Long noncoding RNA ENSRNOG00000037522 is involved in the podocyte epithelial‑mesenchymal transition in diabetic rats

Figure 1. Protein expression of α-SMA, vimentin, PODXL1 and nephrin, as examined by immunohistochemical assay. The α-SMA and vimentin expression levels were almost absent in the control group, but were highly expressed in diabetic kidneys at 1 and 6 weeks. However, PODXL1 and nephrin were intensely expressed in the control rats, but markedly downregulated in the kidneys following STZ injection. All experiments were performed in triplicate, and representative immunohistochemistry images are shown. α-SMA, α-smooth muscle actin; PODXL1, podocalyxin-like 1; STZ, streptozocin.

Figure 2. (A) Serum CysC, (B) serum β3-MG, (C) UmAlb and (D) BUN concentrations were determined by ELISA. These factors were increased in diabetic rats compared with the control rats. All measurements were performed in triplicate, and the results are presented as the mean ± standard deviation. *P<0.05 vs. control group. CysC, cystatin C; β2-MG, β2-microglobulin; UmAlb, urine microalbumin; BUN, blood urea nitrogen.

Figure 3. Morphology of podocytes on days 3 and 5 after incubation, and on day 7 after subculture was observed under a microscope (magnification, ×100). Scale bar, 50 µm. All experiments were performed in triplicate, and representative images are shown.

Figure 4. Nephrin, PODXL1, vimentin and α-SMA protein expression levels in the podocytes of diabetic and control rats were examined with western blot analysis. All experiments were performed in triplicate, and the results are presented as the mean ± standard deviation. *P<0.05 vs. control group. PODXL1, podocalyxin-like 1; α-SMA, α-smooth muscle actin.

Figure 5. Hierarchical clustering heat map revealing the distinct lncRNA expression profiles in the podocytes of the control and STZ-treated diabetic rats. After 6 weeks, the podocytes were obtained from control and STZ-treated diabetic rats and hierarchical clustering analysis was performed. The relative lncRNA expression is depicted according to the color scale. The red column indicates upregulation and the green column indicates downregulation. lncRNA, long noncoding RNA; STZ, streptozocin. Control 1, 2 and 3 indicated three representative podocytes samples from control rats; STZ 1, 2 and 3 indicated three representative podocytes samples from STZ-treated diabetic rats.

Figure 6. GO analysis of differentially expressed lncRNAs between the control and STZ-treated diabetic rats. Three domains were covered, including cellular component, molecular function and biological process. The GO analysis indicated that differentially expressed lncRNAs were mainly involved in the cell, cell part and cellular process. GO, Gene Ontology; lncRNA, long noncoding RNA; STZ, streptozocin.

Figure 7. Kyoto Encyclopedia of Genes and Genomes pathway analysis of differentially expressed lncRNAs between the control and STZ-treated diabetic rats. The three most enriched pathways were 'pathways in cancer', 'PI3K-Akt signaling pathway' and 'microRNAs in cancer'. lncRNA, long noncoding RNA; STZ, streptozocin.

Figure 8. (A) RT-qPCR was used to validate the 7 selected lncRNAs transcripts. *P<0.05 vs. control group. (B) Subsequent to treatment of podocytes with high glucose, RT-qPCR validation was performed for the 7 selected lncRNA transcripts. lncRNA ENSRNOG00000037522 was the most upregulated lncRNA in podocytes from the high glucose treatment group compared with the normal group. *P<0.05 vs. normal cells. All experiments were performed in triplicate, and the results are presented as the mean ± standard deviation. lncRNA, long noncoding RNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 9. (A) The knockdown efficiency of the lncRNA ENSRNOG00000037522 was examined with reverse transcription-quantitative polymerase chain reaction, and a significant abrogation of lncRNA ENSRNOG00000037522 was observed following transfection with siRNA1 or siRNA2. (B) Western blot analysis was conducted to determine the α-SMA, vimentin, nephrin and POXLD1 expression levels in lncRNA ENSRNOG00000037522 knockdown and control podocytes. (C) Protein levels were quantified by ImageJ software and are presented in the histogram. Podocytes transfected with NC siRNA served as the controls. All experiments were performed in triplicate, and the results are presented as the mean ± standard deviation. *P<0.05 vs. NC group. lncRNA, long noncoding RNA; NC, negative control; siRNA, small interfering RNA.