Ocular Surface Disorder In Patients With Chronic Renal Failure And Its Mechanism In Vitro

Purpose:1.To investigate the ocular surface changes and tear dysfunction in patients with chronic renal failure(CRF)by non-invasive method,and to explore the role of corneal nerve in their ocular surface changes.2.To investigate the regulation effect of Substance P on the Akt/GSK-3β anti-apoptotic signaling pathway under high urea intervention of human corneal epithelial cells.Methods:Recruit 30 patients with chronic renal insufficiency and 30 age and sex matched normal subjects;1.Non-invasive ocular surface analysis of all subjects using Oculus Keratograph 5M,including non-invasive tear break up time,tear meniscus height,conjunctival hyperemia grading;Assess tear lipid layer thickness by LipiView;OSDI dry eye questionnaire for symptom evaluation;The cornea and conjunctival staining were performed as well.2.The corneal sensation was measured by Cochet-Bonnet aesthiometer;Heidelberg HRT3-CM in vivo confocal microscope was performed on all subject.Corneal subbasal nerve density,number of corneal nerves,mean nerve length,maximum and minimum nerve length were measured by NeuronJ software,nerve tortuosity,nerve reflectivity were also evaluated;Obtain the tear for substance P ELISA analysis.3.Human corneal epithelial cells were cultured in vitro and identified by immunohistochemistry.The corneal epithelial cells were divided into four groups:blank control group,20mmol/L high urea group,20mmol/L high urea+1μmol/L SP group,20mmol/L high urea+1μmol/L SP+1μmol/L Akt inhibitor group;CCK-8 kit for cell viability test;AnnexinV-FITC apoptotic kit for cell apoptosis test.Western-blot for detecting the expression of P-Akt,P-GSK-3β and Caspase3.Results:1.Shortening NI-BUT and increasing Ocular surface staining score were found in CRF patients,as well as the higher OSDI score and bulbar redness compared with the control group(P<0.05).2.The numeral and morphological changes of sub-basal nerve fibers in CRF were observed by in vivo confocal microscopy,presented as the lower nerve fiber density,lower mean nerve fiber length,lower nerve bundles,and higher nerve tortuosity(P<0.05).The corneal sensation also decreased in CRF patients,and the concentration of substance P in the tear was decreased compared to the control group(P<0.05).Human corneal epithelial cells were successfully cultured in vitro.Immunohistochemical method was used to identify corneal cytokeratin AE5 staining.CCK-8 kit assay showed that 20mmol/L high urea intervention decreased the viability of human corneal epithelial cells(P<0.05).Substance P could increase the cell viability under high urea environment,and Akti could antagonize the effect of substance P(P<0.05).Annexin V-FITC apoptosis assay showed that the apoptosis proportion of high urea group was significantly higher than that of the normal group(P<0.05).Substance P could reduce the cell apoptosis compared to high urea group(P<0.05).Akt inhibitor could antagonize the action of substance P;Western Blot was used to detect the anti-apoptotic effect SP.Increased expression of P-Akt,increased expression of P-GSK-3β,decreased expression of Caspase 3 was found in high urea+SP group,and Akti inhibitor could antagonize the effect of substance P on Akt/GSK-3β pathway.Conclusion:Non-invasive assessment to patients with chronic renal insufficiency and found ocular surface and tear function changes.In vivo confocal microscopy revealed morphological and numeral changes of sub-basal corneal nerves in chronic renal insufficiency.Decreased neurotrophic factor substance P in tears and decreased corneal sensation were detected in in patients with chronic renal insufficiency.In vitro cell experiment showed that substance P can protect corneal epithelial cells under high urea intervention by enhancing Akt/GSK3-β anti-apoptotic signaling pathway.