accumulation. Together, our results demonstrate that AQP1 and AQP5 expression is sensitive to human disc degeneration and that HIF-1α uniquely maintains basal expression of both AQPs in NP cells, independent of oxemic tension and HIF-1 binding to promoter HREs. Diminished HIF-1 activity during degeneration may suppress AQP levels in NP cells, compromising their ability to respond to extracellular osmolarity changes.

Hyperoxia stimulates the transdifferentiation of type II alveolar epithelial cells in newborn rats.

Supplemental oxygen treatment in preterm infants may cause bronchopulmonary dysplasia (BPD), which is characterized by alveolar simplification and vascular disorganization. Despite type II alveolar epithelial cell (AEC II) damage being reported previously, we found no decrease in the AEC II-specific marker, surfactant protein C (SP-C), in the BPD model in our previous study. We thus speculated that AEC II injury is not a unique mechanism of BPD-related pulmonary epithelial repair dysfunction and that abnormal transdifferentiation can exist. Newborn rats were randomly assigned to model (85% oxygen inhalation) and control groups (room air inhalation). Expressions of AEC I (aquaporin 5, T1α) and AEC II markers (SP-C, SP-B) were detected at three levels: 1) in intact lung tissue, 2) in AEC II isolated from rats in the two groups, and 3) in AEC II isolated from newborn rats, which were further cultured under either hyperoxic or normoxic conditions. In the model group, increased AEC I was observed at both the tissue and cell level, and markedly increased transdifferentiation was observed by immunofluorescent double staining. Transmission electron microscopy revealed morphological changes in alveolar epithelium such as damaged AECs, a fused air-blood barrier structure, and opened tight junctions in the model group. These findings indicate that transdifferentiation of AECs is not suppressed but rather is increased under hyperoxic treatment by compensation; however, such repair during injury cannot offset pulmonary epithelial air exchange and barrier dysfunction caused by structural damage to AECs.

Effects of total glucosides of peony on AQP-5 and its mRNA expression in submandibular glands of NOD mice with Sjogren's syndrome.

OBJECTIVE: The aim of this study was to observe the effects of total glucosides of peony (TGP) on pathological change, Aquaporin-5 (AQP-5) and its mRNA expression in submandibular glands of non-obese diabetic (NOD) mice with Sjogren's Syndrome, to investigate its regulation on secretion of salivary glands. MATERIALS AND METHODS: 40 NOD mice were randomly divided into model group, TGP group, hydroxychloroquine group, combination group (n = 10). For TGP group, the mice were intragastrically administrated with 0.4 ml TGP dilution per day in accordance with 300 g/kg dose; for hydroxychloroquine group, the mice were intragastrically administrated with 0.4 ml hydroxychloroquine per day in accordance with 60 mg/kg dose; for the combination group, the mice were intragastrically administrated with 0.4 ml TGP dilution and 0.4 ml hydroxychloroquine. 8 weeks later, the mice were sacrificed, and submandibular glands were collected by anatomy. Pathological changes of submandibular gland were observed under a light microscope; AQP-5 protein in submandibular glands was detected by immunohistochemical staining; and AQP-5 mRNA expression in submandibular glands was detected by RT-PCR. RESULTS: The lymphocytic infiltration score of model mice was significantly higher than that of other groups. The pathological morphology and score of NOD mice were significantly improved after administration, and the combination group was superior to the hydroxychloroquine group and TGP group (p < 0.05). AQP-5 mRNA expression level of the model group was lower than other groups (p < 0.05); the expression levels in the TGP group and the combination group were higher than the hydroxychloroquine group (p < 0.05). CONCLUSIONS: TGP may improve pathological damage of submandibular glands of NOD mouse with Sjogren's syndrome by upregulating AQP-5 and its mRNA expression in submandibular glands.

Expression of aquaporin 1, 5 and 9 in the ovarian follicles of cycling and early pregnant pigs.

Aquaporins (AQPs) are water channel proteins responsible for water homeostasis and important for proper functioning of all body systems, including reproductive structures. This study was designed to determine their localization and quantitative changes in the pig ovary during different stages of the estrous cycle and early pregnancy. The expression of AQP 1, 5 and 9 proteins was determined by immunocytochemistry and Western blot analyses. AQP1 was found in the plasma membranes of capillary endothelium, AQP5 - in the plasma membranes of granulosa cells of developing follicles and flattened follicle cells of the primordial follicles, and AQP9 - in granulosa cells of the developing follicles. In the cyclic pigs, the expression of AQP1 and 5 proteins was the highest on Days 18-20, but did not change significantly between Days 2-4, 10-12 and 14-16 of the cycle. In pregnant pigs (Days 14-16 and 30-32), the expression of AQP1 and 5 did not change and was similar to that observed during Days 10-12 and 14-16. In turn, AQP9 expression did not change between all studied periods. In conclusion, studied AQP are localized in different cells populations, the endothelial and granulosa cells, and AQP1 and 5 seem to be crucial for follicular development in pigs.

Reciprocity in the developmental regulation of aquaporins 1, 3 and 5 during pregnancy and lactation in the rat.

Milk secretion involves significant flux of water, driven largely by synthesis of lactose within the Golgi apparatus. It has not been determined whether this flux is simply a passive consequence of the osmotic potential between cytosol and Golgi, or whether it involves regulated flow. Aquaporins (AQPs) are membrane water channels that regulate water flux. AQP1, AQP3 and AQP5 have previously been detected in mammary tissue, but evidence of developmental regulation (altered expression according to the developmental and physiological state of the mammary gland) is lacking and their cellular/subcellular location is not well understood. In this paper we present evidence of developmental regulation of all three of these AQPs. Further, there was evidence of reciprocity since expression of the rather abundant AQP3 and less abundant AQP1 increased significantly from pregnancy into lactation, whereas expression of the least abundant AQP5 decreased. It would be tempting to suggest that AQP3 and AQP1 are involved in the secretion of water into milk. Paradoxically, however, it was AQP5 that demonstrated most evidence of expression located at the apical (secretory) membrane. The possibility is discussed that AQP5 is synthesized during pregnancy as a stable protein that functions to regulate water secretion during lactation. AQP3 was identified primarily at the basal and lateral membranes of the secretory cells, suggesting a possible involvement in regulated uptake of water and glycerol. AQP1 was identified primarily at the capillary and secretory cell cytoplasmic level and may again be more concerned with uptake and hence milk synthesis, rather than secretion. The fact that expression was developmentally regulated supports, but does not prove, a regulatory involvement of AQPs in water flux through the milk secretory cell.