LncRNA evf‐2 Exacerbates Podocyte Injury in Diabetic Nephropathy by Inducing Cell Cycle Re‐entry and Inflammation Through Distinct Mechanisms Triggered by hnRNPU

Figures

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  Figure 1. Positive correlation between increased evf‐2 expression and podocyte injury in DN patients and STZ‐induced mice models. a) Paraffin‐embedded renal biopsies were used for immunohistochemistry staining of proliferating cell nuclear antigen (PCNA). Immunofluorescence images of Podocin (green), SYNPO (green), and B7‐1 (magenta), Mcp‐1 (magenta), EVF‐2 (magenta dots) in DN kidney tissue specimens. Bar = 20 µm. An enlarged section (dashed rectangle) is presented. Staining intensity was quantified and compared across samples (n = 3). *p < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001 (unpaired t‐test). b) Representative transmission electron microscopy (EM) micrograph illustrating binucleated podocytes in biopsies from DN patients. Bar = 2 µm. c) The urinary albumin/creatinine ratio (uACR) of mice in the STZ group is significantly higher than that of mice in the CON group. **p < 0.01, *** < 0.001 (STZ versus CON), ##p < 0.01, ### < 0.001 (different time points in the STZ group), (two‐way ANOVA plus Dunnett's multiple comparisons test). n = 5 mice/group. d) qRT‐PCR analysis of evf‐2 expression in isolated primary podocytes reveals significant increases in STZ‐induced mice. ***p < 0.001…

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  Figure 2. Podocyte‐specific knockdown of evf‐2 alleviates podocyte injury, inflammation, and proteinuria in STZ combined with HFD‐treated mice. a) The mice were categorized into three groups: saline treatment (STZ + saline), blank lentivirus treatment (STZ + con‐shRNA), and evf‐2 shRNA lentivirus treatment (STZ + evf‐2 shRNA). qRT‐PCR assays showing evf‐2 expression in isolated podocytes from three groups. n = 5 mice/group. b) uACR was assessed at various time points post‐injection for comparative analysis among the three groups. *p < 0.05, STZ + evf‐2 shRNA versus STZ + saline, #p < 0.05, ## < 0.01 (different time point in STZ + saline group), (two‐way ANOVA plus Dunnett's multiple comparisons test). n = 5 mice/group. c) A positive correlation was observed between uACR and evf‐2 expression levels in renal tissues. d and e) Random blood glucose (RBG) and kidney weight/body weight (KW/BW) ratio were compared among the three groups. n = 5 mice/group. f) Kidney tissues from mice treated with evf‐2 shRNA lentivirus were examined using Periodic Acid‐Schiff (PAS) staining and EM scanning after 12 weeks. Statistical analyses demonstrated significant changes in the number of foot processes per…

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  Figure 3. Podocyte‐specific overexpression of evf‐2 promotes podocyte injury, inflammation, and cell cycle re‐entry in control mice. a) Control mice were injected with saline (CON + saline), blank lentivirus (CON + con‐LV), or podocyte‐specific evf‐2 overexpression lentivirus (CON + evf‐2). qRT‐PCR assays showing evf‐2 expression in isolated podocytes from the three groups of mice. n = 5 mice/group. b) uACR was compared among the three groups at different times after injection. ****p < 0.0001, CON + evf‐2 versus CON + saline, #p < 0.05 (different time points in CON + evf‐2 group), (two‐way ANOVA plus Dunnett's multiple comparisons test). n = 5 mice/group. c) Positive correlation between uACR and evf‐2 expression levels in renal tissues. d and e) RBG and KW/BW were compared among the three groups. n = 5 mice/group. f) PAS staining and EM scanning analysis of podocyte injury in kidney tissues after 12 weeks of treatment with evf‐2 overexpression lentivirus. Bar = 20 µm. Red stars indicated thickened basement membrane; black triangles indicated podocyte foot process effacement. The statistical graphs depict the number of foot processes per micrometer GBM and the basement membrane thickness in…

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  Figure 4. Podocyte‐specific evf‐2 knockout alleviates podocyte injury and inflammation in STZ‐induced DN mice model. a) Visual comparison of the appearance of control (flox/flox) and podocyte‐specific evf‐2 knockout (evf‐2 KO) mice after STZ induction. b–d) Comparative analysis of RBG(b), uACR(c) and KW/BW(d) levels between the two groups of mice after 7 weeks of STZ injection. n = 6 mice/group. e) qRT‐PCR used to detect evf‐2 expression in primary cultured podocytes isolated from the glomeruli of evf‐2 KO and flox/flox mice after STZ induction. n = 6 mice/group. f) FISH on primary podocytes isolated from STZ‐treated evf‐2 KO mice and STZ‐treated control mice. Bar = 20 µm. g) PAS staining and EM scanning of kidney tissues revealing reduced damage in the kidney structures of the evf‐2‐KO mice following STZ treatment. The statistical chart compares the number of podocyte foot processes per micrometer GBM and the thickness of basement membrane between the two groups. Bar = 20 µm. h) FISH showing almost no expression of evf‐2 in the KO mouse glomeruli. Bar = 20 µm. The intensity of the staining is quantified and compared in different samples (n = 3). i) Positive staining for PCNA decreased in…

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  Figure 5. Podocyte‐specific knockout of evf‐2 alleviates podocyte injury and inflammation in LPS‐induced mouse model. a) The visual appearance of flox/flox and KO mice after LPS induction. b) qRT‐PCR was employed to detect evf‐2 expression in primary isolated podocytes of the glomeruli of KO or flox/flox mice after LPS induction. n = 3 mice/group. c) The body weight of flox/flox and KO mice after LPS induction. *p < 0.05, LPS + flox/flox versus flox/flox, (two‐way ANOVA plus Sidak's multiple comparisons test). n = 6 mice/group. d and e) Indicators of glomerular injury (d), including uACR and urinary immunoglobulin G/creatinine ratio (uIgG/cr), and indicators of tubular injury (e), such as urinary N‐acetyl‐β‐D‐glucosaminidase/creatinine ratio (uNAG/cr) and urinary retinol‐binding protein/creatinine ratio (uRBP/cr), were analyzed in the KO mice. n = 6 mice/group. f) Kidney images of flox/flox and KO mice after LPS induction. Hematoxylin‐eosin staining (HE) and EM analysis of kidney tissues. The statistical chart compares the number of podocyte foot processes per micrometer GBM between the two groups. Bar = 20 µm. g) FISH analysis of the KO mice glomeruli under LPS treatment. Bar = 20 µm. h)…

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  Figure 6. Overexpression of evf‐2 induces podocyte injury through cell cycle re‐entry and inflammation in vitro. a) Flow cytometry analysis of the cell cycle in the cultured mouse podocyte cell line (MPC5) under blank adenovirus (NG+con‐AD) and evf‐2 overexpression conditions (NG+evf‐2). The percentage of cells in the G2/M phase is presented in the right panel (n = 3). ***p < 0.001, (unpaired t‐test). b) Rhodamine phalloidin labeling revealed cytoskeletal damage in MPC cells in the NG group (5.6 mM), NG+con‐AD group, and NG+evf‐2 group. Representative photomicrographs of immunofluorescence staining for Podocin (green) and binucleation (DAPI) of podocytes. An enlarged section (dashed rectangle) is presented, white arrow indicates binucleated podocytes. Bar = 20 µm. c) The percentage of binucleated/multinucleated cells per visual field is presented (n = 3). **p < 0.01, (one‐way ANOVA plus Tukey's multiple comparisons test). d) Gene Ontology (GO) analysis of differentially expressed (DE) genes from RNA‐seq results performed on MPCs. e) Pathway analysis obtained for the enrichment score dot plot, showcasing the top ten pathways. f) Gene Set Enrichment Analysis (GSEA) of biological processes,…

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  Figure 7. Evf‐2 binds to proteins involved in mRNA processing or alternative splicing to induce podocyte cell cycle re‐entry or inflammation. a) GO and KEGG b) analyses of the RNA binding proteins of lncRNA evf‐2. c) Alternative splicing (AS) events analysis of RNA sequencing results performed on MPCs overexpressing evf‐2. d,e) The top 10 terms of GO (d) or KEGG (e) result of significantly different AS genes. f) The Venn result of significantly upregulated genes of RNA‐seq in mitotic cell cycle and significantly different AS genes. g) qRT‐PCR of splicing variants of Ccnb1 and Tacc3. *p < 0.05, ** < 0.01, *** < 0.001, (unpaired t‐test).

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  Figure 8. Knockdown of hnRNPU alleviates the cell cycle re‐entry and increased expression of inflammatory factors induced by evf‐2 overexpression. a) The predicted structure of hnRNPU protein by AlphaFold. This predicted hnRNPU protein consists of four functional regions: DNA binding region (cyan), ATP binding region (purple), RNA binding region (red), and the unknown functional region (yellow). b) Flow cytometry analysis demonstrated knockdown of hnRNPU partially reversed podocytes entering the G2/M phase in evf‐2 overexpressed podocytes. The quantitative analysis is shown in the right panel. c) Representative photomicrographs of rhodamine phalloidin staining and binucleation (DAPI) of podocytes in the evf‐2 + U‐siRNA (transfected with hnRNPU siRNA) and evf‐2 + NC group (transfected with siRNA NC). An enlarged section (dashed rectangle) is presented, white arrow indicates binucleated podocytes. Bar = 20 µm. d) qRT‐PCR analysis of cell cycle and inflammation‐related mRNAs indicated in Figure 6i. in podocytes overexpressing evf‐2 with or without knockdown of hnRNPU (evf‐2+siRNA NC or evf‐2+U‐siRNA). e and f) qRT‐PCR of two splicing isomers of Ccnb1 (e) and Tacc3 (f) and their respective…

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  Figure 9. HnRNPU directly binds to cell cycle‐related genes and inflammation factors through RNA and/or DNA binding sites. a) The Venn diagram depicts the results of ChIP‐sequencing and RIP‐sequencing conducted on podocytes overexpressing evf‐2 by using hnRNPU antibody. The most significantly upregulated genes in the cell cycle or inflammation, as selected from Figure 6h, are presented. b and c) GO (b) and KEGG pathway analysis (c) is performed on genes overlapping of ChIP and RIP assays. d) The binding regions and peaks of genes binding with hnRNPU in ChIP or RIP assays indicated in panel a. e) The gene plot results from RIP assays illustrate the genes binding with hnRNPU. f,g) The most probable specific motif sequence of RNA binding with hnRNPU was analyzed based on the results (f), revealing similarity to the binding motif predicted by mCrossBase (g). h) The gene plot results from ChIP assays illustrate the genes binding with hnRNPU. i, j) The top two binding motifs of target DNA.

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  Figure 10. Proposed mechanism by which lncRNA evf‐2 induces podocyte injury in DN. Left panel: Under normal conditions, lncRNA evf‐2 expression in renal podocytes is minimal, while hnRNPU is expressed at a basal level in healthy podocytes. Right panel: In DN, lncRNA evf‐2 expression is upregulated, binding with hnRNPU, thereby leading to increased expression of cell cycle‐related genes and inflammatory factors, possibly through transcriptional activation, alternative splicing, or RNA stability and translation regulation.