Caracterización funcional del canal de potasio activado por calcio de conductancia intermedia (KCa3.1) en el endotelio de la córnea en condiciones fisiológicas y en ambientes hiperglúcidos
Thesis
The cornea is the lens that protects the anterior surface of the eye and its transparent nature is a crucial part of the functioning of the eye. This characteristic is mainly determined by the activity of the cells of its deepest layer, the corneal endothelium. This is a monolayer of hexagonal cells whose morpho-physiological characteristics allow it not only to compensate the natural hyperhydration of the superficial layers of the cornea, especially the stroma, but also mean it can be a key point for the entry and distribution of nutrients in the cornea. Since the proliferative potential of human corneal endothelial cells after birth is extremely limited, cell density progressively reduces during life, and to restore the tissue, adjacent cells must spread out and cover the area that has been damaged. Pathologies that directly or indirectly affect the corneal endothelium accelerate cell loss and generate dysfunction that ultimately leads to loss of corneal transparency, severely hampering vision, and the only treatment currently available is a transplant. In recent decades, diabetes mellitus (DM) has been identified as one of the systemic diseases that affect the corneal endothelium. In these patients, the literature has described increases in pachymetry, reductions in the endothelial cell count with respect to healthy people of the same age and sex, and even differences between diabetic patients depending on the time of disease progression. Furthermore, the alteration of the function of this ocular barrier and the stromal edema often persists even after surgical procedures. However, the pathophysiological mechanisms by which DM affects the corneal endothelium are poorly described. DM is one of a group of metabolic disorders whose sine qua non is hyperglycaemia. Although it is not the only cause of hyperglycaemia in humans, it is the one that is related to a persistent increase in glucose levels in the blood and other extracellular liquids. This leads to complications that disproportionally affect cells that, like the corneal endothelium, absorb glucose through glucose transporter 1 (GLUT1), that is to say through transporters that are independent of blood insulin levels, as occurs in erythrocytes, astrocytes, neurons and renal cells, cells which show certain similarity to eye tissue in terms not only of their embryonic origin (the endothelium derives from the neural crest), but also in their physiopathology, since there are clinical similarities between renal and ocular involvement Persistent exposure of cells in all tissues to high levels of glucose induces lesions that, in general, are related to an imbalance in which glucose and other metabolites become substrates for metabolic pathways that usually do not use them, favoring the development of morphological and functional alterations that, once they have occurred, are practically irreversible. However, although lesions related to diabetic microenvironments are well characterized for nervous, cardiovascular and renal tissues, the same is not true for the cornea and even less so for the corneal endothelium. Studies in various populations that have attempted to evaluate the impact of the disease on the endothelium, while consistent in terms of morphological changes, give discordant results in terms of endothelial count and pachymetry. Therefore, we considered it important to evaluate the impact of diabetes on the endothelium by means of statistical models that make it possible to discriminate the age-associated changes described for these cells; in particular, for this analysis it was important to identify the effect of the disease on endothelial cell density and its ability to maintain relative stromal dehydration and control corneal thickness. So, a statistical model was constructed, based on a meta-regression that included type of diabetes and age as modulators, to evaluate the real impact of each type of DM (type 1 DM and type 2 DM) on corneal endothelial density and corneal thickness, as determined by pachymetry. This analysis showed that the cell count was significantly reduced by the disease, especially in patients with type 1 DM in whom the compromise was independent of the duration of the disease, and that the increase in corneal thickness in diabetic patients was greater than expected by age, regardless of the type of diabetes. These results, which clinically demonstrated the impact of hyperglycemia on the endothelium, supported the need to evaluate in vitro the effect of high glucose concentrations on these cells, particularly on their proliferation capacity, their ability to migrate to cover a defect and in the induction of apoptosis, the main type of cell death identified so far in these cells, which has been described within the responses of endothelial cells to oxidative stress, a key condition in diabetes pathophysiology. To evaluate changes in the proliferation capacity of endothelial cells, cell cultures from an immortalized line were exposed to a medium defined for them as basal and to a medium with a high concentration of glucose (55mM). The rate of change in cell density was monitored by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assays. In these experiments, cells with active metabolisms convert MTT into a purple-colored product which, once solubilized, enables any change in the number of viable cells to be evaluated by colorimetry. The tests performed showed an increase in the number of cells in the cultures exposed to high concentrations of glucose (55mM), with a significant difference after 24 hours; however, although the difference remained after 48 hours, after that time the cell count measured indirectly by the colorimetric method progressively reduced until, after 5 days, it equaled the number of viable cells of the other group. The influence of different glucose concentrations on the capacity of the endothelium to close a scratch in the monolayer was performed using the method described by Liang et al. in 2007 (Liang et al., 2007), in which a scratch is created in a cell monolayer, and photographs are then taken at regular intervals from the moment the lesion is generated until the wound closes. This makes it possible to compare the time it takes for the endothelial cells to close the wound under different conditions and to quantify the rate of cell migration. In these experiments, there was a significant delay in closing the defect in cells exposed to high concentrations of glucose compared to cells in basal media. While the latter took approximately 5-6 days to close the scratch, by that time cells exposed to high glucose levels had closed, on average, 50% of the distance. Finally, the evaluation of apoptosis was performed using a commercial kit (Roche Cell Death Detection ELISA PLUS) that uses the sandwich ELISA (enzyme immunoassay) technique with monoclonal antibodies for histones to determine mono- and oligonucleosomes in the cytoplasmic fraction of the cell lysates in each medium. These experiments demonstrated that exposure to high concentrations of glucose for 24 hours induced three times more apoptosis than that occurring in cells under basal conditions. In the pathophysiology of the deleterious processes associated with diabetes, the importance of electrophysiological processes has recently been identified. In renal and microglial cells, it has been shown that calcium-activated potassium channels of intermediate conductance (KCa3.1) seem to play an important role. However, the calcium-activated potassium channel (KCa) family had not previously been described in corneal endothelium and it was necessary to identify which channels of this family were expressed in these cells. We started with a bioinformatic analysis that allowed the in silico identification of these channels, after which the presence of the small conductance calcium-activated potassium channels KCa2.2 and KCa2.3, the intermediate conductance one KCa3.1 and the sodium-activated potassium channel KNa2.1 (Slick) in corneal endothelial cells was verified in vitro by PCR and in some cases by Western blot and immunolabeling. While the present work was being produced, Anumanthan et al. (2018) published a paper evaluating KCa3.1 activity in the corneal stroma that included microphotographs showing KCa3.1 expression in the endothelium, which backed up the results obtained. We proceeded to study what functions KCa3.1 has in the endothelium under basal conditions and whether these were modified when the cells were exposed to high concentrations of glucose. These experiments, which included chemical stimulation and inhibition of the channel, allowed us to identify the involvement of KCa3.1 channels in the processes of migration, proliferation and apoptosis. KCa3.1, the intermediate conductance channel of the calcium-activated potassium channel family, can be activated by 1-ethylbenzimidazolin-2-one (EBIO-1) and can be selectively inhibited by 1-[(2-Chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34). These compounds were used to test the effect of channel stimulation and inhibition on the proliferation, migration and apoptosis of corneal endothelial cells exposed to both basal and hyperglycemic conditions. Channel stimulation with EBIO-1 had a significant inhibitory effect on endothelial cell proliferation when used at concentrations of 50, 100 and 200 µM, sufficient to counteract the proliferative effect identified under high glucose conditions during the first few days. Inhibition of the channel with TRAM-34 at concentrations of 2, 4 and 8 µM had the opposite effect, increasing cell proliferation under basal conditions, especially at the higher concentrations, and enhancing the proliferative effect identified under high glucose conditions, maintaining a higher number of viable cells for a longer time. As for migration, stimulation with EBIO-1 reduced the migration rate by approximately 50% under basal conditions and potentiated the effect seen under hyperglycemic conditions. In contrast, inhibition of KCa3.1 with TRAM-34 at 2 µM accelerated migration and even brought the migration rate of cells under high glucose conditions closer to those of basal cells, with smaller effects at higher concentrations. Finally, under basal conditions none of the concentrations described for EBIO-1 and for TRAM-34 increased the rate of apoptosis; however, this does happen in media with high glucose concentrations, EBIO-1 at concentrations of 100 µM and TRAM_34 at 4 µM. In conclusion, this work allowed us to identify the involvement of diabetes mellitus in the corneal endothelium by determining its role in the reduction of the density of this monolayer more than that physiologically expected by age, and its impact on the increase in pachymetry, in addition to identifying a more severe involvement in patients with type 1 diabetes mellitus compared to those with type 2. Additionally, we described for the first time the presence of small conductance calcium-activated potassium channels, the expression of the intermediate conductance channel and the existence of type 2 high conductance sodium-activated potassium channels in the corneal endothelium. Finally, we explored the participation of KCa 3.1 in the proliferation, migration and apoptosis of these cells and described their role as modulators of these processes in both basal and high glucose conditions, which is relevant in both physiological and pathological conditions, not only in relation to diabetes but probably in responses to other events.
