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

Figures

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  Fig. 1. The expression profiles of lncRNAs in the kidney tissue, plasma, and urine samples of diabetic nephropathy (DN) patients, diabetes mellitus (DM) patients, and normal healthy controls (CON). a Paraffin-embedded sections from renal biopsies were stained and analysed. Immunofluorescence assays showed a decrease in podocin (podocyte marker protein, green) and an increase in claudin-1 (podocyte injury-associated factor, magenta) DN kidney tissue specimens. The two markers are colocalized (yellow). An enlarged section (dashed rectangle) is shown. The intensity of the staining is quantified and compared in the different samples (n = 3). *P < 0.05, **P < 0.01 (unpaired t-test). b Paraffin-embedded sections from renal biopsies were stained and analysed. Immunofluorescence assays showed a decrease in synaptopodin (SYNPO, podocyte marker protein, green) and an increase in IL-17 (inflammatory factor, magenta) in the DN specimens. The two markers are colocalized (yellow). An enlarged section (dashed rectangle) is shown. The intensity of the staining is quantified and compared in the different samples (n = 3). *P < 0.05, **P < 0.01 (unpaired t-test). c–g The lncRNA profiles of the kidney…

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  Fig. 2. A correlation between increased lncRNA Dlx6-os1 expression and increased podocyte injury in the db/db mice model with diabetic nephropathy. a qRT-PCR assays showing a significant increase in lncRNA Dlx6-os1 expression in isolated podocytes from mouse kidney. *P < 0.05 vs. db/m (unpaired t-test). n = 6 mice/group. b FISH-based detection of the expression level and location of lncRNA Dlx6-os1 in paraffin-embedded db/db mouse kidney. The expression of lncRNA Dlx6-os1 in podocyte of db/db mice is upregulated (magenta dots increased), and the expression of podocyte marker protein podocin (green) was reduced. The intensity of the staining is quantified and compared in the different samples (n = 3). *P < 0.05, **P < 0.01, (unpaired t-test). Scale bar = 20 µm. Three visual fields were analyzed per sample. n = 3 mice/group. c Correlation between uACR and lncRNA Dlx6-os1 expression levels in isolated podocyte from db/db and db/m mouse kidney. d Western blots of podocin, B7-1, ccl-2, IL-17, and claudin-1 in isolated glomeruli from mouse kidney. GAPDH was used as the loading control. e Quantification from the western blot data shown in d. The intensity of the bands was analyzed in ImageJ and…

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  Fig. 3. Glomerular podocyte injury induced by lncRNA Dlx6-os1 overexpression in phenotypically normal db/m mice. a Treatment flow chart. Mice were on an adaptive feeding for 4 weeks before the experiment (−4 to 0 weeks). The db/m mice were randomly allocated to three groups: saline control (db/m + saline), blank virus control (db/m + con-LV), and lncRNA Dlx6-os1-overexpressing lentivirus (db/m + Dlx6-os1) groups. b–d The uACR of the (db/m + Dlx6-os1) mice is significantly higher than that of the (db/m + saline) mice (b), but there are no significant differences in the random blood glucose level (c) or kidney weight/ body weight (KW/BW) ratio (d). ****P < 0.0001. n = 5 mice/group (one-way ANOVA plus Dunnett’s multiple comparisons test). e Periodic acid-Schiff (PAS) staining and electron microscopy (EM) analysis of kidney tissues and podocytes from db/m mice 12 weeks after lentivirus injection. The statistical charts show the number of foot processes per micrometer and the thickness of the basement membrane. Five visual fields were analyzed per sample, PAS bar = 20 µm; EM bar = 1 µm). ****P < 0.0001 (unpaired t-test). n = 5 mice/group. f FISH-based detection of the location and level of…

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

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  Fig. 5. Establishment of a podocyte-specific lncRNA Dlx6-os1-knockout (KO) mouse model and development of streptozotocin (STZ)-induced diabetic kidney injury. a A flow chart showing the preparation of lncRNA Dlx6-os1-specific knockout mice. b Verification of gene knockout in knockout mice (Dlx6-os1 KO). Both floxed and cre bands are positive, while the negative results for the wild-type gene band confirmed podocyte-specific knockout of lncRNA Dlx6-os1. c qRT-PCR analysis of primary cultured podocytes isolated from the glomeruli of the knockout mice, compared to the control flox/flox mice, confirms the lack of lncRNA Dlx6-os1. ****P < 0.0001 vs. CON (unpaired t-test). n = 6 mice/group. d FISH on primary podocytes isolated from STZ-treated Dlx6-os1 KO mice and STZ-treated control mice (n = 3). Scale bar = 20 µm. e Flow chart showing the STZ-mediated induction of diabetes in Dlx6-os1 KO mice. Six-week-old male Dlx6-os1 KO mice or CON (flox/flox) mice were fed a high-fat diet. After given intraperitoneal injections of STZ (55 mg/kg, once a day) for five consecutive days, blood and urine samples were collected weekly; animals were sacrificed 7 weeks after the STZ injection. n = 6 mice/group.…

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  Fig. 6. LncRNA Dlx6-os1 expression level, localization, and its relationship with podocyte injury in cultured cells. a FISH analysis of lncRNA Dlx6-os1 expression and colocalization with DAPI in the mouse podocyte cell line MPC5 cultured under normal glucose (NG, 5.6 mM) and high glucose (HG, 30 mM) conditions. Images are representatives of three separate experiments. The intensity of the staining is quantified and compared in the different samples (n = 3). **P < 0.01, (unpaired t-test). b The expression of lncRNA Dlx6-os1 in MPC5 cells as determined by qRT-PCR. NG (5.6 mM), HG (30 mM) and HM (5.6 mM glucose and 24.4 mM mannitol) conditions. ****P < 0.0001 (unpaired t-test). n = 3 independent experiments. c Western blot of podocyte marker protein (podocin), injury-associated factors (claudin-1), and inflammatory factors (B7-1, IL-17, and ccl-2) of MPC5 podocytes under different glucose treatments. d Quantification of the western blot data shown in c. The intensity of the bands was analyzed in ImageJ and normalized to that of GAPDH (n = 3 independent experiments). *P < 0.05 (Two-way ANOVA plus Dunnett’s multiple comparisons test). e, f Immunofluorescence-based detection of the podocyte…

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  Fig. 7. RNA-seq results after lncRNA Dlx6-os1 overexpression in the cultured podocytes. a The RNA-seq and cluster analysis. NG, normal cultured podocytes; NG + OE, lncRNA Dlx6-os1-overexpressing podocytes. b Transcription coverage map. c A correlation coefficient heat map of samples. The shade of blue represents the degree of correlation. d The circle map of differential expression mRNA. e A differential expression mRNA volcano plot. f The BP-enrichment score chart. g The pathway analysis obtained for the enrichment score dot plot. The top ten pathways are shown. h–k Western blots of the GSK-3β protein of both cultured podocytes (MPC5) and isolated glomeruli from db/m and db/db mice under different treatments. Quantification of the western blot data were shown below. The intensity of the bands was analyzed in ImageJ and normalized to that of GAPDH (n = 3). **P < 0.01, ***P < 0.001 (one-way ANOVA plus Dunnett’s multiple comparisons test). l–o qRT-PCR analysis of GSK-3β gene expression in both cultured podocytes (MPC5, n = 3) and primary podocytes isolated from db/m and db/db mice under different treatments (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ##P < 0.01 (one-way…

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  Fig. 8. LncRNA Dlx6-os1 inducing glomerular podocyte injury by regulating the GSK-3β pathway, confirmed using podocyte-specific GSK-3β-knockout (cKO) mice. a Flow chart of the model establishment, monitoring, and sampling. b Comparison of the GSK-3β protein expression levels in glomeruli isolated from control (flox/flox) or GSK-3β-KO mice. c Expression of lncRNA Dlx6-os1 in glomeruli of CON or GSK-3β-KO mice injected with lncRNA Dlx6-os1-overexpression-lentivirus 5 weeks after injection assessed using qRT-PCR, compared to non-injected mice. **P < 0.01 vs. CON; ns no significant difference (one-way ANOVA plus Dunnett’s multiple comparisons test). n = 5. d A comparison of the urinary albumin/creatinine ratio (uACR) values. $P < 0.05 vs. (1-week CON + Dlx6-os1); $$P < 0.01 vs. (1-week CON + Dlx6-os1); #P < 0.05 vs. (1-week GSK-3β-KO + Dlx6-os1); *P < 0.05 vs. (5-week CON + Dlx6-os1). Two-way ANOVA plus Dunnett’s multiple comparisons test or Sidak’s multiple comparisons test (n = 5 mice per group). e Periodic acid-Schiff (PAS) staining and electron microscopy (EM) showed improved pathology in the kidney tissues of GSK-3β-KO mice, compared with the flox/flox control. PAS bar = 20 µm; EM bar =…

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  Fig. 9. The regulatory effects of lncRNA Dlx6-os1 on the expression of GSK-3β through miR-346. a Luciferase reporter gene expression confirming that wild-type (WT) Gsk-3β 3’UTR can bind miR-346-5p. Mu, Mutant. **P < 0.01, one-way ANOVA plus Tukey’s multiple comparisons test; n = 3. b Luciferase reporter gene expression confirming that wild-type (WT) Gsk-3β 3’UTR do not bind miR-346-3p. Mu Mutant, ns no significant difference. One-way ANOVA plus Tukey’s multiple comparisons test; n = 3. c Luciferase reporter gene testing confirming that the wild-type (WT) lncRNA Dlx6-os1 can directly bind miR-346-3p. Mu, Mutant. ***P < 0.001. One-way ANOVA plus Tukey’s multiple comparisons test; n = 3. d Luciferase reporter gene testing confirming that the wild-type (WT) lncRNA Dlx6-os1 cannot directly bind miR-346-5p, Mu Mutant, ns no significant difference (one-way ANOVA plus Tukey’s multiple comparisons test); n = 3. e FISH confirming the colocalization of lncRNA Dlx6-os1 and miR-346-5p in MPC5 podocytes under different glucose conditions. The lncRNA Dlx6-os1 expression level is increased, and the extent of their colocalization (yellow area) is increased in podocytes treated with high glucose (HG),…

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  Fig. 10. Proposed mechanism by which lncRNA Dlx6-os1 induces podocyte injury leading to diabetic neuropathy (DN). Left panel: Under normal circumstances, lncRNA Dlx6-os1 expression in renal podocytes is at a basal level, with no change or a slight reduction in diabetic individuals. Podocyte marker protein expression is normal, and inflammatory and injury-associated factors are rarely expressed. Right panel: In individuals with diabetic nephropathy, lncRNA Dlx6-os1 expression is increased, resulting in GSK-3β upregulation, thus leading to increased expression of inflammatory factors and cell damage-associated factors, podocyte injury and albuminuria.