Regulation of water flow in the ocular lens: new roles for aquaporins

The ocular lens is an important determinant of overall vision quality whose refractive and transparent properties change throughout life. The lens operates an internal microcirculation system that generates circulating fluxes of ions, water and nutrients that maintain the transparency and refractive properties of the lens. This flow of water generates a substantial hydrostatic pressure gradient which is regulated by a dual feedback system that uses the mechanosensitive channels TRPV1 and TRPV4 to sense decreases and increases, respectively, in the pressure gradient. This regulation of water flow (pressure) and hence overall lens water content, sets the two key parameters, lens geometry and the gradient of refractive index, which determine the refractive properties of the lens. Here we focus on the roles played by the aquaporin family of water channels in mediating lens water fluxes, with a specific focus on AQP5 as a regulated water channel in the lens. We show that in addition to regulating the activity of ion transporters, which generate local osmotic gradients that drive lens water flow, the TRPV1/4‐mediated dual feedback system also modulates the membrane trafficking of AQP5 in the anterior influx pathway and equatorial efflux zone of the lens. Since both lens pressure and AQP5‐mediated water permeability ( PH2O${P_{{{\mathrm{H}}_{\mathrm{2}}}{\mathrm{O}}}}$ ) can be altered by changes in the tension applied to the lens surface via modulating ciliary muscle contraction we propose extrinsic modulation of lens water flow as a potential mechanism to alter the refractive properties of the lens to ensure light remains focused on the retina throughout life.


Introduction
The transparent and refractive properties of the ocular lens are key components of the visual pathway that contribute to overall vision quality by ensuring that light is correctly focused on the retina (Donaldson et al., 2017(Donaldson et al., , 2022)).The ability of the lens to focus light on the retina is in turn a product of a specialized cellular architecture that first establishes lens transparency and refractive power, and a unique cellular physiology that utilizes circulating ion and water fluxes to actively maintain these optical properties over many decades of life.However, with increasing age the transparent and refractive properties of the lens change.During middle age the ability of the lens to dynamically change its shape (accommodate) to focus on near objects is lost and we become presbyopic (Glasser & Kaufman, 1999).With advancing age the transparent properties of the lens become gradually compromised and in the elderly this ultimately manifests as age-related cataract (Asbell et al., 2005).Taken together these two lens pathologies of presbyopia and cataract account for the majority of age-related vision loss in the world today (Frick et al., 2015).
As our understanding of how the underlying lens cellular physiology operates to drive water flow in the normal lens grows, it is becoming apparent that dysfunction of water flow regulation plays important roles in the onset of both presbyopia and cataract (Schey et al., 2017(Schey et al., , 2022)).In this review we focus on the roles played by the aquaporin family of water channels in mediating water fluxes in the lens, with a specific focus on the emerging roles played by AQP5 in the regulation of lens water flow.To provide context for this review we first provide an overview of lens structure and how that structure is maintained by the circulating fluxes of ions and water that are generated by the lens internal microcirculation system.
Lens structure and function.In mammals the crystalline lens is an avascular, multi-cellular, biological tissue that is positioned in the optical pathway of the eye immediately posterior to the iris and anterior to the vitreous chamber.It is suspended in place by a ring of ligaments known as the zonules of Zinn (Fig. 1A), which consists of delicate but strong polymerized fibrillin fibres that are interwoven into the components of the elastic capsule that encapsulates the lens (Bassnett, 2021), and hence functionally attach the lens to the ciliary body (Kasturi & Matalia, 2017).Contraction of the ciliary muscle moves the ciliary body towards the lens and causes the tension applied to the lens capsule via the zonular fibres to be reduced, which in turn allows the inherent elasticity of the capsule to alter the lens geometry and surface curvatures to effect a change to the refractive power of the lens.In young human lenses, which are capable of dynamic accommodation, the contraction of the ciliary muscle causes an increase in axial thickness, a decrease in equatorial diameter and a steepening of both lens surface curvatures (Fig. 1A), which results in an increase in the refractive power (Glasser, 2010), while in non-accommodating animals it has been proposed that changes to the zonular tension applied to the lens result in changes in the steady state refractive properties of the lens (Chen et al., 2019).The refractive properties of the lens are, however, not solely set by the surface geometry of the lens, but are also enhanced by an inherent gradient of refractive index (GRIN) that varies from being lowest at the surfaces, gradually increasing to a peak in the central lens core (Smith & Pierscionek, 1998).The GRIN also establishes a negative spherical aberration that compensates for the positive spherical aberration introduced by the cornea, the first refractive Figure 1.Lens structure and function A, when the young emmetropic human eye is in the relaxed state (left panel), the ciliary muscle is relaxed and tension is applied to the lens capsule via taut zonular fibres, causing the lens to adopt a flattened shape that optimizes the eye for far vision.In the accommodated state (right panel), the ciliary muscle contracts, releasing the tension on the zonular fibres and allowing the lens to assume a rounder shape thereby adjusting the eye for near vision.B, schematic diagram of an axial cross section through the lens overlaid with the distribution of AQPs in the different regions of the lens.Left hemisphere shows localization of AQP1 to the anterior epithelium and full length and truncated forms of AQP0 in the outer cortex and lens nucleus, respectively.Right hemisphere shows AQP5 is located throughout all lens regions being predominately cytoplasmic in peripheral fibre cells in the outer cortex but membranous in deeper cells.The expanded view is of an elongated differentiating fibre cell showing the apical and basal tips that form the anterior and posterior sutures, respectively, and the greatly elongated lateral membranes.C-E, equatorial images labelled with the membrane marker wheatgerm agglutin (green) showing the cross-sectional profile of the lateral membranes of fibre cells from the periphery (C), deeper outer cortex (D), and lens nucleus (E) chosen to show how the elongated hexagonal profile of the fibre cells with broad (red) and narrow (orange) sides is replaced by a more circular profile (purple) in deeper mature fibres.Panels A and B reproduced with permission from Donaldson et al. (2017) and (Schey et al. (2022), respectively.
Being transparent is the first prerequisite for the lens to achieve its primary function of light refraction.Typically, light gets absorbed and/or scattered whenever it traverses boundaries of differing refractive index (Bassnett et al., 2011).In biological tissues, this usually occurs at cell borders, and within cells since cellular contents differ in their refractive index.The effects of light absorption and scattering are further compounded by the random distribution of cellular components in most cells, rendering most biological tissues opaque.However, in contrast to other tissues the biomolecular constituents of the lens absorb only weakly in the visible spectrum (Bassnett et al., 2011), and as a consequence the percentage of visible light transmitted by the lens in a middle-aged individual is in the order of 88% (Artigas et al., 2012).This implies that the internal organization of the lens must differ in some fundamental way from that of other tissues.
Structural features that establish the transparent and refractive properties of the lens.At first glance the lens is a relatively simple tissue that consist of just two cell types: an epithelial cell layer that covers the anterior surface of the lens and fibre cells that comprise the bulk of the lens (Fig. 1B) and it is a combination of specialized cellular adaptations that afford the lens its optical properties.The absence of blood vessels, the degradation of cellular organelles in the light path, the orderly arrangement of cortical fibre cells, and the matching of refractive indices of fibre cell components in older fibre cells located in the central lens nucleus have all been shown to be the crucial components that minimize light scattering in the lens and enable this living structure to function as 'biological glass' (Bassnett et al., 2011;Donaldson et al., 2017Donaldson et al., , 2022)).
Fibre cells are derived from epithelial cells, which at the equator of the lens continuously divide to form differentiating fibres that exhibit extensive changes to their protein expression profile, undergo massive elongation and eventually lose their light scattering cellular organelles (Audette et al., 2017;Bassnett, 2009).Since these processes of epithelial cell division and subsequent fibre cell differentiation continue throughout life, newly differentiated secondary fibre cells constantly internalize older mature fibre cells, which in turn have internalized the oldest primary fibre cells that were originally laid down during embryonic development (McAvoy et al., 1999;Sugiyama et al., 2011).Hence, all fibres are retained within the lens and a gradient of fibre cell age is established.During the process of fibre cell elongation the original apical and basal membrane domains of the progenitor epithelial cells migrate along the anterior epithelium and capsule (Fig. 1B), respectively, and their lateral membranes adopt a hexagonal profile that consists of two broadsides and four narrow sides (Fig. 1C and  D), which facilitates the packing of the fibre cells into an orderly array of adjacent cell columns (Bassnett et al., 2011).
This orderly packing arrangement of fibre cells in the outer cortex of the lens serves to reduce the dimensions of extracellular space to below the wavelength of light (Bassnett et al., 2011).In accordance with diffraction theory it has been proposed that this regular spatial order minimizes light scattering produced by the local refractive index variations observed in the cortical lens fibres (Michael et al., 2003).The hexagonal lattice formed by the cortical fibre cells therefore acts as a diffraction grating, where the regular spacing of the individual scattering centres leads to constructive interference in the forward direction and destructive interference at other angles to minimize light scattering from the membranes in the cortex (Bassnett et al., 2011).Hence, retaining the spatial order in the outer cortex of the lens through the regulation of fibre cell volume is critical to the maintenance of transparency in this outer region of the lens (Donaldson et al., 2009(Donaldson et al., , 2022)).
The process of fibre cell elongation continues until the apical and basal tips of fibre cells from the opposing lens hemisphere meet to form the anterior and posterior sutures (Kuszak et al., 2004), respectively.The formation of the sutures tends to coincide with the loss the light scattering nuclei and other cellular organelles (Audette et al., 2017;Bassnett, 2009), and marks the transition from differentiating fibre cells in the outer cortex of the lens to mature fibre cells in the inner cortex and nucleus of the lens.Thus, there is only a restricted zone in the outer cortex of the lens where differentiating fibre cells are capable of aerobic metabolism and de novo protein synthesis, with the remaining bulk of mature fibre being uncapable of de novo protein synthesis cells and reliant on anaerobic glycolysis to meet their energy requirements (Zahraei et al., 2022).This loss of cellular organelles means that internalized mature fibre cells have no processes to repair damaged proteins or generate new proteins and requires that proteins synthesized during lens development and growth must last throughout life (Truscott & Friedrich, 2016).These long lived proteins are therefore exposed to a multitude of age-related post-translational modifications (Bloemendal et al., 2004;Wilmarth et al., 2006), which can result in protein cross-linking and aggregation that eventually compromise lens transparency (Schey et al., 2020).
In the deeper regions of the lens the distinctive regular cellular organization, so apparent in the outer cortex is lost (Bassnett et al., 2011), and mature fibre cells in the lens nucleus have very little spatial order and exhibit irregular membrane profiles (Fig. 1E).In the absence of cellular order, transparency in this region of the lens is due to a matching of the relative refractive indices between the cytoplasm and plasma membranes of fibre cells that removes the physical basis for light scatter in these cells (Michael et al., 2003).Thus, in the deeper regions of the lens it is the establishment and maintenance of the GRIN that is the key factor that needs to be regulated to ensure that the transparency and refractive properties of the lens are maintained.The GRIN is established by having different concentrations of water and extraordinary concentrations of protein in each region of the lens.Protein concentrations are three times higher than that found in typical cells and range from 240 mg/ml in the cortex to more than 450 mg/ml in the nucleus (Slingsby et al., 2013;Takemoto & Sorensen, 2008).In contrast, there is a higher amount of water in the cortex than the nucleus (Vaghefi et al., 2011).Consequently, the nucleus (n = 1.406) has a significantly higher refractive index than the cortex (n = 1.386).
Aside from varying the water-to-protein ratio, the various protein concentrations that determine the GRIN are also generated by expressing different subtypes of water-soluble crystallin proteins with varying refractive index increments during the process of fibre cell differentiation (Pierscionek et al., 1987;Slingsby et al., 2013;Thomson & Augusteyn, 1985;Zhao et al., 2011).Age-dependent post-translational modifications to crystallin proteins can affect the GRIN, since aggregated proteins cannot bind as much water due a smaller net exposed surface area, which renders them less soluble and alters their refractive index increment (Khago et al., 2018).However, it is not just the absolute water content of the lens that is important, but the regional distribution and physical interaction of water with lens proteins that determines their conformational state (Bellissent-Funel et al., 2016), and therefore their relative contributions to the GRIN (Khago et al., 2018).
In summary, while the cellular structure and protein composition of the lens is critical to the establishment of the transparent and refractive properties of the lens, controlling the lens water content to regulate lens volume in the outer cortex and the water to protein ratio (GRIN) in the central nucleus is essential to the maintenance of the overall optical properties of the lens (Donaldson et al., 2017(Donaldson et al., , 2022)).Consistent with this notion of differential regulation of water content in specific regions of the lens, the profile of AQP expression, subcellular location and post-translational modification changes as a function of fibre cell differentiation.
Differential expression of lens AQPs.While at least five aquaporins (AQP0, AQP1, AQP5, AQP7, AQP8) are known to be expressed in the lens (Schey et al., 2017(Schey et al., , 2022)), we will focus on AQPs 0, 1, and 5, which have been studied more extensively and have been shown to make specific contributions to water flow in the different regions of the lens (Fig. 1B).
AQP1.AQP1 is a constitutively active water channel exclusively localized to lens epithelial cells, where it mediates water influx and efflux in the central and equatorial regions of the lens, respectively.Lenses of AQP1-null mice exhibited a mild opacification and a change in water content demonstrating a role for AQP1 in the maintenance of lens transparency (Ruiz-Ederra & Verkman, 2006).Epithelial cells isolated from AQP1 knockout lenses also exhibited a threefold reduction in water permeability (Ruiz-Ederra & Verkman, 2006), indicating that expression of AQP1 in lens epithelial cells is required to promote water influx and efflux across the epithelium and maintain lens transparency especially following exposure to stress conditions such as hyperglycaemia and osmotic imbalance.Recently, it has been reported (Lo et al., 2020) that AQP1 expression in two distinct epithelial regions change as a function of lens development and growth in mice.In younger lenses (P3-P9) AQP1 expression was distributed across the entire lens epithelium.However, in older lenses AQP1 expression was increased specifically in the equatorial epithelium and regions in the central epithelium associated with the anterior suture, suggest that with age AQP1 expression increases at the two major sites associated with water influx and efflux (see Fig. 2).AQP0.Formerly known as MIP26 but renamed upon the discovery of the aquaporin protein family (Borgnia et al., 1999), AQP0 is the most abundant integral membrane protein in the lens making up roughly 50% of the lens membrane proteome (Fitzgerald et al., 1983).AQP0 expression is restricted to the fibre cells and it has been shown to act as a multi-functional protein with specific roles in the cortical and nuclear regions of the lens.In the lens cortex, it functions primarily as a low-permeability water channel (Varadaraj et al., 1999), while in the deeper lens nucleus AQP0's primary role shifts to junction formation and cell-to-cell adhesion (Gonen et al., 2004;Kumari & Varadaraj, 2009) and acting as a structural protein linking the plasma membrane to the cytoskeleton (Nakazawa et al., 2011;Rose et al., 2008;Wang & Schey, 2011).Since AQP0 has so many roles other than just a simply water channel, it is not surprising that the loss of functional AQP0 produces wide ranging deleterious effects on lens development, suture formation (Al-Ghoul et al., 2003) and lens transparency (Berry et al., 2000;Francis et al., 2000;Yu et al., 2014;Zeng et al., 2013).Consistent with this view the replacement of AQP0 with AQP1, which lacks the adhesive and structural functions of AQP0, does not fully rescue the cataract phenotype induced by the deletion of AQP0 (Varadaraj et al., 2010).

AQP5.
Immunolabelling and proteomic studies have showed that AQP5 is distributed across the entire lens, being found in the epithelial cells and both differentiating J Physiol 602.13 and mature fibre cells (Fig. 1B), but with a subcellular localization pattern that varies between the different lens regions (Bassnett et al., 2009;Grey et al., 2013;Wang et al., 2008).Specifically, unlike AQP0, AQP5 does not immediately insert into the membranes of differentiating lens fibre cells (Gletten et al., 2022;Petrova et al., 2015).Importantly, lens vesicles show increased water permeability (P H 2 O ) when AQP5 is present in their membranes (Petrova et al., 2018).This result suggests that P H 2 O changes from the outer cortex to the inner cortex.Further, when combined with evidence of AQP5 trafficking in response to changes in zonular tension (Petrova et al., 2020), the results suggest that AQP5 can dynamically regulate lens fibre cell P H 2 O , at least in the outer cortex of the lens.The fact that AQP5 knockout animals are cataractous (Tang et al., 2021), and are susceptible to osmotic stress-induced cataract (Sindhu Kumari & Varadaraj, 2013), suggests that control of AQP5

Figure 2. Contributions of different expression of AQPs to the lens microcirculation system
A, 3D representation of the microcirculation model showing fluxes of ions and fluid that enter the lens at both poles via an extracellular influx pathway, before crossing fibre cell membranes and exiting via an intracellular outflow pathway mediated by gap junctions, which directs the fluxes to the equatorial efflux zone where the ions and fluid exit the lens.B, the extracellular influx pathway at both poles is thought to localize to the sutures that are formed by the apical and basal tips from adjacent fibre cells which interact to form the anterior and posterior sutures, respectively.The fibre cell tips of both sutures contain AQP0, but only the apical tips of fibre cells in inner cortex contain AQP5.In the inner cortex of this anterior influx zone changes in zonular tension dynamically regulate the trafficking of AQP5 to the apical tips of fibre cells to modulate the flow of water from the anterior suture into fibre cells in this region.In the lens core we propose that water uptake is mediated mainly by AQP5 rather than truncated AQP0.C, from the nucleus water flows out towards the equatorial surface via an intercellular outflow pathway mediated by gap junctions.In the inner cortical region of this outflow pathway we propose that the P H 2 O of fibre cells is reduced relative to the P H 2 O of gap junctions to facilitate the cell-to-cell movement of water.This reduction in plasma membrane P H 2 O is facilitated in part by recruitment of AQP0 to junctional structures that restrict the extracellular space and AQP5 to plaque-like structures on the broad sides of fibre cells that have a similar distribution to gap junction plaques.Having reached the equatorial efflux zone water leaves the lens via AQP1 and AQP5 in epithelial cells and AQP5 and AQP0 in differentiating fibres, and in this zone P H 2 O is also dynamically regulated by changes in AQP5 membrane trafficking in response to changes to the tension applied to the lens via the zones.Reproduced with permission from Schey et al. (2022).
P H 2 O is important in regulating fibre cell volume and water homeostasis.
The differential expression patterns and functional properties of the lens AQPs suggest that they make specific contributions to P H 2 O in the different regions of the lens and hence the transport of water through the lens that is driven by a unique cellular physiology which generates an internal microcirculation system that controls the optical properties of the lens (Donaldson et al., 2017(Donaldson et al., , 2022;;Mathias et al., 1997Mathias et al., , 2007)).

Contributions of AQPs to the water flow generated by
the lens internal microcirculation system.To compensate for the metabolic and physiological constraints imposed by the lack of a blood supply, the loss of cellular organelles and high protein concentration gradient, Mathias et al. (1997) first proposed that the lens operates a unique internal microcirculation (Fig. 2) system that maintains lens homeostasis.In this system, Na + predominately moves into the deeper regions of the lens at both the anterior and posterior poles via an extracellular influx pathway associated with the sutures (Candia & Zamudio, 2002;Vaghefi et al., 2011) that connect the lens nucleus to the surface (Fig. 2B), and crosses an extracellular diffusion barrier formed in the inner cortex (Vaghefi & Donaldson, 2018;Vaghefi et al., 2012).Driven by its transmembrane electrochemical gradient, Na + crosses into the intracellular compartment of deeper fibre cells, before then moving back towards the lens surface via an intercellular outflow pathway mediated by gap junctions (Fig. 2C).This outflow of Na + is directed toward the equatorial surface of the lens due to gap junction coupling between differentiating fibre cells being highest at the lens equator (Le & Musil, 2001;Mathias et al., 2010), where Na + /K + pumps are concentrated to remove Na + from the lens (Candia & Zamudio, 2002;Gao et al., 2000;Tamiya et al., 2003) and complete the circulation of Na + throughout the lens.Since fibre cells in the deeper lens have no Na + /K + pump activity, the entry and exit sites of intracellular Na + are spatially distinct (Fig. 2C), creating a difference in the electromotive potential of the surface cells and the deeper-lying fibre cells that causes the current to flow.This circulating current is accompanied by an isotonic fluid flux that preferentially enters the lens at both poles via an extracellular pathway associated with the sutures (Candia et al., 2012;Vaghefi et al., 2011).
Driven by local osmotic gradients created by the transmembrane Na + flux, water enters fibre cells through AQP water channels (Petrova et al., 2018;Varadaraj et al., 1999) before moving towards the lens surface via the intercellular outflow pathway formed by gap junction channels (Gao et al., 2011).It has been shown that this movement of water through the gap junctions generates a substantial gradient in hydrostatic pressure that is regulated by a dual feedback system (Gao et al., 2015) that responds to changes in cell volume (Shahidullah et al., 2020) and the zonular tension applied to the lens capsule (Chen et al., 2019).At the lens surface local osmotic gradients generated by Na + /K + pumps and NKCC1 then cause water to leave at the equatorial efflux zone again through AQP water channels.
In this system the P H 2 O of epithelial and fibre cell membranes is provided by the differential expression of AQP0, AQP1 and AQP5 (Figs 1B and 2B and C).Since it is exclusively localized to lens epithelial cells, AQP1 mediates water influx and efflux in the central and equatorial regions of the lens, respectively.In contrast, fibre cell P H 2 O is determined by the differentiation-dependent changes in the subcellular localization and post-translational modifications to AQP0 and AQP5, which by altering local P H 2 O contribute to the overall magnitude and directionality of water fluxes that circulate through the lens (Schey et al., 2022).It is proposed that full length AQP0 found in the outer cortex of the lens provides a basal level of P H 2 O in this region of the lens and that AQP0 P H 2 O can be altered by AQP0 phosphorylation/calmodulin binding (Fields et al., 2017;Lindsey Rose et al., 2006).In the lens inner cortex, AQP0 forms cell-cell junctions, which form as either AQP0-AQP0 junctions or AQP0-plasma membrane junctions (Gonen et al., 2004;Zampighi et al., 1989), which potentially contribute the formation of an extracellular diffusion barrier that restricts movement of molecules into the lens nucleus (Grey et al., 2003;Wang et al., 2021).In the lens nucleus, extensive age-related modifications such as truncation of the C-terminus of AQP0 will change the regulation of AQP0 P H 2 O permeability by phosphorylation/calmodulin binding and interaction with binding partners (Schey et al., 2017), while the altered lipid environment in the lens nucleus is predicted to reduce the P H 2 O of AQP0 (Tong et al., 2013).
Unlike AQP0, which is always located in the plasma membrane of fibre cells, AQP5 in peripheral fibre cells in equatorial efflux and anterior influx zones exhibits a membrane localization that can be dynamically regulated by changes in the tension applied to the lens by the zonules (Petrova et al., 2020).This suggests that like in other tissues (D' Agostino et al., 2023), AQP5 can act as a regulated water channel in certain regions of lens, with changes to its trafficking to and from the plasma membrane having been shown to alter fibre cell P H 2 O (Petrova et al., 2018).In deeper regions of the lens AQP5 trafficking from subcellular locations to the plasma membrane is completed (Gletten et al., 2022) and in the inner cortex AQP5 accumulates in plaque-like structures on the broadsides of fibre cells (R. S. Petrova, unpublished data) that resemble the gap junction plaques known to form in this region of the lens (Jacobs et al., 2004).In contrast to AQP0, the majority of the C-terminus of AQP5 is largely intact in the lens nucleus (Grey et al., 2013;Petrova et al., 2015), and hence we have proposed that the bulk of water flow occurs via full length AQP5 in the nucleus rather than via truncated AQP0 water channels.
In addition to these regional changes in the relative expression of AQP0 and AQP5, the anterior and posterior sutures that are formed by the apical and basal tips of the fibre cells, respectively, have been shown to differentially express AQP0 and AQP5 (Petrova et al., 2020).While AQP0 was localized to the fibre cell tips of both sutures, AQP5 was only found to be associated with specific regions of the anterior suture (Fig. 2B).In the outer cortical region of sutural influx zone AQP5 was not associated with the apical tips of fibre cells and hence we have proposed that water enters fibre cells located in this region via AQP0 water channels (Fig. 2B, anterior influxouter cortex).In this inner cortical region of the anterior influx zone, AQP5 is found to be associated with the apical tips of fibre cells, but only in lenses that have tension applied to their surfaces via the zonules.Decreasing zonular tension, either mechanical or pharmacological, resulted in the removal of AQP5 from the apical tips of fibre cells in the anterior influx zone (Fig. 2B, anterior influx -inner cortex), but had no effect on the posterior sutural influx zone.In the core of the lens the water delivered to this zone would then be potentially taken up into fibre cells via both truncated AQP0 and AQP5 (Fig. 2B, influx/outflow -core); however, as mentioned above, we are currently unsure whether truncated AQP0 functions as a water channel or an adhesion protein in this region of the lens.
In summary, it appears that regional differences in the expression, membrane localization and post-translational modification of lens AQPs contribute to the movement of water through the lens.Furthermore, changes to membrane localization of AQP5, and hence the fibre cell P H 2 O in the equatorial efflux and anterior influx zones in response the tension applied to the lens (Petrova et al., 2020), imply that changes in the AQP5-mediated P H 2 O also contribute to the observed dynamic regulation of lens water flow.

Contributions of AQP5 to the regulation of lens water
flow.As briefly mentioned above, the outward flow of water through gap junction channels has been shown to generate a substantial hydrostatic pressure gradient (Gao et al., 2011), which is conserved in all species of lenses studied to date (Gao et al., 2013).It has subsequently emerged that this pressure gradient is subject to regulation by a complex dual feedback regulation system, which by reciprocally modulating the Na + /K + -ATPase (Gao et al., 2015) and NKCC1 (Shahidullah et al., 2020) activity alters the osmotic gradients that drive water flow through the lens (Fig. 3).This feedback system utilizes the mechanosensitive ion channels TRPV1 (Fig. 3A) and TRPV4 (Fig. 3B) to sense decreases and increases, respectively, in lens pressure to activate competing arms of the dual feedback loop system to maintain a constant lens pressure (Fig. 3C).Activation of TRPV1 by capsaicin (Fig. 3A) or hyperosmotic challenge (Shahidullah et al., 2020) caused a biphasic increase in lens pressure which can be sustained by blocking the TRPV4-mediated arm of a dual feedback system (Fig. 4B).Conversely, activation of TRPV4 by GSK (Fig. 3B) or hypoosmotic challenge (Shahidullah et al., 2012) caused a biphasic decrease in lens pressure, which could be sustained by blocking the TRPV1-mediated arm of a dual feedback system in mouse (Gao et al., 2015), rat (Petrova et al., 2023) and bovine (Chen et al., 2022) lenses.TRPV1/4 channels are also capable of sensing changes to the tension applied to the lens by either cutting the zonules that attach the lens to the ciliary muscle or pharmacological modulation of the ciliary muscle by the muscarinic agonist pilocarpine (Fig. 4A) and antagonist tropicamide, which increase or decrease contractility, respectively (Chen et al., 2019;Petrova et al., 2023).Interestingly, while the effects of pilocarpine and tropicamide on zonular tension were also mediated via TRPV1 (Fig. 4A) and TRPV4 (Chen et al., 2019), respectively, they induced sustained changes to the magnitude of the hydrostatic pressure gradient and not the biphasic change observed by the direct pharmacological activation of either TRPV1 or TRPV4 (Chen et al., 2019;Petrova et al., 2023).Furthermore, the effect of the pilocarpine-induced change in zonular tension on hydrostatic pressure could be mimicked by the dual application of TRPV1 activators and inhibitors (Fig. 4B).
The pilocarpine-induced reduction in zonular tension that activates TRPV1 to trigger this sustained increase in lens pressure has subsequently been shown (Petrova et al., 2020;Petrova et al., 2023) to cause AQP5 to be removed from membranes of peripheral fibre cells in the equatorial efflux zone (Fig. 4) and from deeper fibre cells located in the inner cortex of the anterior (Fig. 5D-F), but not the posterior influx pathway (Fig. 5A-C).In both the equatorial efflux zone (Fig. 4F) and the anterior influx pathway (Fig. 5G) this removal of AQP5 by changes in zonular tension was abolished by the addition of the TRPV1 inhibitor A-88, while the removal of AQP5 from the membrane in the equatorial efflux (Fig. 4G) and anterior influx zones (Fig. 5H) could be also be induced by the combination of HC-06 and capsaicin, which by inhibiting TRPV4 and activating TRPV1 mimics the sustained effects of pilocarpine on surface pressure (Fig. 4B).
Since the pilocarpine-induced removal of AQP5 water channels from fibre cell membranes should result in a reduction in P H 2 O , these results suggests that AQP5-mediated changes to fibre cell P H 2 O are also involved in the localized regulation of water flow in the equatorial efflux zone and the anterior influx pathway.In the equatorial efflux zone, TRPV1 activation not only increases lens hydrostatic pressure due to a rapid increase in ion uptake by NKCC1 to increase cellular osmolarity to drive water retention (Shahidullah et al., 2020), but also through the removal of AQP5 water channels from the membrane to decrease the P H 2 O and promote water retention in the peripheral fibre cells located in the efflux zone (Fig. 6).These localized changes in surface pressure have been also shown to alter the hydrostatic pressure gradient and change the magnitude of pressure measured in the central lens nucleus (Chen et al., 2019), which has in turn been proposed to alter the GRIN and therefore lens power (Donaldson et al., 2022).
In the anterior influx pathway, the removal of the AQP5-mediated P H 2 O from the apical tips of fibre cells would be expected to reduce the movement of water from the extracellular space into fibre cells located specifically in the inner cortex of the lens, and could affect the volume of the cells in this region of the lens.In support of his view it has been shown in the human lens that it is the anterior curvature that undergoes the largest changes in shape in response to changes in zonular tension that occur during accommodation (Pierscionek, 1993(Pierscionek, , 1995;;Xiang et al., 2021).In rodent lenses, which do not accommodate, we propose that changes in zonular tension cause localized changes to water content specifically in the anterior influx pathway, which alters the anterior surface curvature of the lens and hence the contribution of lens geometry to steady state optical power of the lens.Taken together the AQP-mediated modulation of P H 2 O in the anterior influx pathway and equatorial efflux zone appear to be part of a regulatory system which by regulating water flow (pressure) and lens volume (geometry) acts to set the overall optical power of the lens (Donaldson et al., 2022).
Water flow actively maintains the transparency and refractive properties of the lens.It is our working hypothesis that the dynamic regulation of water flow is critical to not only the maintenance of the lens transparency in the different regions of the lens but also the refractive properties of the whole lens (Donaldson et al., 2022).In the outer cortex of the lens, where the high local spatial order of peripheral fibre cells is the main Figure 3. Lens pressure is regulated by a dual feedback system A and B, application of the TRPV1 activator capsaicin (A) or the TRPV4 activator GSK (B) causes either a transient biphasic increase or decrease, respectively, in average in surface pressure ( P) in the rat lens.C, lens surface pressure (P set ) is maintained by the competing activities of the two arms of a dual-feedback system that regulate ion transporters that control the intracellular osmolarity of cells at the lens surface.Increases in pressure ( P i ), hypoosmotic stress, increased zonular tension or the TRPV4 agonist GSK all work via TRPV4 to activate a signalling pathway that involves the release of ATP via hemichannels, the subsequent activation of purinergic P2Y receptors, and the Src family of protein tyrosine kinases (SFK) to increase the activity of the Na + /K + -ATPase and decrease lens pressure.Decreases in pressure ( P i ), hyperosmotic stress, decreased zonular tension or the TRPV1 agonist capsaicin all work via TRPV1 to activate the extracellular signal-regulated kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3K)/Akt, kinase with no lysine (WNK), and Ste20-related proline-alanine-rich kinase (SPAK)/oxidative stress-responsive kinase-1 (OSR1) signalling pathways to directly activate the sodium potassium dichloride cotransporter (NKCC) and to eventually reduce the decrease in the activity of the Na + /K + -ATPase to effect an increase in surface pressure.AAH = Artificial Aqueous Humour.This scheme is modified from Nakazawa et al. (2021).
contributor to lens transparency (Michael et al., 2003), any failure to maintain fibre cell volume will disrupt this precise spatial organization and generate light scattering that compromises transparency.Hence, in the outer cortex of the lens, the relative activities of volume-sensitive ion transporters and AQP5-mediated P H 2 O is coordinated by the TRPV1/4-mediated feedback system to ensure that local changes in osmotic pressure (water content) maintain fibre cell volume (Donaldson et al., 2009(Donaldson et al., , 2017) ) and hence the spatial order of peripheral fibre cells that is so critical for establishing the transparency of this region of the lens (Bassnett et al., 2011).In the lens nucleus, where mature fibre cells express high levels of crystallin proteins and are exposed to high intracellular pressures, changes in free water content are buffered by syneretic processes that respond to alterations to the pressure gradient (water flow) by changing the amount of water bound to crystallin proteins (Bettelheim, 1999;Bettelheim et al., 2003).This pressure-sensitive buffering of water content acts as a mechanism to eliminate any local transient mismatches between the refractive index of the membranes and cytoplasm in nuclear fibre cells that would increase light scattering and reduce lens transparency in the unordered nuclear regions of the lens (Donaldson et al., 2022).
As well as actively maintaining the transparent properties of the lens, changes to water flow will also have an impact on lens refractive power through the modulation of lens geometry and GRIN (Lim et al., 2016;Vaghefi et al., 2015), the two key parameters that determine lens optics.Localized changes in free water content induced by changes in ion transport and AQP5-mediated P H 2 O that alter the anterior surface curvature will result in changes to the relative contribution made by lens geometry to overall refractive power (Fig. 6).In addition to their effects on lens geometry, changes to ion transport and AQP5-mediated P H 2 O that alter the magnitude of the pressure gradient will also change the binding of water to crystallin proteins, which by modulating protein hydration and therefore the refractive index increment (dn/dc) of γ -crystallins (Khago et al., 2018;Roskamp et al., 2020), will alter the GRIN specifically in the lens nucleus where the pressure gradient and protein concentrations are the highest.Hence, by modulating the transport of water through the lens, the TRPV1/4-mediated dual feedback system operates to maintain steady-state lens power constant in the face of random fluctuations in water flow.In support of this contention the application of various combinations of TRPV1/4 activators and inhibitors to induce either positive or negative shifts in lens pressure induced reciprocal changes in the geometry and GRIN of bovine lenses, which by counteracting each other had no significant effect on overall lens power measured by laser ray tracing (Chen et al., 2022).These results suggest that within a certain auto-regulatory range, fluctuations in lens pressure are buffered by pressure sensitive syneretic processes that induce lens protein to either release or bind water so that the free water content of the lens, and hence steady state lens power, remains relatively constant in the face of fluctuations in lens water flow.

Figure 5. Effects of pilocarpine on the subcellular localization of AQP5 in the posterior and anterior influx zone of the rat lens
Representative image montages of posterior (A) and anterior (D) sutures spanning from the outer cortex to the core of the lens, which was labelled with the membrane marker WGA (red).The white boxes represent the regions within the sutures from where high magnification images were captured to show the localization of AQP5 protein (green).Arrows indicate the location of the sutures.B, in control lenses AQP5 was found missing from the basal tips of fibre cells that form the posterior sutures in IP1 and IP2 regions but AQP5 did colocalize with the suture in the lens core (IP3) region.C, the addition of pilocarpine alone caused no change to the subcellular distribution of AQP5 along the length of the posterior suture.E, in control lenses AQP5 was found missing from the apical tips of fibre cells that form the anterior suture in outer cortical (IA1) region, but associated with the sutures in the inner cortex (IA2) and core (IA3) regions of the lens.F, in the presence of pilocarpine, AQP5 remained absent from the suture in the outer cortex (IA1), but was missing from the sutures in inner cortex (IA2) and remained present in the core (IA3).G, pre-incubation of lenses in the TRPV1 inhibitor A88 followed by the addition of pilocarpine inhibited the removal of AQP5 from the anterior suture in the inner cortical region (IA2) of the rat lens.H, the pre-incubation of lenses with the TRPV4 inhibitor HC-60 followed by the addition of capsaicin resulted in the removal of AQP5 from the suture in the inner cortex (IA2), but had no effect on AQP5 labelling in outer cortex (IA1) and core (IA3) regions.AAH = Artificial Aqueous Humour.Images for the figure were obtained from Petrova et al. (2023).In this model the pilocarpine-induced decrease in the zonular tension applied to the lens activates a TRPV1-mediated signalling pathway that immediately increases the activity of NKCC to increase the uptake of ions and after a delay reduces the activity of the Na + ,K + -ATPase to reduce the removal of ions from surface fibre cells, to effect an intracellular accumulation of ions.In parallel to these changes in ion transporter activity, the removal of AQP5 from the membrane in the anterior influx pathway and equatorial efflux zone by TRPV1 activation specifically reduces the P H 2 O in these regions of the lens.These changes in transporter activity and P H 2 O combine to increase the intracellular osmolartity and in turn the hydrostatic pressure gradient.Furthermore, based on the observed ability of hyperosmotic challenge to increase NKCC1 activity via a TRPV1-mediated signalling pathway (Shahidullah et al., 2020), we would predict that hyperosmotic challenge would also modulate AQP5 membrane trafficking.Reproduced from Petrova et al. (2023).
However, our discovery that the intrinsic TRPV1/4 dual feedback system can be regulated by changes to zonular tension suggests that the set point around which lens power is auto-regulated can be shifted in response to signals that are extrinsic to the lens.In this regard it is interesting that the process of emmetropization, which ensures that the images are correctly focused on the retina as the eye grows, is associated with major changes to lens geometry and optical power.In mice this process occurs between 4 and 6 weeks of age and results in a change in lens shape from spheroid to lentoid, and a decrease in lens power (Cheng et al., 2019;Kalligeraki et al., 2020;Tkatchenko et al., 2010).It has been shown to be accompanied by an increase in the GRIN (Cheng et al., 2019), which has recently been shown to be driven by a decrease in free water content over this period of lens growth (Pan et al., 2023).In addition preliminary in vivo MRI experiments performed on AQP5 KO mice have showed that the observed age-dependent flattening of the anterior surface of the lens observed in wild-type lenses did not occur (Pan et al., 2023), suggesting that the AQP5-mediated changes to water permeability observed in the anterior influx pathways may be involved in the process of emmetropization in the mouse lens.
In conclusion, several new research opportunities are opened by the growing realization that the lens operates a dual feedback system that can respond to both intrinsic and extrinsic signals to affect changes in ion transport and AQP5-mediated P H 2 O , which alter the hydrostatic pressure gradient that controls lens water content and transport and in turn the optical properties of the lens.Obviously more work needs to be done to understand the signalling pathways involved in the regulation of lens water flow and the remodelling of lens geometry so that therapeutic strategies can be developed to maintain the optical properties of the lens throughout life and prevent or delay the onset of the lens pathologies, presbyopia and cataract.

0
Paul Donaldson obtained his PhD in the field of epithelial transport from the University of Otago in New Zealand and this was followed by post-doctoral fellowships at Yale University and the University of Texas Medical Branch in Galveston.Upon returning to New Zealand he has held various position at the University of Auckland.Currently he is the Head of the School of Medical Sciences.His research involves determining how a variety of ion channels and transporters contribute to the maintenance of lens transparency.Kevin Schey obtained his PhD in Analytical Chemistry from Purdue University.After post-doctoral training at the University of Chicago he took a position at the Medical University of South Carolina before moving to the Mass Spectrometry Research Centre and Department of Biochemistry at Vanderbilt University.His research interests lie in the areas of lens biochemistry, proteomics of ageing, exosome proteomics and imaging mass spectrometry.

Figure 4 .
Figure 4. Effect of pilocarpine on lens surface pressure and AQP5 membrane trafficking in the equatorial efflux zoneA, the pilocarpine-induced decrease in the zonular tension applied to the lens produces a sustained increase in surface pressure ( P) that is abolished in the presence of the TRPV1 inhibitor A-88.B, activation of TRPV1 by capsaicin in lenses pre-incubated in the presence of the TRPV4 inhibitor HC-06 mimics the sustained increase in surface pressure induce by pilocarpine.C, image of the lens equator that encompasses the efflux zone labelled with membrane (WGA, red) and nuclei ( 4 ,6-diamidino-2-phenylindole, blue) markers to show the zone of transition where equatorial epithelial cells differentiate into fibre cells and undergo extensive elongation of their lateral membranes.The white box represents the region from where high magnification images (D-G) were captured to show the localization of AQP5 protein (green).D, in lenses incubated in AAH for 60 min, AQP5 was found localised to the lateral membranes of differentiating fibre cells of the outer cortex.E, in lenses treated with pilocarpine for 60 min the AQP5 distribution shifted to a cytoplasm labelling pattern.F, addition of pilocarpine to lenses pre-incubated with the TRPV1 inhibitor A-88 for 30 min maintained the association of AQP5 with the membrane in this zone of the lens.G, exposure of lenses to the TRPV4 inhibitor HC-06 for 30 min prior to the addition of the TRPV1 activator capsaicin to mimic the effects of pilocarpine on lens pressure caused a shift in AQP5 from the membrane to the cytoplasm.To appreciate the membrane and cytoplasmic localization of AQP5 the magnified images (B-E) are presented as a split image that includes single AQP5 areas and adjacent areas labelled with both AQP5 and WGA.AAH = Artificial Aqueous Humour.Data for the figure were sourced fromPetrova et al. (2023).

JFigure 6 .
Figure 6.Regulation of lens hydrostatic pressure by zonular tensionA simplified model showing only the TRPV1-mediated arm of the dual feedback system that regulates hydrostatic pressure in the lens.In this model the pilocarpine-induced decrease in the zonular tension applied to the lens activates a TRPV1-mediated signalling pathway that immediately increases the activity of NKCC to increase the uptake of ions and after a delay reduces the activity of the Na + ,K + -ATPase to reduce the removal of ions from surface fibre cells, to effect an intracellular accumulation of ions.In parallel to these changes in ion transporter activity, the removal of AQP5 from the membrane in the anterior influx pathway and equatorial efflux zone by TRPV1 activation specifically reduces the P H 2 O in these regions of the lens.These changes in transporter activity and P H 2 O combine to increase the intracellular osmolartity and in turn the hydrostatic pressure gradient.Furthermore, based on the observed ability of hyperosmotic challenge to increase NKCC1 activity via a TRPV1-mediated signalling pathway(Shahidullah et al., 2020), we would predict that hyperosmotic challenge would also modulate AQP5 membrane trafficking.Reproduced fromPetrova et al. (2023).