Material

CCK8 was purchased from Dojindo (Kumamoto, Japan). Malondialdehyde (MDA) and superoxide dismutase (SOD) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) to evaluate oxidative stress. 2′,7′-dichlorofluorescin diacetate (DCFH-DA), AKT, GSK3, caspase-3 and actin antibodies were obtained from Beyotime Institute of Biotechnology (Shanghai, China). P-AKT and P-GSK3β antibodies were purchased from Cell Signaling Technology, Inc (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM/High Glucose) was obtained from Thermo Fisher Scientific (Shanghai, China) as well as DMEM/Low Glucose.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Cells cultures

Rat glomerular mesangial cells were bought from Boster (Wuhan, China). We selected the 5^th–10^th generation of mesangial cells in the present study. The cells were cultured in DMEM (5.6 mmol/l glucose) supplemented with 10% fetal bovine serum (FBS), antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin), at 37°C in 5% CO₂ for 24 h.

The cultured cells were randomly divided into four groups: the normal glucose group (NG, 5.6 mmol/l glucose), the high glucose group (HG, 25 mmol/l glucose, and then normal glucose conditions incubated for the night), the glucose fluctuation group (FG, alternated 5.6 mmol/l glucose and 25 mmol/l glucose every 3 h, repeated 3 times, and then normal glucose conditions incubated for the night) and the mannitol group (MG, 5.6 mM glucose plus 24.4 mM mannitol as an osmotic pressure control).

Effects of fluctuation of blood glucose on mesangial cell viability

Cell viability was assayed by the CCK8 kit according to the manufacturer’s protocol [20]. Briefly, mesangial cells were seeded onto 96-well plates and every well contained 5 × 10³ cells. Then they were grown to 90% confluency. The cells were cultured for 12, 24, 48 and 72 h. Then, we added 10 µl of CCK8 reagent into every well. After 1–4 h of incubation at 37°C, cell viability was assessed by scanning with a microplate reader at 450 nm. There were at least 6 duplicate wells in each group.

Mesangial cell apoptosis using flow cytometry

Mesangial cell apoptosis can be detected using flow cytometry analysis [20]. In brief, the mesangial cells were seeded onto 96-well plates at 37°C in a humidified incubator containing 5% CO₂ for 48 h. The cells were washed once in phosphate-buffered saline (PBS), collected by trypsin digestion with 0.25% trypsin (without EDTA), washed twice in PBS, then 500 µl of binding buffer, 5 µl of annexin V-FITC and 5 µl of propidium iodide were added to each well. Then, the cells were incubated in the dark for 15 min at 37°C and apoptosis was immediately analyzed.

Effects of fluctuating blood glucose on ROS levels in mesangial cells

The level of intracellular ROS was determined by flow cytometry using the peroxide-sensitive fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA) as previously described [21]. In brief, mesangial cells were seeded in a 96-well culture plates and incubated with 10 µM DCFH-DA for 30 min at 37°C. Then, cells were harvested and washed three times with PBS, re-suspended in ice-cold PBS and placed on ice in a dark environment, and washed twice with DMEM deprived of serum to fully remove unbound ROS from the intracellular DCFH-DA. The fluorescent signal was recorded using fluorescence microscopy (488 nm filter; OLYMPUS IX-73, Japan).

Effects of fluctuating blood glucose on MDA level and SOD activity in mesangial cells

The MDA level and SOD activity were analyzed using specific reagents according to the protocols provided by the manufacturer. Briefly, mesangial cells were seeded into 96-well plates and every well contained 5 × 10³ cells. Cultured cells were incubated for 48 h. Then, we collected the supernatant to determine the MDA level and SOD activity.

Western blotting

Glomerular mesangial cells were plated in 100 mm dishes for western blot analysis. After 48 h cells were lysed with cell lysis solution. Lysate was centrifuged at 14 000 g for 30 min at 4°C, and the supernatant was stored at –80°C. The protein was loaded per lane, separated by SDS–PAGE gel and transferred onto an NC membrane and incubated with the primary antibody (1 : 500). After washing, the membranes were incubated for 2 h with secondary antibody (1 : 200). The immunoblots were scanned using Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD, USA).

Statistical analysis

At least six samples were selected from each group for experimental statistical analysis. Image J analysis software was used to analyze the Western blotting results. Data were expressed as mean ± SD. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test using SPSS 13.0 computer software. P < 0.05 was considered statistically significant.

Mesangial cell viability under glucose fluctuation at different time points

In order to elucidate the change of glomerular mesangial cells under glucose fluctuation at different time points, we examined glomerular mesangial cell viability using the CCK-8 method. As shown in Figure 1, mesangial cell viability in both the HG and FG groups was increased significantly in comparison with the NG group at 12 h, but there was no significant difference between the HG group and the FG group. However, mesangial cell viability was significantly decreased in the FG group compared with the NG and HG groups at 24 h, while no significant difference was found between the NG group and the HG group. When compared with the NG group, mesangial cell viability was decreased in the HG and FG groups at 48 h and 72 h; especially, the FG group had lower cell viability than the NG group. Moreover, there was no significant difference in mesangial cell viability between the NG group and the MG group at 12, 24 and 48 h. Nevertheless, mesangial cell viability in the MG group at 72 h was decreased significantly compared to the NG group. In general, these results indicated that fluctuating blood glucose obviously reduced mesangial cell viability from 48 h when compared with the HG group.

Figure 1

Effects of fluctuation blood glucose on mesangial cell proliferation. Cells incubated in different glucose conditions for 12, 24, 48 and 72 h were analyzed by CCK-8

Data are shown as mean ± SD. ^aP < 0.05 vs. the NG group, ^bp < 0.05 vs. the HG group and ^cp < 0.05 vs. the FG group by one-way ANOVA.

Fluctuating blood glucose increased glomerular mesangial cell apoptosis

Next, we detected glomerular mesangial cell apoptosis using flow cytometry. As shown in Figure 2, glomerular mesangial cell apoptosis in the FG group was significantly increased compared with either the NG group or the HG group, even though in the HG group it was higher than in the NG group. There was no significant difference between the NG group and the MG group. These results indicated that blood glucose fluctuation accelerated glomerular mesangial cell apoptosis.

Figure 2

Apoptosis of mesangial cells by flow cytometry

Data are shown as mean ± SD. ^aP < 0.05 vs. the NG group, ^bp < 0.05 vs. the HG group and ^cp < 0.05 vs. the FG group by one-way ANOVA.

Glucose fluctuation aggravated oxidative stress in glomerular mesangial cells

It is reported that oxidative stress plays an important role in glomerular mesangial cell injury in high sugar conditions [22]. In addition, fluctuating blood glucose aggravated renal injury by increasing the generation of oxidative stress [23]. As shown in Figure 3, the level of intracellular ROS in the HG group was increased in comparison with that in the NG group; the increase was more obvious in the FG group, and the ROS level in the FG group was higher than in the HG group. The MDA level in the HG group was significantly raised compared with the NG group; moreover the increase was more marked in the FG group. The SOD activity was significantly lower in the FG group compared with either the NG group or the HG group. These results suggested that glucose fluctuation aggravated the oxidative stress state in glomerular mesangial cells.

Figure 3

Effects of glucose fluctuation on intracellular ROS level, MDA level and SOD activity on mesangial cells

Data are shown as mean ± SD. ^aP < 0.05 vs. the NG group, ^bp < 0.05 vs. the HG group and ^cp < 0.05 vs. the FG group by one-way ANOVA.

The effect of glucose fluctuation on P-AKT, P-GSK-3β and cleaved caspase-3 expression in glomerular mesangial cells

Previous studies indicated that high glucose accelerated the glomerular mesangial cell death via the AKT signaling pathway [24, 25]. To explore the effect of glucose fluctuation on the AKT pathway in glomerular mesangial cells, we examined P-AKT, P-GSK-3β and cleaved caspase-3 expression levels in glomerular mesangial cells exposed to glucose fluctuation. As shown in Figure 4, there was no significant difference in the total AKT expression among the four groups. The level of P-AKT was lower in the HG group compared with that in the NG group, and the P-AKT level was lower in the FG group than that in the HG group. Moreover, high glucose could increase P-GSK-3β and caspase-3 activation in glomerular mesangial cells, but glucose fluctuation increase of P-GSK-3β and cleaved caspase-3 levels was more remarkable than stabilized glucose. These results indicated that glucose fluctuation accelerated glomerular mesangial cell apoptosis, which was linked to inhibiting decreased phosphorylation of AKT and increasing P-GSK-3β and cleaved caspase-3 levels.

Figure 4

Effects of glucose fluctuation on P-AKT, P-GSK-3b and cleaved caspase-3 levels. A – Representative bands of P-AKT and AKT. B – Representative bands of P-GSK-3b and GSK-3b. C – Representative bands of caspase-3. D – Analysis for optical density (OD) value of P-AKT, AKT, P-GSK-3b, GSK-3b and cleaved caspase-3 protein expression

The data are shown as mean ± SD and are expressed as fold difference vs. the NG group, ^bp < 0.05 vs. the HG group and ^cp < 0.05 vs. the FG group by one-way ANOVA.