Protective potential of klotho protein on diabetic retinopathy: Evidence from clinical and in vitro studies

Abstract

Aims/introduction: The purpose of the present study was to observe the relationship between serum α-klotho (KL) protein level and diabetic retinopathy (DR), and to further examine the effects of KL protein on apoptosis induced by palmitic acid (PA) in human retinal endothelial cells.

Materials and methods: A total of 17 healthy people and 60 type 2 diabetes patients were included. According to the results from fundus fluorescein angiography, the diabetes patients were divided into three subgroups: without DR, non-proliferative DR and proliferative DR. Serum KL level was measured by enzyme-linked immunosorbent assay. In vitro, human retinal endothelial cells were exposed to PA with or without KL protein. Apoptosis rates were analyzed by flow cytometry analysis. Apoptotic-related protein expressions were detected by western blotting analysis.

Results: Serum KL level was lower in diabetes patients than that in healthy participants (P = 0.007), and was gradually decreased among the without DR, non-proliferative DR and proliferative DR subgroups (P = 0.045). A logistic regression analysis showed that after adjusting for the other confounding factors, serum KL level was independently and negatively related with DR (P = 0.049). Furthermore, the increased apoptosis rates induced by PA were inhibited with the addition of KL protein. Consistently, KL protein reversed the expression levels of the increased pro-apoptotic protein Bax and the decreased anti-apoptotic protein Bcl-2 induced by PA. However, the anti-apoptotic effect of KL protein was attenuated by LY294002 through the phosphatidylinositol 3 kinase-serine∕threonine kinase pathway.

Conclusions: The data suggested that KL protein was probably a potential protective factor against retinopathy in type 2 diabetes patients.

Keywords: Apoptosis; Diabetic retinopathy; α-Klotho.

Figure 1

Serum klotho (KL) levels in diabetes and diabetic retinopathy (DR) patients. (a) Comparison of serum KL levels between healthy control participants (NC group; n = 17) and diabetes patients (DM group; n = 60). (b) Comparison of serum KL levels among diabetes patients without diabetic retinopathy (NDR; n = 27), diabetes patients with non‐proliferative diabetic retinopathy (NPDR; n = 17) and diabetes patients with proliferative diabetic retinopathy (PDR; n = 16). Data are represented as box plots. A Mann–Whitney U‐test was used for comparisons of continuous variables between two groups. Kruskal–Wallis and stepwise–step‐down tests were used for multiple and pairwise comparisons. Statistical differences were defined by P‐values (two‐tailed) <0.05. ^† P < 0.05 versus NDR group.

Figure 2

Effects of different concentrations of palmitic acid (PA) on cell viability. human retinal endothelial cells were treated with different concentrations of PA (0, 200, 400, 800 μmol/L). After the treatments for 24 h, cell viability was assessed by cell counting kit‐8 assay. Data are represented as the mean ± standard error of the mean (n = 3). Analysis of variance and Student–Newman–Keuls tests were carried out for multiple and pairwise comparisons. Statistical differences were defined by P‐values (two‐tailed) <0.05. ^† P < 0.05 versus 0 μmol/L; ^‡ P < 0.05 versus 200 μmol/L; ^§ P < 0.05 versus 400 μmol/L. OD, optical density.

Figure 3

Effects of klotho (KL) protein on palmitic acid (PA)‐induced apoptosis in human retinal endothelial cells. Cells were pretreated with recombinant human KL protein (400 pmol/L) for 1 h and then treated with PA (400 μmol/L) for 24 h. (a,b) The cell apoptosis rate was detected using flow cytometry analysis. (c,d) Levels of Bcl‐2 and Bax were detected by western blot analysis. Data are represented as the mean ± standard error of the mean (n = 3). Analysis of variance and Student–Newman–Keuls tests were carried out for multiple and pairwise comparisons. Statistical differences were defined by P‐values (two‐tailed) <0.05. ^† P < 0.05 versus control (Ctr) group; ^‡ P < 0.05 versus PA group.

Figure 4

Klotho (KL) protein inhibited palmitic acid (PA)‐induced apoptosis by activating the phosphatidylinositol 3 kinase‐serine∕threonine kinase (PI3K/Akt) pathway in human retinal endothelial cells. Cells were pre‐incubated with the PI3K inhibitor, LY294002 (10 μmol/L), for 1 h, followed by co‐treatment with recombinant human KL protein (400 pmol/L) for 1 h and then with PA (400 μmol/L) for 24 h. (a,b) Level of p‐Akt was detected by western blot analysis. (c,d) Levels of Bcl‐2 and Bax were detected by western blot analysis. Data are represented as the mean ± standard error of the mean (n = 3). Analysis of variance and Student–Newman–Keuls tests were carried out for multiple and pairwise comparisons. Statistical differences were defined by P‐values (two‐tailed) <0.05. ^† P < 0.05 versus control (Ctr) group; ^‡ P < 0.05 versus PA group; ^§ P < 0.05 versus PA + KL group. LY, LY294002.