Inflammation primes the murine kidney for recovery by activating AZIN1 adenosine-to-inosine editing.

The progression of kidney disease varies among individuals, but a general methodology to quantify disease timelines is lacking. Particularly challenging is the task of determining the potential for recovery from acute kidney injury following various insults. Here, we report that quantitation of post-transcriptional adenosine-to-inosine (A-to-I) RNA editing offers a distinct genome-wide signature, enabling the delineation of disease trajectories in the kidney. A well-defined murine model of endotoxemia permitted the identification of the origin and extent of A-to-I editing, along with temporally discrete signatures of double-stranded RNA stress and Adenosine Deaminase isoform switching. We found that A-to-I editing of Antizyme Inhibitor 1 (AZIN1), a positive regulator of polyamine biosynthesis, serves as a particularly useful temporal landmark during endotoxemia. Our data indicate that AZIN1 A-to-I editing, triggered by preceding inflammation, primes the kidney and activates endogenous recovery mechanisms. By comparing genetically modified human cell lines and mice locked in either A-to-I edited or uneditable states, we uncovered that AZIN1 A-to-I editing not only enhances polyamine biosynthesis but also engages glycolysis and nicotinamide biosynthesis to drive the recovery phenotype. Our findings implicate that quantifying AZIN1 A-to-I editing could potentially identify individuals who have transitioned to an endogenous recovery phase. This phase would reflect their past inflammation and indicate their potential for future recovery.

Inflammation primes the kidney for recovery by activating AZIN1 A-to-I editing.

The progression of kidney disease varies among individuals, but a general methodology to quantify disease timelines is lacking. Particularly challenging is the task of determining the potential for recovery from acute kidney injury following various insults. Here, we report that quantitation of post-transcriptional adenosine-to-inosine (A-to-I) RNA editing offers a distinct genome-wide signature, enabling the delineation of disease trajectories in the kidney. A well-defined murine model of endotoxemia permitted the identification of the origin and extent of A-to-I editing, along with temporally discrete signatures of double-stranded RNA stress and Adenosine Deaminase isoform switching. We found that A-to-I editing of Antizyme Inhibitor 1 (AZIN1), a positive regulator of polyamine biosynthesis, serves as a particularly useful temporal landmark during endotoxemia. Our data indicate that AZIN1 A-to-I editing, triggered by preceding inflammation, primes the kidney and activates endogenous recovery mechanisms. By comparing genetically modified human cell lines and mice locked in either A-to-I edited or uneditable states, we uncovered that AZIN1 A-to-I editing not only enhances polyamine biosynthesis but also engages glycolysis and nicotinamide biosynthesis to drive the recovery phenotype. Our findings implicate that quantifying AZIN1 A-to-I editing could potentially identify individuals who have transitioned to an endogenous recovery phase. This phase would reflect their past inflammation and indicate their potential for future recovery.

Orexin receptors regulate hippocampal sharp wave-ripple complexes in ex vivo slices.

Orexin is a neuromodulatory peptide produced by lateral hypothalamic orexin neurons and binds to G-protein-coupled orexin-1 receptor and orexin-2 receptors. Whether orexin modulates learning and memory is not fully understood. Orexin has biphasic effects on learning and memory: promoting learning and memory at homeostatic levels and inhibiting at supra- and sub-homeostatic levels. Hippocampal sharp wave-ripples encode memory information and are essential for memory consolidation and retrieval. The role of orexin on sharp wave-ripples in hippocampal CA1 remains unknown. Here, we used multi-electrode array recordings in acute ex vivo hippocampal slices to determine the effects of orexin receptor antagonists on sharp wave-ripples. Bath-application of either the orexin-1 receptor antagonist N-(2-Methyl-6-benzoxazolyl)-N'-1,5-naphthyridin-4-yl urea (SB-334867) or the orexin-2 receptor antagonist N-Ethyl-2-[(6-methoxy-3-pyridinyl)[(2-methylphenyl)sulfonyl]amino]-N-(3-pyridinylmethyl)-acetamide (EMPA) reduced sharp wave and ripple incidence, sharp wave amplitude, and sharp wave duration. SB-334867 and EMPA effects on sharp wave amplitude and duration were equivalent, whereas EMPA exhibited a greater reduction of sharp wave and ripple incidence. EMPA also increased ripple duration, whereas SB-334867 had no effect. Inhibition of both orexin receptors with a dual orexin receptor antagonist N-[1,1'-Biphenyl]-2-yl-1-[2-[(1-methyl-1H-benzimidazol-2-yl)thio]acetyl-2-pyrrolidinedicarboxamide (TCS-1102) had effects similar to EMPA, however, sharp wave amplitude and duration were unaffected. Region-specific expression of orexin receptors suggests orexin may regulate sharp wave generation in CA3, dentate gyrus-mediated sharp wave modification, sharp wave propagation to CA1, and local ripple emergence in CA1. Our study indicates an orexin contribution to hippocampal sharp wave-ripple complexes and suggests a mechanism by which sub-homeostatic concentrations of orexin may inhibit learning and memory function.

Ascorbic Acid Reduces Neurotransmission, Synaptic Plasticity, and Spontaneous Hippocampal Rhythms in In Vitro Slices.

Ascorbic acid (AA; a.k.a. vitamin C) is well known for its cellular protection in environments of high oxidative stress. Even though physiological concentrations of AA in the brain are significant (0.2-10 mM), surprisingly little is known concerning the role of AA in synaptic neurotransmission under normal, non-disease state conditions. Here, we examined AA effects on neurotransmission, plasticity and spontaneous network activity (i.e., sharp waves and high frequency oscillations; SPW-HFOs), at the synapse between area 3 and 1 of the hippocampal cornu ammonis region (CA3 and CA1) using an extracellular multi-electrode array in in vitro mouse hippocampal slices. We found that AA decreased evoked field potentials (fEPSPs, IC50 = 0.64 mM) without affecting V50s or paired pulse facilitation indicating normal neurotransmitter release mechanisms. AA decreased presynaptic fiber volleys but did not change fiber volley-to-fEPSP coupling, suggesting reduced fEPSPs resulted from decreased fiber volleys. Inhibitory effects were also observed in CA1 stratum pyramidale where greater fEPSPs were required for population spikes in the presence of AA suggesting an impact on the intrinsic excitability of neurons. Other forms of synaptic plasticity and correlates of memory (i.e., short- and long-term potentiation) were also significantly reduced by AA as was the incidence of spontaneous SPW-HFOs. AA decreased SPW amplitude with a similar IC50 as fEPSPs (0.65 mM). Overall, these results indicate that under normal conditions AA significantly regulates neurotransmission, plasticity, and network activity by limiting excitability. Thus, AA may participate in refinement of signal processing and memory formation, as well as protecting against pathologic excitability.

Protective Action of Hydrogen Sulfide-Releasing Compounds against Oxidative Stress-Induced Cataract Formation in Cultured Bovine Lenses.

PURPOSE: The gaseous signalling molecule, hydrogen sulfide (H2S) has antioxidant, anti-inflammatory and anti-apoptotic properties. Since oxidative stress has been implicated in the pathogenesis of cataracts and lenticular hydrogen peroxide (H2O2) is elevated in some cataract patients, the present study investigated the ability of H2S-releasing compounds to prevent H2O2-induced cataract formation in cultured bovine lenses. METHODS: Lenses were cultured in either Dulbecco's Modified Eagle Medium (DMEM; control); H2O2 (50 mM); ascorbic acid (AA; 3 mM) (positive control); and the H2S-releasing compounds (diallyl trisulfide [DATS] or GYY4137) in the presence of H2O2 (50 mM). Lens opacity was determined using a plate reader to measure transmittance. Lens glutathione content (GSH), superoxide dismutase (SOD) activity and lactate dehydrogenase (LDH) cytotoxicity were assessed before and after treatment with the H2S-releasing compounds. RESULTS: Both DATS (10-7M - 10-4M) and GYY4137 (10-7M - 10-4M) significantly (p < .001) attenuated H2O2 (50 mM)-induced loss in transmittance, with DATS (10-4M) and GYY4137 (10-7M) achieving a maximal reversal of opacity by 56.86 ± 0.01% (n = 6) and 8.39 ± 0.11% (n = 6) after 120 hours, respectively. These observations were corroborated by photographic evaluation, where DATS (10-5M - 10-4M) and GYY4137 (10-7M - 10-5M)-treated lenses had relatively clear grids after 120 hours, compared to H2O2 (50 mM)-treated lenses. The H2O2 (50 mM)-induced decline in total GSH content and total SOD activity were significantly (p < .001; n = 5) reversed by DATS (10-4M) and GYY4137 (10-7M). After 24 hours, DATS (10-4M) and GYY4137 (10-7M) significantly (p < .001; n = 4) reduced cytotoxicity of primary bovine lens epithelial cells by 33.88 ± 4.59% and 36.19 ± 10.53%, respectively. CONCLUSION: Both H2S-releasing compounds protected cultured bovine lenses against oxidative stress-induced cataract formation. The slow-releasing H2S compound, GYY4137 was more potent than DATS in restoring lenticular total GSH content and total SOD activity along with reducing H2O2 (50 mM)-induced cytotoxicity.

Carbamazepine Reduces Sharp Wave-Ripple Complexes and Exerts Synapse-Specific Inhibition of Neurotransmission in Ex Vivo Hippocampal Slices.

Higher therapeutic concentrations of the antiseizure medication carbamazepine (CBZ) are associated with cognitive side effects. Hippocampal sharp wave-ripple complexes (SPW-Rs) are proposed to participate in memory consolidation during periods of quiet and slow-wave sleep. SPW-Rs are generated in the CA3 region and are regulated by multiple synaptic inputs. Here, we used a multi-electrode array to determine the effects of CBZ on SPW-Rs and synaptic transmission at multiple hippocampal synapses. Our results demonstrate that CBZ reduced SPW-Rs at therapeutically relevant concentrations (IC50 = 37 µM) and altered the core characteristics of ripples, important for information processing and consolidation. Moreover, CBZ inhibited neurotransmission in a synapse-specific manner. CBZ inhibition was most potent at the medial-perforant-path-to-CA3 and mossy-fiber-to-CA3 synapses (IC50s ~ 30 and 60 µM, respectively) and least potent at medial-perforant-path-to-dentate granule cell synapses (IC50 ~ 120 µM). These results suggest that the synapse-specific CBZ inhibition of neurotransmission reduces SPW-Rs and that the CBZ inhibition of SPW-Rs may underlie the cognitive impairments observed with therapeutic doses of CBZ.

Current Trends in the Pharmacotherapy of Cataracts.

Cataracts, one of the leading causes of preventable blindness worldwide, refers to lens degradation that is characterized by clouding, with consequent blurry vision. As life expectancies improve, the number of people affected with cataracts is predicted to increase worldwide, especially in low-income nations with limited access to surgery. Although cataract surgery is considered safe, it is associated with some complications such as retinal detachment, warranting a search for cheap, pharmacological alternatives to the management of this ocular disease. The lens is richly endowed with a complex system of non-enzymatic and enzymatic antioxidants which scavenge reactive oxygen species to preserve lens proteins. Depletion and/or failure in this primary antioxidant defense system contributes to the damage observed in lenticular molecules and their repair mechanisms, ultimately causing cataracts. Several attempts have been made to counteract experimentally induced cataract using in vitro, ex vivo, and in vivo techniques. The majority of the anti-cataract compounds tested, including plant extracts and naturally-occurring compounds, lies in their antioxidant and/or free radical scavenging and/or anti-inflammatory propensity. In addition to providing an overview of the pathophysiology of cataracts, this review focuses on the role of various categories of natural and synthetic compounds on experimentally-induced cataracts.

Standardization of a new method for assessing the development of cataract in cultured bovine lenses.

PURPOSE: To standardize a new method for assessing cataractogenesis in isolated cultured bovine lenses using L-cysteine as the standard anti-cataract agent. METHODS: Intact bovine lenses were cultured in DMEM with L-cysteine in presence or absence of hydrogen peroxide (H2O2). Lens opacity (transmittance) was determined using a plate reader. Lens homogenate glutathione (GSH) and superoxide dismutase (SOD) contents were measured using enzyme immunoassays kits. RESULTS: DMEM-cultured lenses exhibited a time-dependent loss in transmittance (230-710 nm) up to 120 h, achieving the highest reduction of 38.6 ±â€¯0.09% at 420 nm (p < .001;n = 12). Compared to untreated lenses (time in hours [t] = 0), L-cysteine (10-6 M and 10-5 M) significantly (p < .001;n = 6) increased time-dependent transmittance (420 nm) by 31.6 ±â€¯0.17% and 28.0 ±â€¯0.07%(t = 120), respectively. When compared to DMEM-cultured lenses (t = 0), H2O2 (10 mM, 50 mM and 100 mM) significantly (p < .001;n = 12) reduced transmittance by 57.8 ±â€¯0.1, 57.4 ±â€¯0.04 and 87.7 ±â€¯0.6%(t = 120), respectively. Moreover, L-cysteine significantly (p < .001;n = 6) attenuated H2O2 (50 mM)-induced decrease in transmittance by 12.5 ±â€¯0.05%(10-6 M), 13.0 ±â€¯0.09%(10-5 M), 14.5 ±â€¯0.08%(10-4 M) and 8.6 ±â€¯0.11%(10-3 M)(t = 120), respectively. When compared to untreated lenses (t = 0), the time-dependent decrease (p < .001;n = 5) in lenticular total GSH content and total SOD activity of 46.1 ±â€¯0.06% and 42.0 ±â€¯1.65% (t = 120) was attenuated (p < .001;n = 5) by L-cysteine (10-6 M) by 76.6 ±â€¯0.06% and 7.4 ±â€¯1.98%, respectively. Similarly, the H2O2(50 mM)-induced decline (p < .001; n = 5) in total GSH content and SOD activity of 82.6 ±â€¯0.08% and 86.6 ±â€¯0.66% (t = 120) was attenuated by L-cysteine (10-4 M) by 74.7 ±â€¯1.05% and 161.1 ±â€¯4.9%, respectively. CONCLUSION: Measurement of spectral transmission coupled with assessment of the activity of antioxidant enzymes in bovine cultured lens can provide a useful tool in studies of cataracts in an animal model of this disease.

Preparation, Characterization and Antioxidant Evaluation of Poorly Soluble Polyphenol-Loaded Nanoparticles for Cataract Treatment.

Cataract, one of the leading causes of blindness worldwide, is a condition in which complete or partial opacity develops in the lens of the eyes, thereby impairing vision. This study aimed to examine the potential therapeutic and protective effects of poorly soluble polyphenols like curcumin, resveratrol, and dibenzoylmethane, known to possess significant antioxidant activity. The polyphenols were loaded into novel lipid-cyclodextrin-based nanoparticles and characterized by particle size, polydispersity index, differential scanning calorimetry, thermogravimetric analysis, X-ray diffraction, scanning electron microscopy (SEM), entrapment efficiency, and release studies. Ferric-reducing ability of plasma and 2,2-diphenyl-1-picrylhydrazyl chemical assays were used to evaluate their antioxidant properties based on their free radical quenching ability. Biochemical in vitro assays were used to examine these polyphenols on hydrogen peroxide-induced formation of cataracts in bovine lenses by estimating total glutathione content and superoxide dismutase activity. Nanoparticles were thermostable and amorphous. Particle size of curcumin, resveratrol, and dibenzoylmethane nanoparticles were 331.0 ± 17.9 nm, 329.9 ± 1.9 nm, and 163.8 ± 3.2 nm, respectively. SEM confirmed porous morphology and XRD confirmed physical stability. Entrapment efficiency for curcumin-, resveratrol-, and dibenzoylmethane-loaded nanoparticles was calculated to be 84.4 ± 2.4%, 72.2 ± 1.5%, and 86.4 ± 0.6%, respectively. In vitro release studies showed an initial burst release followed by a continuous release of polyphenols from nanoparticles. Chemical assays confirmed the polyphenols' antioxidant activity. Superoxide dismutase and glutathione levels were found to be significantly increased (p < 0.05) after treatment with polyphenol-loaded nanoparticles than pure polyphenols; thus, an improved antioxidant activity translational into potential anticataract activity of the polyphenols when loaded into nanoparticles was observed as compared to pure polyphenols.

Regulation of Excitatory Amino Acid Transmission in the Retina: Studies on Neuroprotection.

Excitotoxicity occurs in neurons due to the accumulation of excitatory amino acids such as glutamate in the synaptic and extrasynaptic locations. In the retina, excessive glutamate concentrations trigger a neurotoxic cascade involving several mechanisms, including the elevation of intracellular calcium (Ca2+) and the activation of α-amino-3-hydroxy 5-methyl-4-iso-xazole-propionic acid/kainate (AMPA/KA) and N-methyl-d-aspartate (NMDA) receptors leading to retinal degeneration. Both ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs) are present in the mammalian retina. Indeed, due to the abundant expression of GluRs, the mammalian retina is highly susceptible to excitotoxic neurodegeneration. Excitotoxicity has been postulated to present a common downstream mechanism for several stimuli, including hypoglycemia, hypoxia, ischemia, and chronic neurodegenerative diseases. Experimental approaches to the study of neuroprotection in the retina have utilized insults that trigger hypoxia, hypoglycemia, or excitotoxicity. Using these experimental approaches, the neuroprotective potential of GluR agents, including the NMDA receptor modulators (MK801, ifenprodil, memantine); AMPA/KA receptor antagonist (CNQX); Group II and III mGluR agonists (LY354740, quisqualate); and Ca2+-channel blockers (diltiazem, lomerizine, verapamil, ω-conotoxin), and others (pituitary adenylate cyclase activating polypeptide, neuropeptide Y, acetylcholine receptor agonists) have been elucidated. In addition to corroborating the exocytotic role of excitatory amino acids in retinal degeneration, these studies affirm that multiple mechanism/s contribute to the prevention of damage caused by excitotoxicity in the retina. Therefore, it is feasible that several pathways are involved in protecting the retina from toxic insults in ocular neurodegenerative conditions such as glaucoma and retinal ischemia. Furthermore, these experimental models are viable tools for evaluating therapeutic candidates in ocular neuropathies.

Regulation of Aqueous Humor Dynamics by Hydrogen Sulfide: Potential Role in Glaucoma Pharmacotherapy.

Hydrogen sulfide (H2S) is a gaseous transmitter with well-known biological actions in a wide variety of tissues and organs. The potential involvement of this gas in physiological and pathological processes in the eye has led to several in vitro, ex vivo, and in vivo studies to understand its pharmacological role in some mammalian species. Evidence from literature demonstrates that 4 enzymes responsible for the biosynthesis of this gas (cystathionine ß-synthase, CBS; cystathionine γ-lyase, CSE; 3-mercaptopyruvate sulfurtransferase, 3MST; and d-amino acid oxidase) are present in the cornea, iris, ciliary body, lens, and retina. Studies of the pharmacological actions of H2S (using several compounds as fast- and slow-releasing gas donors) on anterior uveal tissues reveal an effect on sympathetic neurotransmission and the ability of the gas to relax precontracted iris and ocular vascular smooth muscles, responses that were blocked by inhibitors of CSE, CBS, and KATP channels. In the retina, there is evidence that H2S can inhibit excitatory amino acid neurotransmission and can also protect this tissue from a wide variety of insults. Furthermore, exogenous application of H2S-releasing compounds was reported to increase aqueous humor outflow facility in an ex vivo model of the porcine ocular anterior segment and lowered intraocular pressure (IOP) in both normotensive and glaucomatous rabbits. Taken together, the finding that H2S-releasing compounds can lower IOP and can serve a neuroprotective role in the retina suggests that H2S prodrugs could be used as tools or therapeutic agents in diseases such as glaucoma.