Müller cell intracellular edema was detected in diabetic rat retina, which was alleviated by ranibizumab
In order to evaluated Müller cell intracellular edema in vivo, we adopted the published method by using semithin sections of the retina[19]. As shown in Fig. 1, Compared to normal control group, the fluid accumulation was detected between nuclei of the outer nuclear layer as strip-like morphology, indicating Müller cell apical processes swollen or dilated. The edema of Müller cells was alleviated after ranibizumab treatment (Fig. 1C).
The expression of Kir4.1 was down-regulated in rat retina with diabetes progression
The examination of protein expression of Kir4.1 in diabetic rat retinas showed that, compared with the control, the Kir4.1 level in diabetic rat retinas was decreased by 21.0% in 6-week (n = 6, p > 0.05, Fig. 2A) and 46.7% in 12-week (n = 4, p < 0.05, Fig. 2B), respectively.
The decreased expression of Kir4.1 in 12-week diabetic rat retina was also confirmed with immunofluorescence. As shown in Fig. 2C, in normal control, Kir4.1 is mainly expressed in the inner limiting membrane (ILM) and co-localized with GS, a specific marker for Müller cells. However, in diabetic retinas, the distribution of Kir4.1 was largely disrupted, extending from ILM to the outer limiting membrane (OLM), with weak immunostaining especially in ILM and around retinal blood vessels.
Ranibizumab increased the expressions of Kir4.1 and AQP4 in diabetic rat retina
To test the effect of ranibizumab on the expressions of Kir4.1 and AQP4, western blot was performed in 12-week diabetic rat retinas treated with or without ranibizumab. As showed in Fig. 3A, after ranibizumab treatment, the protein level of Kir4.1 was up-regulated by 47.5% (n = 7, p < 0.05) compared with that in diabetic rat. Similarly, the protein level of AQP4 in diabetic group was decreased significantly by 43.3% (n = 7, p < 0.05) compared to that in normal control group, which was up-regulated by 30.9% (n = 7, p < 0.05) after ranibizumab treatment (Fig. 3B). The protein expressions of GS and GFAP in the Müller cells in diabetic retinas were also evaluated and the data showed that the GS expression in diabetic retinas was decreased by 23.7% (n = 7, p < 0.05, Fig. 3C), while GFAP was increased by 222.7% (n = 7, p < 0.05, Fig. 3D), as compared with the control, indicating the activation of Müller cells with decreased function in metabolizing glutamate. However, ranibizumab has no effect on the expressions of GS and GFAP.
To further confirm the effect of ranibizumab on Kir4.1 and GFAP, we performed double immunostaining of both proteins in diabetic rat retinas treated with or without ranibizumab. As shown in Fig. 3E, in normal control, Kir4.1 was mainly expressed in the ILM and around the vessels, which co-localized with GFAP, another marker of Müller cells. However, in 12-week diabetic rat retinas, the decreased expression of Kir4.1 with its altered distribution was detected, attenuated staining pattern especially in ILM and around vessels. While GFAP immunostaining in Müller cells was increased in 12-week diabetic rat retinas with its characteristic radial immunostaining pattern. Ranibizumab treatment increased the expression of Kir4.1 as well as maintained its distribution to nearly normal level, but showed no effect on GFAP (Fig. 3E).
Ranibizumab decreased VEGF-A and increased the protein expressions of Kir4.1, AQP4 and Dp71 in glyoxal-treated rMC-1 cells
To further confirm above observation, we adopted glyoxal-treated rMC-1 cells to mimic diabetic condition. As shown in Fig. 4, the rMC-1 cells were treated with different doses of glyoxal (Fig. 4A) for different time points (Fig. 4B) to optimize the glyoxal treatment conditions. Cell viability was decreased dose-dependently by 0.3% (0.1 mM, p > 0.05), 5.2% (0.25 mM, p > 0.05), 15.3% (0.5 mM, p < 0.05), 23.4% (1 mM, p < 0.05) and 59.2% (2 mM, p < 0.05) when treated with different doses of glyoxal for 24 hours (n = 12). When the cells were treated with glyoxal (1 mM), the cell viability was slightly increased by 2% (1 hour, p > 0.05), then decreased by 3.1% (3 hours, p > 0.05), 6.4% (6 hours, p > 0.05), 16.1% (12 hours, p < 0.05), 23.4% (24 hours, p < 0.05) and 46.0% (36 hours, p < 0.05) at different time points (n = 12). Based on the result of cell viability, we chose 1 mM of glyoxal and 24 hours’ treatment for the following study.
When rMC-1 cells treated with glyoxal (1 mM) for 24 hours, the mRNA expression level of Kir4.1 was about 118.1% (p > 0.05, at 1 hour), 93% (p > 0.05, at 3 hour), 25.6% (p < 0.05, at 6 hour), 11.5% (p < 0.05, at 12 hour), 17.7% (p < 0.05, at 24 hour) of that in normal control (n = 6, Fig. 4C). The changes of Kir4.1 was also confirmed with WB, which showed that the protein level was decreased by 27.2% and 51.0%, separately, at 12 and 24 hours after glyoxal treatment (Fig. 4D).
To study the effect of ranibizumab on rMC-1 cells, glyoxal-treated rMC-1 cells were treated with or without ranibizumab and the changes of VEGF-A, Kir4.1, AQP4, Dp71 and GS were examined at both mRNA and protein levels. Although the cell viability was decreased in a time-dependent manner with glyoxal treatment (Fig. 4B), VEGF expression was increased at both 12 and 24 hours (Fig. 5A and B). The mRNA level of VEGF-A was increased by 54.4% (n = 6, p < 0.05) and 26.4% (n = 6, p < 0.05) at 12 and 24 hours in glyoxal-treated group (Fig. 5A). VEGF-A protein level was increased by 44.4% (n = 3, p < 0.05) and 78.9% (n = 3, p < 0.05), separately, at the same time points (Fig. 5B). VEGF-A level in the supernatant of cell culture was decreased significantly after ranibizumab treatment (n = 4, p < 0.05, Fig. 5C).
The mRNA levels of Kir4.1, AQP4, Dp71 and GS were also decreased significantly in glyoxal-treated group, i.e., decreased by 82.2% (Kir4.1, n = 8, p < 0.05, Fig. 6A), 71.1% (AQP4, n = 8, p < 0.05, Fig. 6D), 52.6% (Dp71, n = 8, p < 0.05, Fig. 7A) and 53.6% (GS, n = 8, p < 0.05, Fig. 7C), respectively; which were increased by 210.4% (Kir4.1,n = 8, p < 0.05, Fig. 6A), 65.0% (AQP4, n = 8, p < 0.05, Fig. 6D), 36.9% (Dp71, n = 8, p < 0.05, Fig. 7A) and decreased by 5.7% (GS, n = 8, p > 0.05, Fig. 7C), respectively, by ranibizumab. The changes of protein expression followed a similar pattern. The protein levels of Kir4.1, AQP4, Dp71, and GS were decreased by 36.0% (Kir4.1, n = 4, p < 0.05, Fig. 6B), 42.2% (AQP4, n = 4, p < 0.05, Fig. 6E), 41.4% (Dp71, n=4, p < 0.05, Fig. 7B) and 26.9% (GS, n=4, p < 0.05, Fig. 7D), respectively, in glyoxal-treated group, which were increased by 39.5% (Kir4.1, n=4, p < 0.05, Fig. 6B), 70.5% (AQP4, n=4, p < 0.05, Fig. 6E), 34.9% (Dp71, n=4, p < 0.05, Fig. 7B) and 2.4% (GS, n=4, p > 0.05, Fig. 7D), respectively, after treatment of ranibizumab. The changes of Kir4.1 (Fig. 6C) and AQP4 (Fig. 6F) were also confirmed with immunofluorescence.
Exogenous VEGF-A decreased the expression of Kir4.1 in rMC-1 cells
To study whether the increased VEGF-A in glyoxal-treated rMC-1 cells could decrease Kir4.1 expression, we treated rMC-1 cells with recombinant human VEGF-A (rh-VEGF-A). In Fig. 8A, cell viability was increased significantly with different doses of rh-VEGF-A treatment, e.g., the cell viability increased by 29.8% (n = 12, p < 0.05, 1 ng/mL), 32.9% (n = 12, p < 0.05, 10 ng/mL) and 32.4% (n=12, p < 0.05, 100 ng/mL). The protein expressions of Kir4.1 was decreased dose-dependently by rh-VEGF-A, i.e., decreased by 23.1% (n=4, p < 0.05, 50 ng/mL) and 38.6% (n=4, p < 0.05, 100 ng/mL), indicating the down-regulation of Kir4.1 might be partially caused by increased VEGF-A in glyoxal-treated rMC-1 cells (Fig. 8B). Since ranibizumab has no effect on cell viability (Fig. 8C), the increased Kir4.1 by ranibizumab further confirmed the causal effect of VEGF-A on Kir4.1. We also detected the changes of AQP4 and Dp71 under the treatment of rh-VEGF-A and found no significant change for these 2 proteins (Data not shown).
Ranibizumab decreased intracellular osmotic pressure by sodium efflux
To test whether ranibizumab could prevent Müller cell from intracellular edema through decreasing the osmotic pressure, we detected the intracellular potassium and sodium level with their corresponding indicators (PBFI and SBFI). After treatment with glyoxal (1mM) for 24 hours, the intracellular potassium level is increased significantly (n=10, p < 0.05), while the intracellular sodium level remained relatively unchanged (n=10, p > 0.05) compared with that in normal control group (Fig. 9). However, when treated with ranibizumab, intracellular sodium level, but not potassium, was decreased significantly (n=10, p < 0.05). This result indicated that, besides up-regulation of Kir4.1, decreasing intracellular osmotic pressure might be another mechanism for ranibizumab to prevent the cellular edema of Müller cells in DR. To further explore the possible reasons, we performed the western blot to detect the protein expression of Na+-K+-ATPase in glyoxal-treated rMC-1 cells with or without ranibizumab treatment. The data in Fig. 9C showed that, compared with that in normal control, the expression of Na+-K+-ATPase in glyoxal-treated group remained unchanged, while ranibizumab treatment increased the expression of Na+-K+-ATPase by 20.6% (n=4, p < 0.05). The detailed mechanisms need further exploration.