Marc H. Levin

FUNCTIONS OF AQUAPORINS IN THE EYE

The aquaporins (AQPs) are integral membrane proteins whose main function is to transport water across cell membranes in response to osmotic gradients. At the ocular surface, AQP1 is expressed in corneal endothelium, AQP3 and AQP5 in corneal epithelium, and AQP3 in conjunctival epithelium. AQPs are also expressed in lens fiber cells (AQP0), lens epithelium (AQP1), ciliary epithelium (AQP1, AQP4) and retinal Müller cells (AQP4). Mutations in AQP0 produce congenital cataracts in humans. Analysis of knockout mice lacking individual AQPs suggests their involvement in maintenance of corneal and lens transparency, corneal epithelial repair, intraocular pressure (IOP) regulation, retinal signal transduction and retinal swelling following injury. The mouse phenotype findings implicate AQPs as potential drug targets for therapy of elevated IOP and ocular disorders involving the cornea, lens and retina. However, much research remains in defining cell-level mechanisms for the ocular AQP functions, in establishing the relevance to human eye disease of conclusions from knockout mice, and in developing AQP-modulating drugs.

Urearetics: a small molecule screen yields nanomolar potency inhibitors of urea transporter UT-B

Functional studies in knockout mice indicate a critical role for urea transporters (UTs) in the urinary concentrating mechanism and in renal urea clearance. However, potent and specific urea transport blockers have not been available. Here, we used high‐throughput screening to discover high‐affinity, small molecule inhibitors of the UT‐B urea transporter. A collection of 50,000 diverse, drug‐like compounds was screened using a human erythrocyte lysis assay based on UT‐B‐facilitated acetamide transport. Primary screening yielded ~30 UT‐B inhibitors belonging to the phenylsulfoxyoxazole, benzenesulfonanilide, phthalazinamine, and aminobenzimidazole chemical classes. Screening of ~700 structurally similar analogs gave many active compounds, the most potent of which inhibited UT‐B urea transport with an EC50 of ~10 nM, and 100% inhibition at higher concentrations. Phenylsulfoxyoxazoles and phthalazinamines also blocked rodent UT‐B and had good UT‐B vs. UT‐A specificity. The UT‐B inhibitors did not reduce aquaporin‐1 (AQP1)facilitated water transport. In AQP1‐null erythrocytes, “chemical UT‐B knockout” by UT‐B inhibitors reduced by ~3‐fold UT‐B‐mediated water transport, supporting an aqueous pore pathway through UT‐B. UT‐B inhibitors represent a new class of diuretics, “urearetics,” which are predicted to increase renal water and solute clearance in water‐retaining states.—Levin, M. H., de la Fuente, R., Verkman, A. S. Urearetics: a small molecule screen yields nanomolar potency inhibitors of urea transporter UT‐B. FASEB J. 21, 551–563 (2007)

A lack of delayed intraocular pressure elevation in patients treated with intravitreal injection of bevacizumab and ranibizumab.

Purpose: The purpose of this study was to report the rate of intraocular pressure (IOP) elevation after intravitreal injections of anti–vascular endothelial growth factor agents for exudative age-related macular degeneration. Methods: Retrospective chart review of all patients receiving intravitreal ranibizumab and/or bevacizumab injections for exudative age-related macular degeneration from November 2005 to June 2010. Delayed ocular hypertension (OHT) was defined as either an IOP ≥22 mmHg on 2 consecutive visits (with an increase from baseline >6 mmHg) or an IOP >26 mmHg on a single visit with a concomitant initiation or augmentation of IOP-lowering treatment. Noninjected fellow eyes served as controls. Incidence of delayed OHT was analyzed using survival analyses, with risk assessed by Cox proportional hazards regression models. Eyes with glaucoma were evaluated separately. Results: Three hundred and two treated eyes and 226 control eyes met inclusion criteria. In eyes with exudative age-related macular degeneration without glaucoma, 3 of 270 injected eyes (0.51% incidence per eye-year) developed delayed OHT compared with 4 of 195 control eyes (1.00% incidence per eye-year), a difference that was not statistically significant (hazard ratio = 0.48; 95% confidence interval: 0.11–2.23). In eyes with exudative age-related macular degeneration and glaucoma, 2 of 32 injected eyes developed delayed OHT (3.1% incidence per eye-year) compared with 3 of 31 control eyes (5.7% incidence per eye-year), a difference that was not statistically significant (hazard ratio = 0.59; 95% confidence interval: 0.10–3.60). Conclusion: The incidence of delayed OHT after intravitreal anti–vascular endothelial growth factor injections was low and did not differ between injected and control eyes, including eyes with glaucoma. These results argue against a significant risk of IOP elevation because of repeated anti–vascular endothelial growth factor therapy.

Hypertonic saline therapy in cystic fibrosis : Evidence against the proposed mechanism involving aquaporins

Recent data indicate the clinical benefit of nebulized hypertonic saline in cystic fibrosis lung disease, with a proposed mechanism involving sustained increase in airway surface liquid volume. To account for the paradoxical observation that amiloride suppresses the beneficial effect of hypertonic saline, it has been previously concluded (Donaldson, S. H., Bennett, W. D., Zeman, K. L., Knowles, M. R., Tarran, R., and Boucher, R. C. (2006) N. Engl. J. Med. 354, 241-250) that amiloride-inhibitable aquaporin (AQP) water channels in airway epithelia modulate airway surface liquid volume. Here, we have characterized water permeability and amiloride effects in well differentiated, primary cultures of human airway epithelial cells, stably transfected Fisher rat thyroid epithelial cells expressing individual airway/lung AQPs, and perfused mouse lung. We found high transepithelial water permeability (Pf, 54 ± 5 μm/s) in airway epithelial cells that was weakly temperature-dependent and inhibited by >90% by reduced pH in the basal membrane-facing solution. Reverse transcription-PCR and immunofluorescence suggested the involvement of AQPs 3, 4, and 5 in high airway water permeability. Experiments using several sensitive measurement methods indicated that amiloride does not inhibit water permeability in non-cystic fibrosis (non-CF) or CF airway epithelia, AQP-transfected Fisher rat thyroid cells, or intact lung. Our data provide evidence against the mechanism proposed by Donaldson et al. to account for the effects of amiloride and hypertonic saline in CF lung disease, indicating the need to identify alternate mechanisms.

Incidence of visual improvement in uveitis cases with visual impairment caused by macular edema.

PURPOSE Among cases of visually significant uveitic macular edema (ME), to estimate the incidence of visual improvement and identify predictive factors. DESIGN Retrospective cohort study. PARTICIPANTS Eyes with uveitis, seen at 5 academic ocular inflammation centers in the United States, for which ME was documented to be currently present and the principal cause of reduced visual acuity (<20/40). METHODS Data were obtained by standardized chart review. MAIN OUTCOME MEASURES Decrease of ≥ 0.2 base 10 logarithm of visual acuity decimal fraction-equivalent; risk factors for such visual improvement. RESULTS We identified 1510 eyes (of 1077 patients) with visual impairment to a level <20/40 attributed to ME. Most patients were female (67%) and white (76%), and had bilateral uveitis (82%). The estimated 6-month incidence of ≥ 2 lines of visual acuity improvement in affected eyes was 52% (95% confidence interval [CI], 49%-55%). Vision reduced by ME was more likely to improve by 2 lines in eyes initially with poor visual acuity (≤ 20/200; adjusted hazard ratio [HR] 1.5; 95% CI, 1.3-1.7), active uveitis (HR, 1.3; 95% CI, 1.1-1.5), and anterior uveitis as opposed to intermediate (HR, 1.2), posterior (HR, 1.3), or panuveitis (HR, 1.4; overall P = 0.02). During follow-up, reductions in anterior chamber or vitreous cellular activity or in vitreous haze each led to significant improvements in visual outcome (P <0.001 for each). Conversely, snowbanking (HR, 0.7; 95% CI, 0.4-0.99), posterior synechiae (HR, 0.8; 95% CI, 0.6-0.9), and hypotony (HR, 0.2; 95% CI, 0.06-0.5) each were associated with lower incidence of visual improvement with respect to eyes lacking each of these attributes at a given visit. CONCLUSIONS These results suggest that many, but not all, patients with ME causing low vision in a tertiary care setting will enjoy meaningful visual recovery in response to treatment. Evidence of significant ocular damage from inflammation (posterior synechiae and hypotony) portends a lower incidence of visual recovery. Better control of anterior chamber or vitreous activity is associated with a greater incidence of visual improvement, supporting an aggressive anti-inflammatory treatment approach for ME cases with active inflammation.

OPTIC NEURITIS IN NEUROMYELITIS OPTICA

Neuromyelitis optica (NMO) is an autoimmune demyelinating disease associated with recurrent episodes of optic neuritis and transverse myelitis, often resulting in permanent blindness and/or paralysis. The discovery of autoantibodies (AQP4-IgG) that target aquaporin-4 (AQP4) has accelerated our understanding of the cellular mechanisms driving NMO pathogenesis. AQP4 is a bidirectional water channel expressed on the plasma membranes of astrocytes, retinal Müller cells, skeletal muscle, and some epithelial cells in kidney, lung and the gastrointestinal tract. AQP4 tetramers form regular supramolecular assemblies at the cell plasma membrane called orthogonal arrays of particles. The pathological features of NMO include perivascular deposition of immunoglobulin and activated complement, loss of astrocytic AQP4, inflammatory infiltration with granulocyte and macrophage accumulation, and demyelination with axon loss. Current evidence supports a causative role of AQP4-IgG in NMO, in which binding of AQP4-IgG to AQP4 orthogonal arrays on astrocytes initiates complement-dependent and antibody-dependent cell-mediated cytotoxicity and inflammation. Immunosuppression and plasma exchange are the mainstays of therapy for NMO optic neuritis. Novel therapeutics targeting specific steps in NMO pathogenesis are entering the development pipeline, including blockers of AQP4-IgG binding to AQP4 and inhibitors of granulocyte function. However, much work remains in understanding the unique susceptibility of the optic nerves in NMO, in developing animal models of NMO optic neuritis, and in improving therapies to preserve vision.

Experimental mouse model of optic neuritis with inflammatory demyelination produced by passive transfer of neuromyelitis optica-immunoglobulin G.

BackgroundAlthough optic neuritis (ON) is a defining feature of neuromyelitis optica (NMO), appropriate animal models of NMO ON are lacking. Most NMO patients are seropositive for immunoglobulin G autoantibodies (NMO-IgG) against the astrocyte water channel aquaporin-4 (AQP4).MethodsSeveral approaches were tested to develop a robust, passive-transfer mouse model of NMO ON, including NMO-IgG and complement delivery by: (i) retrobulbar infusion; (ii) intravitreal injection; (iii) a single intracranial injection near the optic chiasm; and (iv) 3-days continuous intracranial infusion near the optic chiasm.ResultsLittle ON or retinal pathology was seen using approaches (i) to (iii). Using approach (iv), however, optic nerves showed characteristic NMO pathology, with loss of AQP4 and glial fibrillary acidic protein immunoreactivity, granulocyte and macrophage infiltration, deposition of activated complement, demyelination and axonal injury. Even more extensive pathology was created in mice lacking complement inhibitor protein CD59, or using a genetically modified NMO-IgG with enhanced complement effector function, including significant loss of retinal ganglion cells. In control studies, optic nerve pathology was absent in treated AQP4-deficient mice, or in wild-type mice receiving control (non-NMO) IgG and complement.ConclusionPassive transfer of NMO-IgG and complement by continuous infusion near the optic chiasm in mice is sufficient to produce ON with characteristic NMO pathology. The mouse model of NMO ON should be useful in further studies of NMO pathogenesis mechanisms and therapeutics.

In Situ Fluorescence Measurement of Tear Film [Na+], [K+], [Cl-], and pH in Mice Shows Marked Hypertonicity in Aquaporin-5 Deficiency

PURPOSE Tear film composition depends on water and ion transport across ocular surface epithelia and on fluid secretion by lacrimal glands. The purpose of this study was to establish in situ fluorescence methods to measure tear film ionic concentrations and pH in mice and to determine whether tear film composition is sensitive to deficiency of the major ocular surface aquaporin water channels. METHODS Tear film ionic concentrations and pH were measured in anesthetized mice by ratio imaging fluorescence microscopy after topical application of ion/pH-sensing, dual-wavelength fluorescent indicators. [Na(+)], [K(+)], and [Cl(-)] were measured with membrane-impermeant indicators developed by our laboratory, and pH was measured with bis(carboxyethyl)-carboxyfluorescein fluorescence-conjugated dextran. Measurements were performed on wild-type mice and on knockout mice lacking aquaporins AQP1, AQP3, and AQP5. RESULTS In wild-type mice, tear film [Na(+)] was 139 +/- 8 mM, [K(+)] was 48 +/- 1 mM, [Cl(-)] was 127 +/- 4 mM, and pH was 7.59 +/- 0.2 (SE; n = 5-8). pH did not differ significantly in the AQP knockout mice. [Na(+)] was increased by approximately twofold in AQP5 null mice (230 +/- 20 mM) and was greatly reduced after exposure of the ocular surface to a humidified atmosphere. [K(+)] was mildly reduced in AQP1 null mice. CONCLUSIONS These results establish an in situ optical methodology to measure tear film [Na(+)], [K(+)], [Cl(-)], and pH in living mice, without the need for fluid sampling. Tear film hypertonicity in AQP5 deficiency is likely caused by reduced transcorneal water secretion in response to evaporative water loss.

CFTR activator increases intestinal fluid secretion and normalizes stool output in a mouse model of constipation.

Background & Aims Constipation is a common clinical problem that negatively impacts quality of life and is associated with significant health care costs. Activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the primary pathway that drives fluid secretion in the intestine, which maintains lubrication of luminal contents. We hypothesized that direct activation of CFTR would cause fluid secretion and reverse the excessive dehydration of stool found in constipation. Methods A cell-based, high-throughput screen was performed for 120,000 drug-like, synthetic small molecules. Active compounds were characterized for mechanism of action and one lead compound was tested in a loperamide-induced constipation model in mice. Results Several classes of novel CFTR activators were identified, one of which, the phenylquinoxalinone CFTRact-J027, fully activated CFTR chloride conductance with an half-maximal effective concentration (EC50) of approximately 200 nmol/L, without causing an increase of cytoplasmic cyclic adenosine monophosphate. Orally administered CFTRact-J027 normalized stool output and water content in a loperamide-induced mouse model of constipation with a 50% effective dose of approximately 0.5 mg/kg; CFTRact-J027 was without effect in cystic fibrosis mice lacking functional CFTR. Short-circuit current, fluid secretion, and motility measurements in mouse intestine indicated a prosecretory action of CFTRact-J027 without direct stimulation of intestinal motility. Oral administration of 10 mg/kg CFTRact-J027 showed minimal bioavailability, rapid hepatic metabolism, and blood levels less than 200 nmol/L, and without apparent toxicity after chronic administration. Conclusions CFTRact-J027 or alternative small-molecule CFTR-targeted activators may be efficacious for the treatment of constipation.

Asymmetric bilateral demyelinating optic neuropathy from tacrolimus toxicity

OBJECTIVE To report the first histopathologic description of optic nerve demyelination from tacrolimus (FK 506) toxicity in the absence of toxic levels of tacrolimus in a patient presenting with asymmetric bilateral visual loss after 5 years of tacrolimus therapy. PATIENTS We report a patient status post cardiac and renal transplantation who developed severe, progressive and asynchronous bilateral visual loss after prolonged treatment with tacrolimus. Orbital MRI showed an enlarged left optic nerve that enhanced with gadolinium. CONCLUSION After extensive negative work up, biopsy of one optic nerve was performed. Microscopic analysis showed extensive demyelination in the absence of vasculitis, neoplastic or infectious etiologies. Our patient illustrates that demyelination of the optic nerve causing asynchronous vision loss can be associated with tacrolimus toxicity in the absence of toxic drug levels.