Upregulations of Clcn3 and P-Gp Provoked by Lens Osmotic Expansion in Rat Galactosemic Cataract

Hindawi Journal of Diabetes Research Volume 2017, Article ID 3472735, 8 pages https://doi.org/10.1155/2017/3472735 Research Article Upregulations of Clcn3 and P-Gp Provoked by Lens Osmotic Expansion in Rat Galactosemic Cataract 1 2 1 Lixia Ji, Lixia Cheng, and Zhihong Yang 1Department of Pharmacology, School of Pharmacy, Qingdao University, Qingdao, China 2Department of Endocrinology, People’s Hospital of Weifang, Weifang, China Correspondence should be addressed to Lixia Ji; [email protected] Received 21 September 2017; Accepted 1 November 2017; Published 21 November 2017 Academic Editor: Hiroshi Okamoto Copyright © 2017 Lixia Ji et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective. Lens osmotic expansion, provoked by overactivated aldose reductase (AR), is the most essential event of sugar cataract. Chloride channel 3 (Clcn3) is a volume-sensitive channel, mainly participating in the regulation of cell fundamental volume, and P-glycoprotein (P-gp) acts as its modulator. We aim to study whether P-gp and Clcn3 are involved in lens osmotic expansion of galactosemic cataract. Methods and Results. In vitro, lens epithelial cells (LECs) were primarily cultured in gradient galactose medium (10–60 mM), more and more vacuoles appeared in LEC cytoplasm, and mRNA and protein levels of AR, P-gp, and Clcn3 were synchronously upregulated along with the increase of galactose concentration. In vivo, we focused on the early stage of rat galactosemic cataract, amount of vacuoles arose from equatorial area and scattered to the whole anterior capsule of lenses from the 3rd day to the 9th day, and mRNA and protein levels of P-gp and Clcn3 reached the peak around the 9th or 12th day. Conclusion. Galactosemia caused the osmotic stress in lenses; it also markedly leads to the upregulations of AR, P-gp, and Clcn3 in LECs, together resulting in obvious osmotic expansion in vitro and in vivo. 1. Introduction Although galactosemic cataract and diabetic cataract are together called “sugar cataract” [7], most researchers prefer Sugar cataract is the major ocular complication of diabetes, to use galactose-induced cataract model to study the patho- which seriously impairs the ocular function of diabetic logical mechanism of lens osmotic expansion [1, 9–11]. subjects [1]. Hyperglycemia is thought to be the underlying In lenses, AR is primarily located in LECs, and its biologic factor of diabetic cataractogenesis [2]. Lens osmotic expan- activity in LECs is approximately 21-fold of that in cortical sion, a crucial and reversible process, greatly triggers the fibers [12]. In physiological conditions, almost no glucose rapid onset and accelerates the maturation of true cataract enters polyol pathway; but it will be markedly activated by [3]; thus, this is the critical stage for the prevention of diabetic hyperglycemia and even more than 30% glucose enters cataract. Cell swelling is the main feature of lens osmotic polyol pathway when blood glucose level is over 11.1 mmol/l expansion; several proteins such as AR, P-gp, and Clcn3 are [4, 13]. Activated AR converts amount of glucose to sorbitol, proposed to participate in this pathological mechanism. and the later does not readily diffuse across cell membrane AR catalyzes the NADPH-dependent reductions of a and is poorly further reduced by sorbitol dehydrogenase broad variety of aldehydes or ketones to their corresponding [4, 13]. Therefore, more and more hypertonic sorbitol alcohols [4–6]. AR is the rate-limiting enzyme of polyol path- accumulates in cytoplasm and absorbs water, leading to grad- way; its overactivation induced by hyperglycemia is the blast- ual swelling of lenticular cells [13]. In lenses, perhaps AR ing fuse of diabetic cataract [6]. Both galactose and glucose activation originally acts as homeostatic regulation against belong to AR substrates, but AR has higher affinity to galac- the high osmolality of extracellular glucose; nothing but the tose than to glucose, and galactitol is even more difficult to be overactivation of AR causes the accumulation of a large further metabolized than sorbitol, so galactosemic cataract majority of sorbitol accompanied by influx of much water, formed more rapidly than diabetic cataract model [3, 7, 8]. so LECs gradually swell and eventually cause cell rupture. 2 Journal of Diabetes Research Modulation of cell volume is highly conservative in evo- later, we observed that many LECs were growing below or lution to maintain constant cellular volume by regulating around the capsule tissue fractions, the 3rd to 8th passages internal ingredients to cope with the changed osmolarity of LECs were used in following studies. [14, 15]. Despite a matter of debate existed, most investiga- tors thought that modulation of cell volume mainly depended 2.2. Osmotic Expansion of LECs Induced by Gradient on the coordination of P-gp and Clcn3 [15, 16]. Galactose. The LECs were harvested and seeded on 6-well P-gp, a highly conserved membrane protein, is a member plates (20,000/well); 24 hrs later, galactose was added to final of ATP-binding cassette (ABC) transporter protein super concentrations of 0, 10, 20, 30, 40, 50, and 60 mM. After family [17–19]. In humans, P-gp is encoded by the multidrug 48-hour cultivation, we observed LECs’ morphology under resistance gene 1 (MDR 1); in rodents, mdr1a and mdr1b inverted microscope and harvested them. One part was genes are homologous to human MDR1 [17–19]. P-gp has used to extract total mRNA and detect mRNA expression already been approved to have bimodal functions; it not only by real-time RT-PCR; another part was used to extract serves as the transmembrane transporter being responsible total protein and measure targeted genes’ protein level by for the efflux of multidrugs in isotonic condition but also Western blot. For the assessment of cell viability, LEC cul- switches to a modulator of Clcn3 in hyperosmotic environ- tivation was carried out on 96-well plates, and cell viability ment [20, 21]. Reversible phosphorylation is the possible was monitored by using CCK-8 reagent (Dojindo, Japan). mechanism for functional shuttle of P-gp [16]. Upregulation of P-gp is closely linked to overactivated Clcn3 [10, 16], 2.3. Induction of Rat Galactosemic Cataract and Assessment which is a volume-regulated chloride channel. Clcn3 is highly of Lens Opacification [26]. Male Sprague-Dawley rats (21 associated with the regulation of fundamental volume in days old) with average body weight of 40 ± 4.07 g were pur- response to cell swelling or shrinking [15]. The opening of chased from the laboratory animal center at Qingdao Food Clcn3 allows the efflux of diverse osmolytes such as several and Drug Administration (SCXK-Lu-20140001; Qingdao, amino acids and polyols, leading to concomitant decrease China). They were treated according to the ARVO statement of cell volume [16, 22]. Anti-P-gp monoclonal antibody could for the Use of Animals in Ophthalmic and Vision Research. directly combine distinct epitopes of P-gp and obviously All the rats were randomized into two groups (n =24), and − inhibited Cl conductance induced by cell swelling [23]. they had free access to AIN-93 stock diet. While animals P-gp also expressed in lens [24] and was markedly upreg- drank different water, rats in normal group drank pure water, ulated in LEC layer isolated from 30% galactose-fed rats but rats in control group drank 12.5% galactose solution in on the 4th and 7th days [10]. Besides, P-gp transgenic the first 7 days and 10% galactose solution from the 8th day hamsters could develop cataracts resembling those in mice to the end; the whole duration was 18 days. Every 3 days, lens with congenital osmotic cataract [25]. P-gp should be con- opacification was evaluated (without anesthesia) by using a sidered as a modulator in lens osmotic expansion of sugar hand-held slit lamp (SL-15; Cowa, Japan) proceeded after full cataract [24]. However, administration of AR inhibitors to mydriasis with tropicamide hydrochloride. Cataract severity galactose-fed rats could reverse the upregulation of P-gp, was scored by the same experienced observer who was indicating a potential pathophysiological linkage between blinded to the identity of each individual animal. Lens the osmotic stress and P-gp stimulation [10]. opacification was graded into five stages as follows [8, 26]: On the basis of the previous theories and results, we grade 0, clear normal lenses; grade 1, vacuoles, located in hypothesize that lens osmotic expansion, imposed by AR the cortex, cover less than one-third of the lens anterior overactivation, would stimulate Clcn3 and its modulator segment, forming a subcapsular cataract; grade 2, vacuoles P-gp to delay the lenticular swelling. cover approximately two-thirds of the lens anterior segment; grade 3, diffuse opacity in cortex with/without some vacu- 2. Materials and Methods oles; grade 4, diffuse opacity in cortex and moderate nuclear opacity; grade 5, mature milky cataract is observed as a dense 2.1. Primary Culture of Beagle Dog LECs. The whole eyeballs opacity in both cortex and nucleus. After assessment of sugar fi were immediately enucleated from beagle dogs after being cataract, four rats of each group were sacri ced with CO2 on sacrificed; we soaked them into 75% ethanol for 10 minutes, the day of 3, 6, 9, 12, 15, and 18, respectively. Each time, we carefully isolated the lens by posterior method, and cut the promptly isolated the intact lenses by posterior approach, lens capsule into two parts at the equatorial zone. Due to weighed them, and observed the lens shape and transparency the specific distribution of monolayer LECs below the in vitro. At last, all the lens samples were thrown into lipid ° anterior capsule of lens, all anterior capsules were collected, nitrogen and stored at −80 C until further assay.

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