Water homeostasis in the ischaemic retina: is aquaporin‐4 involved? (2005)

Control of extracellular volume is important in any tissue, including the retina. The retina incorporates two very different extracellular spaces: the subretinal space and the extracellular space in the inner retina. The subretinal space is bounded externally by the inner surface of the retinal pigment epithelium (RPE) and internally by the external limiting membrane. It is a loosely organized, extracellular space, sometimes with several microns between the cellular elements, and it contains an extracellular matrix, the so-called interphotoreceptor matrix. Fluid accumulation in the subretinal space does not necessarily lead to major loss of function, as evidenced by the relatively good visual acuity we often find in patients with central serous chorioretinopathy. Due to the spacious nature of the subretinal matter, fluid, protein and particulate matter can easily reach the surface of the retinal pigment epithelium, which serves as the scavenger in this region. The extracellular space of the inner retina is very different from the subretinal space, and consists of the narrow clefts between the tightly packed cellular elements (Hamann 2002). Because of this organization of the extracellular space in the inner retina, the hydraulic conductivity is very low, particular in the plexiform layers (Antcliff et al. 2001). An excess of extracellular fluid leaking out from damaged inner retinal capillaries can hardly escape through the extracellular route and may only accumulate by displacing retinal cellular elements. Any extracellular fluid accumulation in the inner retina would therefore tend to be cystic in nature and disrupt the normal anatomy of the neuronal connections. Yet from clinical experience, we know that the retina can protect itself from cystoid macular oedema despite considerable inner retinal leakage. A hypothesis of transcellular movement of water, and ions, such as the one proposed in this issue of Acta by Bringmann et al., is therefore entirely justified. Some 13 years ago, our understanding of transcellular movement of water was greatly advanced by the group led by Professor Peter Agre, who discovered the first molecular water channel, later termed aquaporin (AQP)-1 (Preston et al. 1992). In recognition of this discovery, Peter Agre was rewarded with the Nobel Prize in 2003 (Agre 2004). The AQPs constitute a family of at least 13 water channels, which mediate the transcellular osmotic movement of fluid in tissues with a need for rapid water transport. The AQPs provide important building blocks in constructing explanations for the secretion and absorption of cerebrospinal fluid in the brain, the secretion of aqueous humour in the eye, the secretion of tears, sweat, saliva and bile, and for the concentration of urine in the kidneys (Agre 2004). In the inner retina, the only water channel with a distinct membrane localization is AQP-4, which is present in Müller cell membranes, with extensive expression in processes facing the retinal capillaries and the vitreous body (Hamann et al. 1998; Nagelhus et al. 1998). This is consistent with potential roles for Müller cells and AQP-4 in water transport between the vitreous body and the retinal capillaries, and for the maintenance of extracellular ion concentration and osmolarity during neuronal activity. Aquaporin-4 is present in a variety of neurosupportive and epithelial cells, and the expression pattern in the retina is very similar to the extensive polarized AQP-4 expression in glial cells in the brain (Nielsen et al. 1997). Studies of mice in which the gene encoding AQP-4 has been knocked out point to important roles for AQP-4 in neural signal transduction and cellular swelling. AQP-4 knock-out mice have expanded extracellular spaces in the brain (Binder et al. 2004a), altered seizure thresholds (Binder et al. 2004b), impaired auditory brain-stem-evoked potentials (Li & Verkman 2001), and slightly impaired light-induced retinal potentials (Li et al. 2002). A colocalization of the K+ channel Kir4.1 and AQP-4 in specific membrane domains of rat Müller cells has been found (Nagelhus et al. 1999), and it has been suggested that altered K+ recycling by AQP-4-associated Kir4.1 channels may be responsible for these abnormalities (Nagelhus et al. 1999; Amiry-Moghaddam et al. 2003). On the other hand, brain swelling and neurological deterioration in AQP-4-deficient mice are reduced in cytotoxic brain injury caused by acute water intoxication and ischaemic stroke (Manley et al. 2000), and recently it has been found that AQP-4 deletion in mice is neuroprotective in a transient ischaemia model of retinal injury (Verkman et al. 2000). In this issue of Acta, Andreas Bringmann et al. present a review offering the interesting hypothesis that Müller cells are important for neuronal cell swelling and apoptosis in the ischaemic inner retina. The paper proposes a mechanism for (post)ischaemia-induced retinal swelling that involves the glutamate receptors on the Müller cells. Glutamate, the major excitatory neurotransmitter within the retina and brain (Massey 1990), is liberated during reperfusion (and to a smaller degree during ischaemia), which causes increased Na+ influx into neurons. This is assumed to be followed by osmotic water movement through Müller cell AQP-4, causing the neurons to swell. At the same time overexcited neurons (by glutamate) release K+ into the extracellular space. This presumably starts a vicious cycle leading to further neuronal overexcitation, additional neuronal swelling, Ca2+ influx via voltage-gated Ca2+ channels and eventual neuronal apoptosis (and loss of function). During the first few days post-ischaemia, the Müller cells then down-regulate their endfeet Kir4.1. This is put forward to be the cause of the Müller cell swelling, observed in previous studies, due to intracellular K+ overload leading to osmotic water movement from the blood and vitreous into the cells. It is further hypothesized that inhibition of Müller cell AQP-4 might be effective in reducing the water permeability of the Müller cells, and thereby preventing cytotoxic (cellular) oedema. However, the authors wisely state that AQP-4 inhibition might at the same time impair the retinal response to vasogenic (extracellular) oedema. In addition, as shown by Li & Verkman (2001), AQP-4-deficient mice are deaf, a rather unpleasant side-effect of systemic AQP-4 inhibition. Therefore, it is proposed that it might be possible to stimulate water transport in Müller cells, as it has proven possible in RPE cells, using INS37217, a synthetic P2Y2 receptor agonist, as the stimulating agent (Maminishkis et al. 2002; Meyer et al. 2002). The idea that Müller cell water transport is important in neuronal cell swelling, and shrinkage, and that pharmacological modification of it holds potential is very interesting. It provides an important building block in the understanding of the complex machinery of neuronal versus glial cell swelling in the retina. However, a few points regarding the water homeostasis of the retina are worth keeping in mind. The hypothesis presented in the paper requires the build-up of local hypo- and hyperosmolarities in the intercellular synaptic clefts, while no such ‘standing osmotic gradient’ has ever been measured, despite the launching of this hypothesis almost 40 years ago (Zeuthen 1983, 2002). During the last two decades it has become well known that fluid transport in tissues cannot be explained in purely osmotic terms (Zeuthen 2002). In addition to the aquaporins, which serve as facilitators of osmotic-driven water movements, there are cotransport proteins, which act as molecular water pumps, capable of transporting significant amounts of water without the need for any osmotic gradient, or even against an osmotic gradient (Zeuthen & MacAulay 2002). This seems to be of particular importance in the retina, where vascular leakage is accompanied by both extracellular fluid and protein accumulation. In diabetic maculopathy, for example, we recognize vascular leakage sources by the circinate hard exudates that surround them. However, in the early stages, no or minimal retinal thickening is usually present despite the leakage. Apparently, the retina can remove extracellular water and precipitate the protenaceous solutes as crystals. There is no way this can be explained solely by osmotic-driven water transport. Water pumps that can transport water against an osmotic gradient seem necessary. In the eye, the existence of molecular water pumps has been demonstrated in the retinal pigment epithelium of bullfrogs and pigs, allowing the RPE cells to transport water against an osmotic gradient (Zeuthen et al. 1996; Hamann et al. 2003). Another example of a molecular water pump is the Na+-glutamate cotransporter EAAT1, which is abundantly expressed in Müller cell processes in all retinal layers (Lehre et al. 1997). The Müller cell EAAT1 is responsible for the removal of most of the extracellular glutamate within the retina, and defects in its function cause a major deterioration in visual function (Harada et al. 1998; Rauen et al. 1998). EAAT1 has two modes of water transport, one of which is passive while the other is active (MacAulay et al. 2004). When exposed to an osmotic gradient, EAAT1 works as a regular water channel, but during the cotransport of glutamate additional water is cotransported along with the substrates. The coupling of water and glutamate transport within EAAT1 is entirely independent of external parameters, such as ion concentrations and osmotic gradients. Thus, EAAT1 likely constitutes an important building block in Müller cell water transport, and would need to be incorporated in a complete model. Of course, EAAT1, AQP-4 and K+ channels constitute only three types of several transport proteins present in the Müller cell membrane that might be important for water transport. Numerous other cotransporters, ion channels, and, perhaps, as yet undiscovered AQP isoforms, may be directly or indirectly involved in retinal transcellular water movements. The review by Bringmann et al. incorporates results from different exciting studies regarding Müller cell water transport into a unifying complete model of water transport, explaining neuronal cell swelling and apoptosis in the ischaemic retina. It is, however, too early to end this chapter, and further experiments on a variety of transport proteins, expressed in Müller cells and neuronal cells, will in the future lead to a more detailed understanding of water homeostasis in ischaemic retinas.