Fig. 4: Effects of lncRNA Dlx6-os1 knockdown on podocyte injury, inflammation, and proteinuria in db/db mice. | Cell Death & Disease

Fig. 4: Effects of lncRNA Dlx6-os1 knockdown on podocyte injury, inflammation, and proteinuria in db/db mice.

From: Long non-coding RNA DLX6-AS1 is the key mediator of glomerular podocyte injury and albuminuria in diabetic nephropathy by targeting the miR-346/GSK-3β signaling pathway

Fig. 4

a Treatment flow chart. The 10-week-old db/db mice were treated with lncRNA Dlx6-os1 shRNA; then, blood, urine, and kidney tissue samples were analyzed at 4- and 12- weeks post-injection. b qRT-PCR assays showing that lncRNA Dlx6-os1 expression is significantly reduced in the shRNA-treated db/db mice compared with that of the controls in isolated podocytes from mouse kidney. **P < 0.01 (unpaired t-test). n = 5 mice/group. c–e The mice were injected with saline (db/db + saline), blank lentivirus (db/db + con-shRNA), or lncRNA Dlx6-os1 shRNA lentivirus (db/db + Dlx6-os-shRNA). The urinary albumin/creatinine ratio (uACR), random blood glucose, and kidney weight/body weight (KW/BW) ratio were compared among the three groups at different times after injection. **P < 0.01 (one-way ANOVA plus Dunnett’s multiple comparisons test). n = 5 mice/group. f Periodic acid-Schiff (PAS) staining and electron microscopy (EM) analysis of podocyte injury in kidney tissues 12 weeks after treatment with lncRNA Dlx6-os1 shRNA lentivirus. The below statistical graphs show the number of foot processes per micrometer and the basement membrane thickness of the db/db mice under different treatments. Five observation fields are collected per sample, PAS bar = 20 µm; EM bar = 1 µm. ****P < 0.0001 (unpaired t-test). n = 5 mice/group. g FISH analysis in paraffin-embedded kidney tissue samples. The lncRNA Dlx6-os1 shRNA-treated mice show decreased lncRNA Dlx6-os1 expression in podocytes (magenta dots) and increased podocin (green) and WT-1 expressions (green) (n = 3). Scale bar = 20 µm. h Western blot to detect the expression levels of podocin, B7-1, ccl-2, IL-17, and claudin-1 of isolated glomeruli from mouse kidney. GAPDH was used as the loading control. n = 3 mice/group. i Quantification from the western blot data shown in h. The intensity of the bands was analyzed in ImageJ and normalized to that of GAPDH (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way ANOVA, plus Dunnett’s multiple comparisons test). j–l Comparison of podocin (j, magenta), SYNPO (synaptopodin, k and l, magenta), claudin-1 (j, green), B7-1 (k, green), and IL-17 (l, green) expression levels and localization sites in OCT-embedded frozen kidney tissues (n = 3). Scale bar = 20 µm.

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