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.

Efficient generation of a stable CHO-K1 cell line overexpressing the human water channel aquaporin-5 as tool to generate therapeutic antibodies.

Aquaporins (AQPs) are a family of water permeable channels expressed on the plasma membrane with AQP5 being the major channel expressed in several human tissues including salivary and lacrimal glands. Anti-AQP5 autoantibodies have been observed in patients with Sjögren's syndrome who are characterised by dryness of both salivary and lacrimal glands, and they have been implicated in the underlying mechanisms of glandular dysfunction. AQP5 is formed by six transmembrane helices linked with three extracellular and two intracellular loops. Develop antibodies against membrane protein extracellular loops can be a challenge due to the difficulty in maintaining these proteins as recombinant in their native form. Therefore, in this work we aimed to generate an efficient stable-transfected cell line overexpressing human AQP5 (CHO-K1/AQP5) to perform primarily cell-based phage display biopanning experiments to develop new potential recombinant antibodies targeting AQP5. We also showed that the new CHO-K1/AQP5 cell line can be used to study molecular mechanisms of AQP5 sub-cellular trafficking making these cells a useful tool for functional studies.

Retroductal dexamethasone administration promotes the recovery from obstructive and inflammatory salivary gland dysfunction.

Introduction: Salivary gland dysfunction, often resulting from salivary gland obstruction-induced inflammation, is a prevalent condition. Corticosteroid, known for its anti-inflammatory and immunomodulatory properties, is commonly prescribed in clinics. This study investigates the therapeutic implications and potential side effects of dexamethasone on obstructive sialadenitis recovery using duct ligation mice and salivary gland organoid models. Methods: Functional and pathological changes were assessed after administering dexamethasone to the duct following deligation 2 weeks after maintaining ligation of the mouse submandibular duct. Additionally, lipopolysaccharide- and tumor necrosis factor-induced salivary gland organoid inflammation models were established to investigate the effects and underlying mechanisms of action of dexamethasone. Results: Dexamethasone administration facilitated SG function restoration, by increasing salivary gland weight and saliva volume while reducing saliva lag time. Histological evaluation revealed, reduced acinar cell atrophy and fibrosis with dexamethasone treatment. Additionally, dexamethasone suppressed pro-inflammatory cytokines IL-1ß and TNF expression. In a model of inflammation in salivary gland organoids induced by inflammatory substances, dexamethasone restored acinar markers such as AQP5 gene expression levels, while inhibiting pro-inflammatory cytokines TNF and IL6, as well as chemokines CCL2, CXCL5, and CXCL12 induction. Macrophages cultured in inflammatory substance-treated media from salivary gland organoid cultures exhibited pro-inflammatory polarization. However, treatment with dexamethasone shifted them towards an anti-inflammatory phenotype by reducing M1 markers (Tnf, Il6, Il1b, and Cd86) and elevating M2 markers (Ym1, Il10, Cd163, and Klf4). However, high-dose or prolonged dexamethasone treatment induced acino-ductal metaplasia and had side effects in both in vivo and in vitro models. Conclusions: Our findings suggest the effectiveness of corticosteroids in treating obstructive sialadenitis-induced salivary gland dysfunction by regulating pro-inflammatory cytokines.

Application of anti-vascular endothelial growth factor antibody restores the function of saliva secretion in a type 2 diabetes mouse model.

OBJECTIVES: Xerostomia, a common complication of type 2 diabetes, leads to an increased risk of caries, dysphagia, and dysgeusia. Although anti-vascular endothelial growth factor (VEGF) antibodies, such as ranibizumab (RBZ), have been used to treat diabetic retinopathy, their effects on the salivary glands are unknown. This study evaluated the effects of RBZ on salivary glands to reduce inflammation and restore salivary function in a mouse model of type 2 diabetes. METHODS: Male KK-Ay mice with type 2 diabetes (10-12 weeks old) were used. The diabetes mellitus (DM) group received phosphate-buffered saline, while the DM + RBZ group received an intraperitoneal administration of RBZ (100 µg/kg) 24 h before the experiment. RESULTS: Ex vivo perfusion experiments showed a substantial increase in salivary secretion from the submandibular gland (SMG) in the DM + RBZ group. In addition, the mRNA expression levels of TNF-α and IL-1ß were considerably lower in this group. In contrast, those of aquaporin 5 were substantially higher in the DM + RBZ group, as revealed by quantitative reverse transcription PCR. Furthermore, the number of lymphocyte infiltration spots in the SMG was notably lower in the DM + RBZ group. Finally, intracellular Ca2+ signaling in acinar cells was considerably higher in the DM + RBZ group than that in the DM group. CONCLUSION: Treating a type 2 diabetic mouse model with RBZ restored salivary secretion through its anti-inflammatory effects.

Downregulated GPX4 in salivary gland epithelial cells contributes to salivary secretion dysfunction in Sjogren's syndrome via lipid ROS/pSTAT4/AQP5 axis.

Sjogren's syndrome (SS) is an autoimmune disease characterized by dysfunction of exocrine glands, such as salivary glands. However, the molecular mechanism of salivary secretion dysfunction in SS is still unclear. Given the significance of glutathione peroxidase 4 (GPX4) in cellular redox homeostasis, we hypothesized that dysregulation of GPX4 may play a pivotal role in the pathogenesis of salivary secretion dysfunction observed in SS. The salivary gland of SS patients and the SS mouse model exhibited reduced expression of the ferroptosis inhibitor GPX4 and the important protein aquaporin 5 (AQP5), which is involved in salivary secretion. GPX4 overexpression upregulated and GPX4 knockdown downregulated AQP5 expression in salivary gland epithelial cells (SGECs) and salivary secretion. Bioinformatics analysis of GSE databases from SS patients' salivary glands revealed STAT4 as a key intermediary regulator between GPX4 and AQP5. A higher level of nuclear pSTAT4 was observed in the salivary gland of the SS mouse model. GPX4 overexpression inhibited and GPX4 knockdown promoted STAT4 phosphorylation and nuclear translocation in SGECs. CHIP assay confirmed the binding of pSTAT4 within the promoter of AQP5 inhibiting AQP5 transcription. GPX4 downregulation accumulates intracellular lipid ROS in SGECs. Lipid ROS inhibitor ferrostatin-1 treatment during in vitro and in vivo studies confirmed that lipid ROS activates STAT4 phosphorylation and nuclear translocation in SGECs. In summary, the downregulated GPX4 in SGECs contributes to salivary secretion dysfunction in SS via the lipid ROS/pSTAT4/AQP5 axis. This study unraveled novel targets to revitalize the salivary secretion function in SS patients.

Botulinum toxin type a blocks aquaporin 5 trafficking by decreasing synaptosomal-associated protein 23 in submandibular acinar cells.

The trafficking of aquaporin 5 (AQP5) is critical for salivary secretion. Synaptosomal-associated protein 23 (SNAP23) is an important regulator in the process of membrane fusion. However, the role of SNAP23 on AQP5 trafficking has not been explored. Botulinum toxin type A (BoNT/A) is a bacterial toxin that effectively treats sialorrhea. We previously reported that BoNT/A induced AQP5 redistribution in cultured acinar cells, but the mechanism remained unclear. In this study, SNAP23 was predominantly localized to the plasma membrane of acinar cells in the rat submandibular gland (SMG) and colocalized with AQP5 at the apical membrane of acinar cells. In stable GFP-AQP5-transfected SMG-C6 cells, the acetylcholine receptor agonist carbachol (CCh) induced trafficking of AQP5 from intracellular vesicles to the apical membrane. Furthermore, SNAP23 knockdown by siRNA significantly inhibited CCh-induced AQP5 trafficking, whereas this inhibitory effect was reversed by SNAP23 re-expression, indicating that SNAP23 was essential in AQP5 trafficking. More importantly, BoNT/A inhibited salivary secretion from SMGs, and the underlying mechanism involved that BoNT/A blocked CCh-triggered AQP5 trafficking by decreasing SNAP23 in acinar cells. Taken together, these results identified a crucial role for SNAP23 in AQP5 trafficking and provided new insights into the mechanism of BoNT/A in treating sialorrhea and thereby a theoretical basis for clinical applications.

Methazolamide Reduces the AQP5 mRNA Expression and Immune Cell Migration-A New Potential Drug in Sepsis Therapy?

Sepsis is a life-threatening condition caused by the dysregulated host response to infection. Novel therapeutic options are urgently needed and aquaporin inhibitors could suffice as aquaporin 5 (Aqp5) knockdown provided enhanced sepsis survival in a murine sepsis model. Potential AQP5 inhibitors provide sulfonamides and their derivatives. In this study, we tested the hypothesis that sulfonamides reduce AQP5 expression in different conditions. The impact of sulfonamides on AQP5 expression and immune cell migration was examined in cell lines REH and RAW 264.7 by qPCR, Western blot and migration assay. Subsequently, whether furosemide and methazolamide are capable of reducing AQP5 expression after LPS incubation was investigated in whole blood samples of healthy volunteers. Incubation with methazolamide (10-5 M) and furosemide (10-6 M) reduced AQP5 mRNA and protein expression by about 30% in REH cells. Pre-incubation of the cells with methazolamide reduced cell migration towards SDF1-α compared to non-preincubated cells to control level. Pre-incubation with methazolamide in PBMCs led to a reduction in LPS-induced AQP5 expression compared to control levels, while furosemide failed to reduce it. Methazolamide appears to reduce AQP5 expression and migration of immune cells. However, after LPS administration, the reduction in AQP5 expression by methazolamide is no longer possible. Hence, our study indicates that methazolamide is capable of reducing AQP5 expression and has the potential to be used in sepsis prophylaxis.

Goji Berry Juice Prevents Tumor Necrosis Factor Alpha-Induced Xerostomia in Human Salivary Gland Cells.

Sjögren's syndrome (SS) is an autoimmune disorder characterized by oral dryness that is primarily attributed to tumor necrosis factor alpha (TNF-α)-mediated reduction in saliva production. In traditional Chinese medicine, goji berries are recognized for their hydrating effect and are considered suitable to address oral dryness associated with Yin deficiency. In the present study, we used goji berry juice (GBJ) to investigate the potential preventive effect of goji berries on oral dryness caused by SS. Pretreatment of human salivary gland cells with GBJ effectively prevented the decrease in aquaporin-5 (AQP-5) mRNA and protein levels induced by TNF-α. GBJ also inhibited histone H4 deacetylation and suppressed the generation of intracellular reactive oxygen species (ROS). Furthermore, GBJ pretreatment reserved mitochondrial membrane potential and suppressed the upregulation of Bax and caspase-3, indicating that GBJ exerted an antiapoptotic effect. These findings suggest that GBJ provides protection against TNF-α in human salivary gland cells and prevents the reduction of AQP-5 expression on the cell membrane. Altogether, these results highlight the potential role of GBJ in preventing oral dryness caused by SS.

Involvement of aquaporin 5 and Na-K-2Cl cotransporter 1 in the pathogenesis of primary focal hyperhidrosis: evidence from the primary sweat gland cell culture.

People with primary focal hyperhidrosis (PFH) usually have an overactive sympathetic nervous system, which can activate the sweat glands through the chemical messenger of acetylcholine. The role of aquaporin 5 (AQP5) and Na-K-2Cl cotransporter 1 (NKCC1) in PFH is still unknown. The relative mRNA and protein levels of AQP5 and NKCC1 in the sweat gland tissues of three subtypes of patients with PFH (primary palmar hyperhidrosis, PPH; primary axillary hyperhidrosis, PAH; and primary craniofacial hyperhidrosis, PCH) were detected with real-time PCR (qPCR) and Western blot. Primary sweat gland cells from healthy controls (NPFH-SG) were incubated with different concentrations of acetylcholine, and the relative mRNA and protein expression of AQP5 and NKCC1 were also detected. NPFH-SG cells were also transfected with si-AQP5 or shNKCC1, and acetylcholine stimulation-induced calcium transients were assayed with Fluo-3 AM calcium assay. Upregulated AQP5 and NKCC1 expression were observed in sweat gland tissues, and AQP5 demonstrated a positive Pearson correlation with NKCC1 in patients with PPH (r = 0.66, P < 0.001), patients with PAH (r = 0.71, P < 0.001), and patients with PCH (r = 0.62, P < 0.001). Upregulated AQP5 and NKCC1 expression were also detected in primary sweat gland cells derived from three subtypes of patients with PFH when compared with primary sweat gland cells derived from healthy control. Acetylcholine stimulation could induce the upregulated AQP5 and NKCC1 expression in NPFH-SG cells, and AQP5 or NKCC1 inhibitions attenuated the calcium transients induced by acetylcholine stimulation in NPFH-SG cells. The dependence of ACh-stimulated calcium transients on AQP5 and NKCC1 expression may be involved in the development of PFH.NEW & NOTEWORTHY The dependence of ACh-stimulated calcium transients on AQP5 and Na-K-2Cl cotransporter 1 (NKCC1) expression may be involved in the development of primary focal hyperhidrosis (PFH).

Regulation of Axon Guidance by Slit2 and Netrin-1 Signaling in the Lacrimal Gland of Aqp5 Knockout Mice.

Purpose: Dry eye disease (DED) is multifactorial and associated with nerve abnormalities. We explored an Aquaporin 5 (AQP5)-deficiency-induced JunB activation mechanism, which causes abnormal lacrimal gland (LG) nerve distribution through Slit2 upregulation and Netrin-1 repression. Methods: Aqp5 knockout (Aqp5-/-) and wild-type (Aqp5+/+) mice were studied. LGs were permeabilized and stained with neuronal class III ß-tubulin, tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), and calcitonin gene-related peptide (CGRP). Whole-mount images were acquired through tissue clearing and 3D fluorescence imaging. Mouse primary trigeminal ganglion (TG) neurons were treated with LG extracts and Netrin-1/Slit2 neutralizing antibody. Transcription factor (TF) prediction and chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) experiments verified the JunB binding and regulatory effect on Netrin-1 and Slit2. Results: Three-dimensional tissue and section immunofluorescence showed reduced LG nerves in Aqp5-/- mice, with sympathetic and sensory nerves significantly decreased. Netrin-1 was reduced and Slit2 increased in Aqp5-/- mice LGs. Aqp5+/+ mice LG tissue extracts (TEs) promoted Aqp5-/- TG neurons axon growth, but Netrin-1 neutralizing antibody (NAb) could inhibit that promotion. Aqp5-/- mice LG TEs inhibited Aqp5+/+ TG axon growth, but Slit2 NAb alleviated that inhibition. Furthermore, JunB, a Netrin-1 and Slit2 TF, could bind them and regulate their expression. SR11302, meanwhile, reversed the Netrin-1 and Slit2 shifts caused by AQP5 deficiency. Conclusions: AQP5 deficiency causes LG nerve abnormalities. Persistent JunB activation, the common denominator for Netrin-1 suppression and Slit2 induction, was found in Aqp5-/- mice LG epithelial cells. This affected sensory and sympathetic nerve fibers' distribution in LGs. Our findings provide insights into preventing, reversing, and treating DED.

Ablation of AQP5 gene in mice leads to olfactory dysfunction caused by hyposecretion of Bowman's gland.

Smell detection depends on nasal airflow, which can make absorption of odors to the olfactory epithelium by diffusion through the mucus layer. The odors then act on the chemo-sensitive epithelium of olfactory sensory neurons (OSNs). Therefore, any pathological changes in the olfactory area, for instance, dry nose caused by Sjögren's Syndrome (SS) may interfere with olfactory function. SS is an autoimmune disease in which aquaporin (AQP) 5 autoantibodies have been detected in the serum. However, the expression of AQP5 in olfactory mucosa and its function in olfaction is still unknown. Based on the study of the expression characteristics of AQP5 protein in the nasal mucosa, the olfaction dysfunction in AQP5 knockout (KO) mice was found by olfactory behavior analysis, which was accompanied by reduced secretion volume of Bowman's gland by using in vitro secretion measure system, and the change of acid mucin in nasal mucus layer was identified. By excluding the possibility that olfactory disturbance was caused by changes in OSNs, the result indicated that AQP5 contributes to olfactory functions by regulating the volume and composition of OE mucus layer, which is the medium for the dissolution of odor molecules. Our results indicate that AQP5 can affect the olfactory functions by regulating the water supply of BGs and the mucus layer upper the OE that can explain the olfactory loss in the patients of SS, and AQP5 KO mice might be used as an ideal model to study the olfactory dysfunction.

TGF-ß1 Triggers Salivary Hypofunction via Attenuating Protein Secretion and AQP5 Expression in Human Submandibular Gland Cells.

Aging-related salivary gland degeneration usually causes poor oral health. Periductal fibrosis frequently occurs in the submandibular gland of the elderly. Transforming growth factor ß1 (TGF-ß1) is the primary driving factor for fibrosis, which exhibits an increase in the fibrotic submandibular gland tissue. This study aimed to investigate the effects of TGF-ß1 on the human submandibular gland (HSG) cell secretory function and its influences on aquaporin 5 (AQP5) expressions and distribution. We found that TGF-ß1 reduces the protein secretion amount of HSG and leads to the abundance alteration of 151 secretory proteins. Data are available via ProteomeXchange with the identifier PXD043185. The majority of HSG secretory proteins (84.11%) could be matched to the human saliva proteome. Meanwhile, TGF-ß1 enhances the expression of COL4A2, COL5A1, COL7A1, COL1A1, COL2A1, and α-SMA, hinting that TGF-ß1 possesses the potential to drive HSG fibrosis-related events. Besides, TGF-ß1 also attenuates the AQP5 expression and its membrane distribution in HSGs. The percentage for TGF-ß1-induced AQP5 reduction (52.28%) is much greater than that of the TGF-ß1-induced secretory protein concentration reduction (16.53%). Taken together, we concluded that TGF-ß1 triggers salivary hypofunction via attenuating protein secretion and AQP5 expression in HSGs, which may be associated with TGF-ß1-driven fibrosis events in HSGs.

PAI1 inhibits the pathogenesis of primary focal hyperhidrosis by targeting CHRNA1.

BACKGROUND: Primary focal hyperhidrosis (PFH) may be attributed to the up-regulation of the cholinergic receptor nicotinic alpha 1 subunit (CHRNA1) in eccrine glands. Plasminogen activator inhibitor-1 (PAI1, encoded by SERPINE1) is reported to inhibit the expression of CHRNA1, while the role of PAI1 in hyperhidrosis is unknown. METHODS: Serpine1 KO mice, Serpine1-Tg mice, and wild type BALB/c mice were intraperitoneally injected with pilocarpine hydrochloride to induce PFH. Cisatracurium (CIS, antagonist of CHRNA1) or PAI-039 (small-molecule inhibitor of PAI1) was pre-administrated before the induction of hyperhidrosis. On the other hand, Chrna1-expressing AAV was constructed and administered to Serpine1-Tg mice with hydrochloride stimulation. Hydrochloride-related biomarkers, such as acetylcholine (ACH) in the serum, calcium voltage-gated channel subunit alpha1 C (CACNA1C), and aquaporin 5 (AQP5) in sweat glands of mice were assayed with ELISA, RT-PCR, and Western blot. RESULTS: The administration of PAI-039 or Pai1 knock-out increased Chrna1 expression, sweat secretion, and hydrochloride-related biomarkers (ACH, CACNA1C, and AQP5) expression. On the other hand, CIS administration diminished the strengthened hyperhidrosis phenotype induced by Pai1 knock-out with decreased sweat gland secretion. CONCLUSION: PAI1 inhibits CHRNA1-mediated hydrochloride-induced hyperhidrosis, with decreased sweat gland secretion and diminished ACH, AQP5, and CACNA1C expression. These results indicate the potential to utilize PAI1 to alleviate PFH.

Upregulation of ITGB6 in primary palmar hyperhidrosis.

BACKGROUND: The regulatory effect of integrin ß6 (ITGB6) on sweat gland cells in primary palmar hyperhidrosis (PPH) remains unclear. OBJECTIVES: This study investigated the involvement of ITGB6 in the pathogenesis of PPH. MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Role of aquaporin 5 and glandular blood flow in the acetylcholine-induced secretion of saliva in rats.

To clarify the role of the aquaporin 5 (AQP5) in salivary secretion, we evaluated acetylcholine (ACh)-induced secretion in Sprague-Dawley (SD) rats, rats expressing a low level of AQP5 protein (AQP5/low SD) which developed from SD rats, and Wistar/ST rats. The salivary secretion in AQP5/low SD rats in response to infusions of low-dose ACh (60-120 nmol/min) was 27-42% of that in SD rats. By contrast, Wistar/ST rats exhibited comparable secretion to that of SD rats in response to low-doses ACh, despite their low-level expression of AQP5. Experiments using spectrofluorometry and RT-PCR demonstrated no differences in the ACh-induced Ca2+ responses or the mRNA expression of muscarinic receptor, Cl- channel, or cotransporter between these strains. These findings imply that factors other than the function of salivary acinar cells regulates the secretion in response to weak stimuli. Monitoring of the hemodynamics in the submandibular gland revealed that low-doses ACh induced different patterns of the fluctuations in the blood flow in these strains. The blood flow decreased below the resting level in AQP5/low SD rats, but remained mostly above the resting level in Wistar/ST rats. The present study reveals that the contribution of AQP5-dependent transport of water is altered by stimulus intensity and blood flow.

Aquaporin 5 maintains lens transparency by regulating the lysosomal pathway using circRNA.

The lens is transparent, non-vascular, elastic and wrapped in a transparent capsule. The lens oppacity of AQP5-/- mice was increased more than that of wild-type (AQP5+/+ ) mice. In this study, we explored the potential functional role of circular RNAs (circRNAs) and transcription factor HSF4 in lens opacity in aquaporin 5 (AQP5) knockout (AQP5-/- ) mice. Autophagy was impaired in the lens tissues of AQP5-/- mice. Autophagic lysosomes in lens epithelial cells of AQP5-/- mice were increased compared with AQP5+/+ mice, based on analysis by transmission electron microscopy. The genetic information of the mice lens was obtained by high-throughput sequencing, and then the downstream genes were analysed. A circRNA-miRNA-mRNA network related to lysosomal pathway was constructed by the bioinformatics analysis of the differentially expressed circRNAs. Based on the prediction of the TargetScan website and the validation by dual luciferase reporter assay and RNA immunoprecipitation-qPCR, we found that circRNA (Chr16: 33421321-33468183+) inhibited the function of HSF4 by sponging microRNA (miR-149-5p), and it downregulated the normal expression of lysosome-related mRNAs. The accumulation of autophagic lysosome may be one of the reasons for the abnormal development of the lens in AQP5-/- mice.

Aquaporin-5 Dynamic Regulation.

Aquaporin-5 (AQP5), belonging to the aquaporins (AQPs) family of transmembrane water channels, facilitates osmotically driven water flux across biological membranes and the movement of hydrogen peroxide and CO2. Various mechanisms have been shown to dynamically regulate AQP5 expression, trafficking, and function. Besides fulfilling its primary water permeability function, AQP5 has been shown to regulate downstream effectors playing roles in various cellular processes. This review provides a comprehensive overview of the current knowledge of the upstream and downstream effectors of AQP5 to gain an in-depth understanding of the physiological and pathophysiological processes involving AQP5.

Loss of aquaporin 5 contributes to the corneal epithelial pathogenesis via Wnt/ß-catenin pathway.

AQP5 plays a crucial role in maintaining corneal transparency and the barrier function of the cornea. Here, we found that in the corneas of Aqp5-/- mice at older than 6 months, loss of AQP5 significantly increased corneal neovascularization, inflammatory cell infiltration, and corneal haze. The results of immunofluorescence staining showed that upregulation of K1, K10, and K14, and downregulation of K12 and Pax6 were detected in Aqp5-/- cornea and primary corneal epithelial cells. Loss of AQP5 aggravated wound-induced corneal neovascularization, inflammation, and haze. mRNA sequencing, western blotting, and qRT-PCR showed that Wnt2 and Wnt6 were significantly decreased in Aqp5-/- corneas and primary corneal epithelial cells, accompanied by decreased aggregation in the cytoplasm and nucleus of ß-catenin. IIIC3 significantly suppressed corneal neovascularization, inflammation, haze, and maintained corneal transparent epithelial in Aqp5-/- corneas. We also found that pre-stimulated Aqp5-/- primary corneal epithelial cells with IIIC3 caused the decreased expression of K1, K10, and K14, the increased expression of K12, Pax6, and increased aggregation in the cytoplasm and nucleus of ß-catenin. These findings revealed that AQP5 may regulate corneal epithelial homeostasis and function through the Wnt/ß-catenin signaling pathway. Together, we uncovered a possible role of AQP5 in determining corneal epithelial cell fate and providing a potential therapeutic target for corneal epithelial dysfunction.