LncRNA PVT1 induces mitochondrial dysfunction of podocytes via TRIM56 in diabetic kidney disease

Figures

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  Fig. 1. DKD is associated with increased expression of PVT1 and mitochondria damage. A Levels of PVT1 in the plasma and kidney tissues from controls and patients with DKD. B The correlation between PVT1 levels and UACR (r = 0.535, P < 0.01) or Scr (r = 0.318, P = 0.029). C RNA-FISH combined with immunofluorescence illustrated the distribution of PVT1 and its relative position to podocin by confocal laser scanning microscope (n = 3). The podocytes were labeled with arrows. D Levels of Pvt1 in the kidney tissues from CTL mice and STZ mice at 6 weeks (n = 7), 8 weeks (n = 7), 12 weeks (n = 7), and 20 weeks (n = 5) after the onset of diabetes. E RNA-FISH combined with immunofluorescence illustrated the distribution of Pvt1 and its relative position to podocin in paraffin-embedded mouse sections by confocal laser scanning microscope (n = 5). The podocytes were labeled with arrows. F PAS staining of glomeruli from CTL mice and STZ mice at 20 weeks after DKD initiation (n = 5). G TEM image of podocytes, GBM, and mitochondria in primary podocytes from CTL mice and STZ mice at 20 weeks after DKD initiation (n = 5). H Measurement of the mitochondrial OCR of primary podocytes from CTL mice and STZ…

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  Fig. 2. PVT1 deletion ameliorated podocyte injury in STZ-induced diabetic mice. A Temporal changes in body weights were measured respectively at 4 weeks (n = 6), 6 weeks (n = 6), 8 weeks (n = 5), 12 weeks (n = 5), and 20 weeks (n = 5) after DKD initiation. *P < 0.05 vs CTL-Cre+/Pvt1+/+, #P < 0.05 vs STZ-Cre+/Pvt1+/+. B Kidney weight to body weight ratio was elevated in mice at 20 weeks after DKD initiation (n = 5). C Temporal urinary albumin-to-creatinine ratio (ACR) of mice were detected respectively at 6 weeks (n = 6), 8 weeks (n = 5), 12 weeks (n = 5), 16 weeks (n = 5), and 20 weeks (n = 5). *P < 0.05 vs CTL-Cre+/Pvt1+/+, #P < 0.05 vs STZ-Cre+/Pvt1+/+. D Representative fluorescence images of WT-1 in glomeruli isolated from the four groups at 20 weeks after DKD initiation (n = 5). Scale bar = 50 μm. E PAS staining of glomeruli from the four groups at 20 weeks after DKD initiation (n = 5). Scale bar = 50 μm. F TEM image of podocytes and, GBM, and mitochondria in primary podocytes from the four groups at 20 weeks after DKD initiation (n = 5). G Expression of TFAM, BAX, DRP1, and mitochondrial OXPHOS proteins in primary podocytes from the four groups at 20 weeks after DKD initiation (n =…

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  Fig. 3. Silence of PVT1 alleviated mitochondrial dysfunction and inflammation in high glucose-stimulated podocytes. A The RNA levels of PVT1 in podocytes under different conditions (n = 5). B Measurement of the mitochondrial OCR of podocytes under different conditions (n = 5). C Energy phenotype profile (EPP) was mentioned. D Production of mtROS in podocytes (n = 5). Scale bar = 50 μm. E Representative fluorescence images of MitoTracker in podocytes cultured under the indicated conditions (n = 4). Scale bar =50 μm. F qRT-PCR measurement of the mRNA levels of BAX in podocytes (n = 5) and levels of mtDNA in cytoplasm of podocytes (n = 4). G Representative fluorescence images of podocytes with anti-Tom 20 (red) and anti-dsDNA (green) antibodies (n = 4). Scale bar = 50 μm. H Expression of TFAM, BAX, and mitochondrial OXPHOS proteins in podocytes under different conditions by western blotting (n = 4). I Expression of p-p65, p65, IL-6, TNF-α, and CXCL10 in podocytes under different conditions. J Representative fluorescence images of podocytes with anti-p65 antibody (n = 3). Scale bar = 50 μm. K Expression of p-p65, p65, IL-6, and TNF-α in podocytes in the presence of EtBr (1.0 μg/ml) to block…

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  Fig. 4. PVT1 participated in high glucose-induced mitochondrial dysfunction and inflammation through promoting TRIM56 expression. A RNA-FISH revealed the location and expression of PVT1 under LG or HG conditions in podocytes (n = 4). Scale bar = 25 μm. B Silver staining of RNA pull-down in podocytes. The differentially expressed proteins have been indicated by arrows. C The eluted proteins from RNA pull-down assay were detected by SDS-PAGE by using anti-TRIM56 antibody (n = 3). D RIP assay using TRIM56 antibody was carried out in podocytes (n = 3). E PVT1 truncations (FL, ∆1, ∆2, and ∆3) were constructed, identified by PCR, and RNA pull-down assays were performd to map the binding region of PVT1 to TRIM56 in podocytes after transfection of Flag-TRIM56. F TRIM56 truncations (FL, #1, #2, and #3) were constructed and RNA pull-down assays were performd to map the binding region of TRIM56 to PVT1 in podocytes after transfection of indicated vectors. G Western blotting detected the expression of TRIM56 in podocytes under different conditions. H Western blotting detected protein levels of TRIM56 in podocytes after treatment with CHX (20 mg/ml) at the indicated time points and relative TRIM56…

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  Fig. 5. PVT1 enhances TRIM56 binding to AMPKα to promote its degradation via ubiquitin-dependent manner. A The interaction between endogenous TRIM56 and AMPKα in podocytes was evaluated by co-IP (n = 3). B Representative fluorescence images of PLA foci (red) represent the proximity of TRIM56 and AMPKα in podocytes (n = 7). Scale bar = 50 μm. C Expression of AMPKα in podocytes under different conditions (n = 3). D Western blotting detected the effect of TRIM56 on the expression of AMPKα in podocytes (n = 3). E Expression of AMPKα in podocytes under different conditions (n = 3). F The ubiquitination of AMPKα was evaluated in podocytes under different conditions (n = 3). G The ubiquitination of AMPKα was evaluated in the presence or absence of HA tagged WT or site-specific ubiquitin mutants of podocytes. H The ubiquitination of Myc-WT, -K60R, -K379R, or -K470R mutants of AMPKα was detected in podocytes under different conditions (n = 3). Error bars represent the mean± S.D, **P < 0.01, and ***P < 0.001.

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  Fig. 6. TRIM56 deficiency reversed podocytes injury and restored the AMPKα expression of glomeruli in STZ-induced diabetic mice. A. Expression of Trim56 and AMPKα were detected by western blotting in glomerulus isolated from Cre+/Trim56flox/flox mice and littermate controls (n = 3). B Temporal changes in body weights of the mice were measured respectively at 4 weeks (n = 6), 6 weeks (n = 6), 8 weeks (n = 6), 12 weeks (n = 6), and 20 weeks (n = 5) after DKD initiation. *P < 0.05 vs CTL-Cre+/Trim56+/+, #P < 0.05 vs STZ-Cre+/Trim56+/+. C Kidney weight to body weight ratio was elevated in mice at 20 weeks after DKD initiation (n = 5). D Temporal ACR of mice were determined respectively at 6 weeks (n = 6), 8 weeks (n = 6), 12 weeks (n = 6), 16 weeks (n = 6), and 20 weeks (n = 5). *P < 0.05 vs CTL-Cre+/Trim56+/+, #P < 0.05 vs STZ-Cre+/Trim56+/+. E Representative fluorescence images of WT-1 in glomeruli isolated from the four groups at 20 weeks after DKD initiation (n = 5). Scale bar = 50 μm. F PAS staining of glomeruli from the four groups at 20 weeks after DKD initiation (n = 5). Scale bar = 50 μm. G TEM image of podocytes, GBM, and mitochondria in primary podocytes from the four groups at 20…

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  Fig. 7. TRIM56-mediated podocytes mitochondrial dysfunction and inflammation via AMPKα in diabetic kidney disease. A. Measurement of the mitochondrial OCR of podocytes under different conditions (n = 3). B Representative fluorescence images of mtROS in podocytes (n = 3). Scale bar = 50 μm. C Representative fluorescence images of ΔΨm in podocytes (n = 3). Scale bar =50 μm. D Relative mRNA levels of IL-6 (n = 4), TNF-α (n = 3), and CXCL10 (n = 3) in podocytes cultured under the indicated conditions. E Expression of TFAM, PGC-1α, BAX, DRP1, and mitochondrial OXPHOS proteins in podocytes with different concentration of compound C (n = 3). F Expression of DRP Ser616 in podocytes with different concentration of compound C (n = 3). G Representative fluorescence images of MitoTracker in podocytes (n = 3). Scale bar =50 μm. H Representative fluorescence images of podocytes cultured under the indicated conditions with anti-Tom 20 (red) and anti-dsDNA (green) antibodies (n = 3). Scale bar =50 μm. I Levels of mtDNA in cytoplasm of podocytes under different conditions (n = 4). J Representative fluorescence images of podocytes with anti-p65 antibody (red) (n = 3). Scale bar =50 μm. K PAS staining of…