This study was conducted to determine how Aquaporin 0 (AQP0) CatTohm natural mutation, which causes congenital cataracts and smaller eyes (microphthalmia) in mice, affects eye development, lens clarity, physiology, and mechanics. Eyes of wild-type (WT) and CatTohm heterozygous (Cat+/Tohm) mutant mice were examined; Cat+/Tohm showed significantly smaller eyes, indicating microphthalmia. Lenses from wild-type (WT), AQP0 heterozygous (AQP0+/-), and Cat+/Tohm mutant mice were imaged, and transparency was quantified. In Cat+/Tohm, transparency was greatly reduced compared with that of WT and AQP0+/- (P < 0.0001). Cell-to-cell adhesion studies using a cell aggregation assay on L-cells transfected with either WT or CatTohm AQP0 showed a significant decrease in cell aggregation in CatTohm AQP0 compared with WT-expressing cells (P < 0.0001). Lens stiffness was determined using compression stress testing, which revealed Cat+/Tohm lenses substantially less stiff than WT lenses (P < 0.001). Gap Junction Coupling (GJC) and Hydrostatic Pressure (HP) were assessed to evaluate the physiological effects on lens microcirculation and homeostasis; Cat+/Tohm lenses displayed significantly increased GJC (P < 0.0001) and reduced HP (P < 0.0001) relative to those in WT lenses. In summary, the results indicate that the mutation has altered cell-to-cell adhesion, biomechanics, GJC, and HP; together, these changes lead to the development of dominant cataracts and microphthalmia. AQP0 serves as a multifunctional protein in the lens, modulating transparency, fiber cell-to-fiber cell adhesion, GJC, HP, biomechanics, microcirculation, and homeostasis.

Purpose:The lens has a feedback control system made by transient receptor potential vanilloid 4 (TRPV4) and TRPV1 to sense pressure and activate ion transport to maintain intracellular hydrostatic pressure close to zero at the lens surface. We have recently reported the presence of another mechanosensitive channel, Piezo1, in the mouse lens epithelium. The purpose of the current study was to characterize the response to Piezo1 activation and test whether it is activated by changes in hydrostatic pressure. We also examined the radial pressure gradient following activation of either TRPV4 or Piezo1. Methods:Intracellular hydrostatic pressure in mouse lenses was measured with a microelectrode-manometer system. The pressure was determined at the lens surface, and the radial pressure gradient between the surface and the lens center was also determined. Results:Activation of Piezo1 by the agonist Yoda1 caused an immediate reduction of hydrostatic pressure in cells near the lens surface. The response was transient, and after ∼25 minutes pressure gradually recovered. Pressure recovery did not occur when TRPV1 was inhibited. TRPV4 activation by the agonist GSK1016790A (GSK) added after Yoda1 caused an additional reduction in pressure, suggesting that Piezo1 and TRPV4 are independent. In keeping with this notion, the inactivation of TRPV4 by the antagonist HC067047 did not prevent the pressure response to Yoda1. In some experiments, the pressure was elevated by subjecting the lens to partial Na/K pump inhibition by strophanthidin and simultaneously inhibiting the TRPV4-dependent feedback recovery loop. Under these conditions, pressure did not recover and remained elevated for over 2 hours even though it was confirmed that the activation of Piezo1 by Yoda1 was able to restore pressure to zero. The findings suggest that lens Piezo1 is not activated by positive pressure. The radial hydrostatic pressure gradient was determined in lenses exposed to Yoda1 in the presence of the TRPV1 antagonist A889425 added to prevent pressure recovery. The slope of the pressure gradient was reduced by ∼50% in the differentiating fiber (DF) region but central and surface pressure were unchanged. Under the same condition, the DF pressure gradient response was similar in lenses exposed to the TRPV4 agonist GSK. The pressure gradient responses to Yoda1 and GSK were absent in lenses subjected to inhibition of protein tyrosine phosphatase 1B (PTP1B) by a selective inhibitor, TCS401. Conclusions:The lens responds with a negative change of intracellular hydrostatic pressure in cells near the lens surface when Piezo1 is activated. Piezo1 and TRPV4 appear to be independent because they cause additive pressure reduction responses. However, unlike TRPV4, Piezo1 likely plays no role in feedback control of surface pressure because it does not respond to positive pressure changes. When TRPV1-dependent pressure recovery was blocked, activation of either Piezo1 or TRPV4 caused a pressure change that appears consistent with phosphatase-dependent activation of latent Na/K pumps in DF cells.

Genes regulate, maintain, and fine-tune the structural organization and physiological homeostasis of the lens and therefore influence lens transparency. RNAseq profiling of the mouse lens revealed that the Cd24a gene, which encodes the mucin-like GPI-linked membrane protein CD24, is abundantly expressed in the lens. Immunolocalization revealed that CD24 protein is abundant at mouse lens fiber cell membranes from early lens development into adulthood, while in adult human lenses, CD24 protein was detected in both the lens epithelium and fibers. Analysis of mice lacking the Cd24a gene revealed that the lens develops normally and is transparent with normal morphology until 2 months of age. However, older Cd24a null mice have smaller than normal lenses which exhibit abnormal fiber cell structure, actin filament disorganization, and refractive defects that lead to premature cataract development by 1 year of age. By integrating RNA sequencing, immunofluorescence, and magnetic resonance imaging, we found that the aquaporin 1 gene that regulates lens epithelial water transport is downregulated and the protein gradient that mediates the lenses refractive properties is altered in aged Cd24a null lenses that exhibit cataract. However, experiments on intracellular gap junction coupling and hydrostatic pressure in 2 month old lenses found no differences between control and Cd24a null lenses, suggesting that the later lens defects do not arise from primary issues with the lens circulation. Overall, our study found that CD24 plays a key role in maintaining the structural organization and refractive properties of the adult lens.

A natural AQP0 mutation, CatTohm, resulted in smaller eyes, and lenses with bilateral dominant cataracts in mice. Our objective was to characterize this mutation and explore the possible reasons for CatTohm causing dominant cataracts. We studied lens morphology, transparency, functional alterations and cytotoxicity. Lens morphology and nuclear fiber cell organization were severely affected. Water permeability (Pw) of oocytes expressing CatTohm-AQP0 cRNA (12 ± 2 μm/s) reduced markedly (P < 0001) compared with WT-cRNA-expressing oocytes (25 ± 2 μm/s); co-expression of both cRNAs decreased the Pw significantly (20 ± 3 μm/s; P < 0.001). Pw of membrane vesicles of heterozygous (16 ± 4 μm/s), or homozygous (7 ± 3 μm/s) fiber cells was considerably lower (P < 0.001) than that of the WT (37 ± 6 μm/s). The hydrogen peroxide permeability of the CatTohm lens was remarkably lesser (P < 0.0001) than that in the WT. The oxidative stress test revealed a significant (P < 0.001) increase in Reactive Oxygen Species in CatTohm lenses. In oocytes and cultured cells, transfected WT-AQP0 trafficked and expressed at the plasma membranes; mutant CatTohm-AQP0 protein remained in the cytoplasm, and partly co-localized with the WT-AQP0. Cells transfected with CatTohm-AQP0 showed more necrosis than apoptosis. The cultured cells expressing mutant AQP0, or ex vivo cultured lenses of CatTohm displayed a substantial (P < 0.001) rise in the discharge of lactate dehydrogenase in the culture medium, corroborating necrosis. A transgenic mouse lens expressing CatTohm mutant AQP0 along with the WT-AQP0 had more severe microphthalmia than that of CatTohm mouse. Overall, the CatTohm mutation exerted a dominant negative effect affecting protein localization and functionality, and causing cellular stress, necrosis, lens cataracts and microphthalmia.

Clouding of the eye lens or cataract is an age-related anomaly that affects middle-aged humans. Exploration of the etiology points to a great extent to oxidative stress due to different forms of reactive oxygen species/metabolites such as Hydrogen peroxide (H2O2) that are generated due to intracellular metabolism and environmental factors like radiation. If accumulated and left unchecked, the imbalance between the production and degradation of H2O2 in the lens could lead to cataracts. Our objective was to explore ex vivo the effects of H2O2 on lens physiology. We investigated transparency, intracellular pH (pH(i)), intercellular gap junction coupling (GJC), hydrostatic pressure (HP) and membrane water permeability after subjecting two-month-old C57 wild-type (WT) mouse lenses for 3 h or 8 h in lens saline containing 50 mu M H2O2; the results were compared with control lenses incubated in the saline without H2O2. There was a significant decrease in lens transparency in H2O2-treated lenses. In control lenses, pH(i) decreases from similar to 7.34 in the surface fiber cells to 6.64 in the center. Experimental lenses exposed to H2O2 for 8 h showed a significant decrease in surface pH (from 7.34 to 6.86) and central pH (from 6.64 to 6.56), compared to the controls. There was a significant increase in GJC resistance in the differentiating (12-fold) and mature (1.4-fold) fiber cells compared to the control. Experimental lenses also showed a significant increase in HP which was similar to 2-fold higher at the junction between the differentiating and mature fiber cells and similar to 1.5-fold higher at the center compared to these locations in control lenses; HP at the surface was 0 mm Hg in either type lens. Fiber cell membrane water permeability significantly increased in H2O2-exposed lenses compared to controls. Our data demonstrate that elevated levels of lens intracellular H2O2 caused a decrease in intracellular pH and led to acidosis which most likely uncoupled GJs, and increased AQP0-dependent membrane water permeability causing a consequent rise in HP. We infer that an abnormal increase in intracellular H2O2 could induce acidosis, cause oxidative stress, alter lens microcirculation, and lead to the development of accelerated lens opacity and age-related cataracts.

Purpose:Glutathione peroxidase 1 (GPX1) and catalase are expressed in the lens epithelial cells and cortical fiber cells, where they detoxify H2O2 to reduce oxidative stress, which is a major cause for cataractogenesis. We sought to find out, between these two enzymes, which is critical for transparency and homeostasis in the aging lens by investigating alterations in the lens's refractive property, transparency, and gap junction coupling (GJC) resistance.Methods:Wild-type (C57BL/6J), GPX1 knockout (GPX1-/-) and catalase knockout (CAT-/-) mice were used. Lens transparency was quantified using dark-field images and ImageJ software. For optical aberration evaluation, each lens was placed over a copper electron microscopy specimen grid; the grid image was captured through the lens using a digital camera attached to a dark-field binocular microscope. Optical aberrations were assessed by the quality of the magnified gridlines. Microelectrode-based intact lens intracellular impedance was measured to determine GJC resistance.Results:In contrast to wild-type (WT) and CAT-/- lenses, GPX1-/- lenses developed accelerated age-related cataracts. While two-month-old lenses were normal, at nine months of age, GPX1-/- mice started to show the development of abnormal optical distortion aberrations and loss of transparency. At 12 months of age, GPX1-/- lenses developed significant opacity and abnormal optical distortion aberrations compared to CAT-/- and WT (p<0.001); these aberrations gradually increased with age and matured into cataracts by 24 months of age. There was also a significant increase (p<0.001) in GJC resistance in the differentiating and mature fiber cells of GPX1-/- lenses at 12 months of age compared to that in similar areas of age-matched CAT-/- and WT lenses.Conclusions:Changes in the refractive and physiological properties of the lens occurred before cataract formation in GPX1-/- lenses but not in CAT-/- lenses. GPX1 is more critical than catalase for lens transparency, optical quality, and homeostasis in the aging lens under normal physiological conditions. GPX1 could be a promising therapeutic target for developing potential strategies to reduce adverse oxidative stress and delay/treat/prevent age-related cataracts.

The endothelial layer of the cornea plays a critical role in regulating its hydration by actively controlling fluid intake in the tissue via transporting the excess fluid out to the aqueous humor. A damaged corneal endothelial layer leads to perturbations in tissue hydration and edema, which can impact corneal transparency and visual acuity. We utilized a non-contact terahertz (THz) scanner designed for imaging spherical targets to discriminate between ex vivo corneal samples with intact and damaged endothelial layers. To create varying grades of corneal edema, the intraocular pressures of the whole porcine eye globe samples (n = 19) were increased to either 25, 35 or 45 mmHg for 4 h before returning to normal pressure levels at 15 mmHg for the remaining 4 h. Changes in tissue hydration were assessed by differences in spectral slopes between 0.4 and 0.8 THz. Our results indicate that the THz response of the corneal samples can vary according to the differences in the endothelial cell density, as determined by SEM imaging. We show that this spectroscopic difference is statistically significant and can be used to assess the intactness of the endothelial layer. These results demonstrate that THz can noninvasively assess the corneal endothelium and provide valuable complimentary information for the study and diagnosis of corneal diseases that perturb the tissue hydration.

Lens-specific beaded filament (BF) proteins CP49 and filensin interact with the C-terminus of the water channel protein Aquaporin 0 (AQP0). Previously we have reported that a C-terminally end-deleted AQP0-expressing transgenic mouse model AQP0ΔC/ΔC developed abnormal optical aberrations in the lens. This investigation was undertaken to find out whether the total loss of the BF structural proteins alter the optical properties of the lens and cause optical aberrations similar to those in AQP0ΔC/ΔC lenses; also, to map the changes in the optical quality as a function of age in the single or double BF protein knockouts as well as to assess whether there is any significant change in the water channel function of AQP0 in these knockouts. A double knockout mouse (2xKO) model for CP49 and filensin was developed by crossing CP49-KO and filensin-KO mice. Wild type, CP49-KO, filensin-KO, and 2xKO lenses at different ages, and AQP0ΔC/ΔC lenses at postnatal day-17 were imaged through the optical axis and compared for optical quality and focusing property. All three knockout models showed loss of transparency, and development of abnormal optical distortion aberration similar to that in AQP0ΔC/ΔC. Copper grid focusing by the lenses at 6, 9 and 12 months of age showed an increase in aberrations as age advanced. With progression in age, the grid images produced by the lenses of all KO models showed a transition from a positive barrel distortion aberration to a pincushion distortion aberration with the formation of three distinct aberration zones similar to those produced by AQP0ΔC/ΔC lenses. Water permeability of fiber cell membrane vesicles prepared from CP49-KO, filensin-KO and 2xKO models, measured using the osmotic shrinking method, remained similar to that of the wild type without any statistically significant alteration (P > 0.05). Western blotting and quantification revealed the expression of comparable quantities of AQP0 in all three BF protein KOs. Our study reveals that loss of single or both beaded filament proteins significantly affect lens refractive index gradient, transparency and focusing ability in an age-dependent manner and the interaction of BF proteins with AQP0 is critical for the proper functioning of the lens. The presence of BF proteins is necessary to prevent abnormal optical aberrations and maintain homeostasis in the aging lens.

Congenital cataracts are associated with gene mutations, yet the underlying mechanism remains largely unknown. Here we reported an embryonic chick lens model that closely recapitulates the process of cataract formation. We adopted dominant-negative site mutations that cause congenital cataracts, connexin, Cx50E48K, aquaporin 0, AQP0R33C, αA-crystallin, CRYAA R12C and R54C. The recombinant retroviruses containing these mutants were microinjected into the occlusive lumen of chick lenses at early embryonic development. Cx50E48K expression developed cataracts associated with disorganized nuclei and enlarged extracellular spaces. Expression of AQP0R33C resulted in cortical cataracts, enlarged extracellular spaces and distorted fiber cell organization. αA crystallin mutations distorted lens light transmission and increased crystalline protein aggregation. Together, retroviral expression of congenital mutant genes in embryonic chick lenses closely mimics characteristics of human congenital cataracts. This model will provide an effective, reliable in vivo system to investigate the development and underlying mechanism of cataracts and other genetic diseases.

High levels of reactive oxygen species such as hydrogen peroxide (H2O2) cause oxidative stress in the lens and lead to cataractogenesis. The present investigation was undertaken to find out whether the mammalian lens aquaporins (AQPs) 0, 1, and 5 perform H2O2 transport across the plasma membrane to reduce oxidative stress. Our in vitro cell culture and ex vivo lens experiments demonstrated that in addition to the established water transport role, mouse AQP0, AQP1 and AQP5 facilitate transmembrane H2O2 transport and function as peroxiporins. Human lens epithelial cells expressing AQP1, AQP5 and AQP8, when treated with 50 mM HgCl2 water channel inhibitor showed a significant reduction in H2O2 transport. Data obtained from the experiments involving H2O2-degrading enzyme glutathione peroxidase 1 (GPX1) knockout lenses showed H2O2 accumulation, suggesting H2O2 transport level by AQPs in the lens is regulated by GPX1. Under hyperglycemic conditions, there was an increased loss of transparency, and enhanced production and retention of H2O2 in AQP5(-/-) lenses compared to similarlytreated WT lenses. Overall, the results show that lens AQPs function as peroxiporins and cooperate with GPX1 to maintain lens H2O2 homeostasis to prevent oxidative stress, highlighting AQPs and GPX1 as promising therapeutic drug targets to delay/treat/prevent age-related lens cataracts. (C) 2020 Elsevier Inc. All rights reserved.

The purpose of this investigation was to find out whether C-terminally end-cleaved aquaporin 0 (AQP0), that is present predominantly in the lens mature fiber cells of the WT, functions as a water channel and a cell-to-cell adhesion (CTCA) protein in a knockin (KI) mouse model (AQP0ΔC/ΔC) that does not express intact AQP0. A genetically engineered KI mouse model, AQP0ΔC/ΔC, expressing only end-cleaved AQP0 was developed. This model expresses 1–246 amino acids of AQP0, instead of the full length 1–263 amino acids. Lens transparency of postnatal day 10 (P10) was analyzed qualitatively by dark field imaging. WT, AQP0+/− and AQP0+/ΔC lenses were transparent; AQP0−/− and AQP0ΔC/ΔC mouse lenses displayed loss of transparency. Lens fiber cell membrane vesicles (FCMVs) were prepared from wild type (WT), AQP0 heterozygous (AQP0+/−), AQP0 knockout (AQP0−/−), AQP0+/ΔC and AQP0ΔC/ΔC; water permeability (Pf) was measured using the osmotic shrinking method. CTCA assay was performed using adhesion-deficient L-cells and FCMVs prepared from the abovementioned genotypes. FCMVs of AQP0+/− and AQP0−/− showed a statistically significant reduction (P < 0.001) in Pf and CTCA compared to those of WT. AQP0+/ΔC and AQP0ΔC/ΔC FCMVs exhibited no statistically significant alteration (P > 0.05) in Pf compared to those of WT. However, CTCA of AQP0+/ΔC AQP0ΔC/ΔC FCMVs was significantly higher (P < 0.001) than that of WT FCMVs. Our experiments clearly show that C-terminally end-cleaved AQP0 can function both as a water channel and a CTCA molecule in the lens fiber cell membranes. Also, end-truncation plays an important role in increasing the CTCA between fiber cells.

This investigation was undertaken to find out whether the positive charges in the Extracellular Loops A (ELA) and C (ELC) of Aquaporin 0 (AQP0) are involved in lens fiber cell-to-cell adhesion (CTCA), and the possible mechanism of CTCA. AQP0 ELA or ELC was substituted with the corresponding AQP1 loop via Polymerase Chain Reaction. Positively charged arginine (R) and histidine (H) of mouse AQP0 ELA and ELC were substituted individually with glutamine (Q) to create R33Q, H40Q, R113Q and H122Q by mutagenesis. cRNA expression, immunostaining, Förster Resonance Energy Transfer (FRET) studies and protein analyses showed localization of all mutants except AQP0-AQP1ELC chimera (AQP0 ELC substituted with AQP1 ELC) at the plasma membrane. Osmotic Swelling Assay revealed comparable water permeability (Pf) among AQP0-AQP1ELA, R33Q, R113Q, and WT. CTCA assay demonstrated a significant reduction in adhesion in all mutants compared to the WT (14–73%) suggesting the importance of the conserved positively charged residues of ELA and ELC for adhesion. Studies involving AQP0-transfected L-cells, and lipid vesicles indicated that CTCA was due to the electrostatic interaction between the positively charged amino acids of AQP0 extracellular loops and the negative charges of the plasma membrane. Schematic models are provided to illustrate the mechanism.

Citation: Varadaraj K, Kumari S. Deletion of seventeen amino acids at the cterminal end of aquaporin 0 causes distortion aberration and cataract in the lenses of AQP0 mice. Invest Ophthalmol Vis Sci. 2019;60:858– 867. https://doi.org/10.1167/ iovs.18-26378 PURPOSE. Investigate the effects of the absence of 17 amino acids at the C-terminal end of Aquaporin 0 (AQP0) on lens transparency, focusing property, and homeostasis. METHODS. A knockin (KI) mouse model (AQP0) was developed to express AQP0 only as the end-cleaved form in the lens. For this, AQP0 was genetically engineered as C-terminally end-cleaved with amino acids 1 to 246, instead of the full length 1 to 263 of the wild type (WT). After verifying the KI integration into the genome and its expression, the mouse model was bred for several generations. AQP0 KI homozygous (AQP0) and heterozygous (AQP0þ/DC) lenses were imaged and analyzed at different developmental stages for transparency. Correspondingly, aberrations in the lens were characterized using the standard metal grid focusing method. Data were compared with age-matched WT, AQP0 knockout (AQP0 / ), and AQP0 heterozygous (AQP0þ/ ) lenses. RESULTS. AQP0 lenses were transparent throughout the embryonic development and until postnatal day 15 (P15) in contrast to age-matched AQP0 / lenses, which developed cataract at embryonic stage itself. However, there was distortion aberration in AQP0 lens at P5; after P15, cataract began to develop and progressed faster surpassing that of agematched AQP0 / lenses. AQP0þ/DC lenses were transparent even at the age of 1 year in contrast to AQP0þ/ lenses; however, there was distortion aberration starting at P15.

Purpose:We reported previously that aquaporin 0 (AQP0) modulates lens fiber cell gap junction (GJ) channel function. The present study was conducted to find out whether the C-terminal end of AQP0 is involved in this regulation.Methods:A mouse model, AQP0ΔC/ΔC, was genetically engineered to express AQP0 with 1-246 amino acids, without the normal intact AQP0 (1-263 amino acids) in the lens. Transparency and focusing of the lens were assessed. Intracellular impedance was measured to determine GJ coupling resistance. Intracellular hydrostatic pressure (HP) was also determined. Western blotting was performed to determine connexin (Cx46 and Cx50) expression levels.Results:At postnatal day 10, AQP0ΔC/ΔC mouse lenses relative to age-matched wild-type lenses showed loss of transparency and abnormal optical distortion; GJ coupling resistance increased in the differentiating (1.6-fold) and mature (8-fold) fiber cells; lens HP increased approximately 1.5-fold at the junction between the differentiating and mature fiber cells and approximately 2.0-fold in the center; there was no significant change (P > 0.05) in expression levels of Cx46 or Cx50.Conclusions:The increase in GJ coupling resistance was not associated with reduced connexin expression, suggesting either a reduction in the open probability or some physical change in plaque location. The increase in resistance was significantly greater than the increase in HP, suggesting less pressure-driven water flow through each open GJ channel. These changes may lead to a loss of transparency and abnormal optical distortion. Overall, our data demonstrate the C-terminal end of AQP0 is involved in modulating GJ coupling to maintain lens transparency and homeostasis.

The purP0se of this investigation was to find out whether C-terminally end-cleaved aquaP0rin 0 (AQP0), that is present predominantly in the lens mature fiber cells of the WT, functions as a water channel and a cell-to-cell adhesion (CTCA) protein in a knockin (KI) mouse model (AQP0(Delta C/Delta C)) that does not express intact AQP0. A genetically engineered KI mouse model, AQP0(Delta C/Delta C), expressing only end-cleaved AQP0 was developed. This model expresses 1-246 amino acids of AQP0, instead of the full length 1-263 amino acids. Lens transparency of P0stnatal day 10 (P10) was analyzed qualitatively by dark field imaging. WT, AQP0(+/-) and AQP0(+/Delta C) lenses were transparent; AQP0(-/-) and AQP0(Delta C/Delta C) mouse lenses displayed loss of transparency. Lens fiber cell membrane vesicles (FCMVs) were prepared from wild type (WT), AQP0 heterozygous (AQP0(+/-)), AQP0 knockout (AQP0(-/-)), AQP0(+/Delta C) and AQP0(Delta C/Delta C); water permeability (p(f)) was measured using the osmotic shrinking method. CTCA assay was performed using adhesion-deficient L-cells and FCMVs prepared from the abovementioned genotypes. FCMVs of AQP0(+/-) and AQP0(-/-) showed a statistically significant reduction (P < 0.001) in P-f and CTCA compared to those of WT. AQP0(Delta C/Delta C) and AQP0(Delta C/Delta C) FCMVs exhibited no statistically significant alteration (P > 0.05) in P-f compared to those of WT. However, CTCA of AQP0(+/Delta C) AQP0(Delta C/Delta C) FCMVs was significantly higher (P < 0.001) than that of WT FCMVs. Our experiments clearly show that C-terminally end-cleaved AQP0 can function both as a water channel and a CTCA molecule in the lens fiber cell membranes. Also, end-truncation plays an important role in increasing the CTCA between fiber cells. (C) 2019 Elsevier Inc. All rights reserved.

Purpose Investigate the effects of the absence of 17 amino acids at the C-terminal end of Aquaporin 0 (AQP0) on lens transparency, focusing property, and homeostasis. Methods A knockin (KI) mouse model (AQP0ΔC/ΔC) was developed to express AQP0 only as the end-cleaved form in the lens. For this, AQP0 was genetically engineered as C-terminally end-cleaved with amino acids 1 to 246, instead of the full length 1 to 263 of the wild type (WT). After verifying the KI integration into the genome and its expression, the mouse model was bred for several generations. AQP0 KI homozygous (AQP0ΔC/ΔC) and heterozygous (AQP0+/ΔC) lenses were imaged and analyzed at different developmental stages for transparency. Correspondingly, aberrations in the lens were characterized using the standard metal grid focusing method. Data were compared with age-matched WT, AQP0 knockout (AQP0−/−), and AQP0 heterozygous (AQP0+/−) lenses. Results AQP0ΔC/ΔC lenses were transparent throughout the embryonic development and until postnatal day 15 (P15) in contrast to age-matched AQP0−/− lenses, which developed cataract at embryonic stage itself. However, there was distortion aberration in AQP0ΔC/ΔC lens at P5; after P15, cataract began to develop and progressed faster surpassing that of age-matched AQP0−/− lenses. AQP0+/ΔC lenses were transparent even at the age of 1 year in contrast to AQP0+/− lenses; however, there was distortion aberration starting at P15. Conclusions A specific distribution profile of intact and end-cleaved AQP0 from the outer cortex to the inner nucleus is required in the lens for establishing refractive index gradient to enable proper focusing without aberrations and for maintaining transparency.

Aquaporins (AQPs), ordinarily regarded as water channels, have recently been shown to participate in other cellular functions such as cell-to-cell adhesion, cell migration, cell proliferation etc. The current investigation was undertaken to find out whether AQP5 water channel plays a role in corneal epithelial wound healing. Expression of AQP5 in mouse cornea and transfected Madin-Darby canine kidney (MDCK) cells was detected using immunofluorescence or EGFP tag. Cell migration and proliferation, the two major events in wound healing, were studied in vitro using cell culture scratch-wound healing model and cell proliferation assay, in vivo by conducting wound healing experiments on corneas of wild-type and AQP5 knockout mouse model and ex vivo on corneal epithelial cells isolated from wild type and AQP5 knockout mice. MDCK cells stably expressing AQP5 showed significantly higher levels of cell migration and proliferation compared to control cells. Likewise, corneal epithelial cells of wild type mouse with innate AQP5 exhibited faster wound healing than those of AQP5 knockout in vivo and under ex vivo culture conditions. In vitro, in vivo and ex vivo studies showed that presence of AQP5 improved cell migration, proliferation and wound healing. The data collected suggest that AQP5 plays a significant role in corneal epithelial wound healing.