Figure 4.

VD-VDR restored defective autophagy induced by high glucose in HK-2 cells. (A) Expression of LC3 and SQSTM1 in HK-2 cells treated with low levels of glucose (LG, 5 mM), high levels of glucose (HG, 30 mM) with/without chloroquine (CQ, 40 μM) for 3 days. ACTB was used as the loading control. n = 3; a representative image is shown. *p < 0.05, **p < 0.01. (B) Expression of LC3 and SQSTM1 in HK-2 cells treated with LG, HG with/without paricalcitol (pari, 0.5 ng/ml) for 3 days. ACTB was used as the loading control. n = 3; a representative image is shown. *p < 0.05, **p < 0.01. (C) HK-2 cells were transfected with tfLC3, and then treated with LG, HG, HG+CQ or HG+pari. The autophagosomes were shown as yellow puncta with both mcherry (red) and GFP (green) labels, autolysosome were shown as red only puncta because after fusion with lysosomes, GFP loses its fluorescence in acidic PH. DAPI (blue) was used to stain nuclei. At least 10 microscopy fields were assessed in each experiment. The images are representative of three independent experiments. Scale bar, 5 μm. *p < 0.05, **p < 0.01. (D) Expression of CCL2, TNF and IL6 mRNA levels in HK-2 cells post treated with LG, HG, HG+CQ or HG+pari. n = 3. *p < 0.05, **p < 0.01.