In silico and in vitro analysis of cation-activated potassium channels in human corneal endothelial cells.

The corneal endothelium is the inner cell monolayer involved in the maintenance of corneal transparence by the generation of homeostatic dehydration. The glycosaminoglycans of the corneal stroma develop a continuous swelling pressure that should be counteracted by the corneal endothelial cells through active transport mechanisms to move the water to the anterior chamber. Protein transporters for sodium (Na+), potassium (K+), chloride (Cl-) and bicarbonate (HCO3-) are involved in this endothelial "pump function", however despite its physiological importance, the efflux mechanism is not completely understood. There is experimental evidence describing transendothelial diffusion of water in the absence of osmotic gradients. Therefore, it is important to get a deeper understanding of alternative models that drive the fluid transport across the endothelium such as the electrochemical gradients. Three transcriptomic datasets of the corneal endothelium were used in this study to analyze the expression of genes that encode proteins that participate in the transport and the reestablishment of the membrane potential across the semipermeable endothelium. Subsequently, the expression of the identified channels was validated in vitro both at mRNA and protein levels. The results of this study provide the first evidence of the expression of KCNN2, KCNN3 and KCNT2 genes in the corneal endothelium. Differences among the level of expression of KCNN2, KCNT2 and KCNN4 genes were found in a differentially expressed gene analysis of the dataset. Taken together these results underscore the potential importance of the ionic channels in the pathophysiology of corneal diseases. Moreover, we elucidate novel mechanisms that might be involved in the pivotal dehydrating function of the endothelium and in others physiologic functions of these cells using in silico pathways analysis.

Diabetes Mellitus Type 1 has a Higher Impact on Corneal Endothelial Cell Density and Pachymetry than Diabetes Mellitus Type 2, Independent of Age: A Meta-Regression Model.

PURPOSE: Patients with diabetes mellitus (DM) often have keratopathy. However, the compromise of the corneal endothelium in type 1 DM (T1DM) and type 2 DM (T2DM) has so far not been well characterized. METHODS: We performed a systematic literature search to find articles on humans combining T1DM and/or T2DM and the corneal endothelium. The period was from inception to June 2020. The meta-regression evaluated the role of each type of DM on corneal endothelial cell density (CED) and pachymetry. The statistical models included age as a modulator to discriminate between the normal changes due to age and the effect of the disease and to determine the impact of the disease duration. RESULTS: The initial search identified 752 records, of which 17 were included in the meta-regression. Patients with T1DM had, on average, 193 cells/mm 2 lesser than control patients ( P < 0.00001). Patients with T2DM had 151 cells/mm 2 less compared with control patients ( P < 0.00001). The loss of corneal endothelial cells was expected because the aging was similar in patients with T1DM and T2DM and their control groups. Patients with T1DM and T2DM showed an increase in pachymetry versus control patients, and in both groups, it was associated with the duration of the disease. CONCLUSIONS: Both types of DM reduced CED and increased pachymetry. These differences were higher in patients with T1DM versus control patients than patients with T2DM versus control patients. In T1DM, CED reduction was not correlated with the time from diagnosis. In both groups, patients had CED reduction due to aging similar to that of their matched control patients.

Proteolytic hydrolysis and purification of the LRP/alfa-2-macroglobulin receptor domain from alpha-macroglobulins.

A new, easier and efficient purification method, using Sephacryl and DEAE-Sephacel, of the C-terminal fragment of two alpha-macroglobulins, alpha(2)-M and PZP, is presented. Two larger peptides were identified for each protein as the C-terminal fragment, with molecular weights of approximately 30 kDa and the N-terminal sequences were determined to be SSTQDTV for alpha(2)-M and VALHLS for PZP. The smaller peptides with molecular weights of 18 kDa correspond to a shorter C-terminal sequence of these proteins, and they were determined to be EEFPFA for alpha(2)-M and ALKVQTV for PZP, with no interfering sequences detected. The results confirmed the discriminatory capacity of the purification procedure and the purity of the fragments. This new methodology facilitates biological studies of alpha-macroglobulins, and will enable elucidation of the role the C-terminal region may exert to eliminate alpha-macroglobulin-proteinases complexes from the circulation by the LRP/receptor.