Increased long noncoding RNA maternally expressed gene 3 contributes to podocyte injury induced by high glucose through regulation of mitochondrial fission

Increased long noncoding RNA maternally expressed gene 3 contributes to podocyte injury induced by high glucose through regulation of mitochondrial fission

lncRNA Meg3 expression is enhanced in human podocytes cultured with high glucose and podocytes of STZ-induced miceA lncRNA array was used to identify lncRNAs implicated in the regulation of human podocytes with high glucose (HG). The expression of Meg3 was higher in human podocytes treated with HG relative to control (Fig. 1a); this was confirmed by real-time PCR analysis (Fig. 1b). Quantitative real-time PCR (qRT-PCR), fluorescence in situ hybridization (FISH), and immunofluorescence (IF) analyses showed increased expression of Meg3 in podocytes from STZ-induced diabetic mice compared with control mice. Meg3 was primarily localized in the cytoplasm of podocytes (Fig. 1c, d). Furthermore, WT1 IF staining was performed to quantify podocyte number per glomerular cross-section18. STZ-induced mice had a significantly decreased number of podocytes per glomerular section compared with control mice, and a negative correlation between the number of podocytes and the expression of Meg3 in podocytes was found (Fig. 1e–g). These data suggest that expression of Meg3 in podocytes is increased and might be involved in podocyte injury in diabetic mice.Fig. 1: LncRNA Meg3 expression is increased in podocytes in diabetic kidney disease.a Heat map of expression of lncRNAs in human podocytes cultured with NG or HG for 72 h. b Expression of lncRNA Meg3 in human podocytes was upregulated by HG in a time-dependent manner. c Expression of lncRNA Meg3 in podocytes isolated from mice. d Confocal FISH + IF images showing localization and expression of lncRNA Meg3 in podocytes of kidney glomeruli from diabetic (STZ-induced) mice compared with glomeruli from non-diabetic (wild-type) control mice (original magnification, ×630, scale bar, 20 μm). e Confocal IF images showing WT1-stained kidney cortex sections (original magnification, ×630, scale bar, 20 μm). f The number of podocytes per glomerular cross-section was detected using WT1 antibody in 50 randomly selected glomeruli per mouse in each group. g Correlation of lncRNA Meg3 with the number of podocytes in a partial correlation analysis. Data were shown as mean ± SD in all statistical graphs (n = 6 mice per group), *P < 0.05, **P < 0.01, ****P < 0.0001.Generation of podocyte-specific lncRNA Meg3 knockdown miceTo assess the contribution of lncRNA Meg3 in mitochondrial fission and podocyte damage in vivo, podocyte-specific Meg3 knockdown mice were generated using the Cre-loxP system (Fig. 2a–c). To further confirm conditional knockdown of Meg3 in podocytes, FISH, IF, and qRT-PCR analyses were performed. Meg3 expression was decreased in podocytes of Cre+Meg3fl/fl mice relative to Meg3+/+ mice (Fig. 2d, e), while no difference on Meg3 expression was found in tubular cells, liver, heart, and spleen between Cre+Meg3fl/fl and Meg3+/+ mice (Supplementary Fig. 1).Fig. 2: Generation of podocyte-specific knockdown of LncRNA Meg3 mice.a Schematic diagram of target strategy of Meg3 deficiency in podocytes using the Cre-loxP system. b The breeding strategy to generate podocyte-specific Meg3-deficient mice (Cre+Meg3fl/fl mice) and the actual appearance of Meg3+/+ mice and Cre+Meg3fl/fl mice were shown. c PCR genotyping of Meg3+/+ mice and Cre+Meg3fl/fl mice by tail RNA at the age of 3 weeks. d Representative images of immunofluorescence (IF) staining for Synaptopodin (green) and fluorescence in situ hybridization (FISH) for Meg3 (red) in glomeruli from Meg3+/+ mice and Cre+Meg3fl/fl mice (original magnification, ×630, scale bar, 20 μm) (n = 6 mice per group). e Total RNA was extracted from primary podocytes isolated from Cre+Meg3fl/fl mice and Meg3+/+ mice, and real-time PCR was performed for Meg3 expression (n = 6 mice per group). Data were presented as mean ± SD in all statistical graphs (n = 6 mice per group). ****P < 0.0001.Knockdown of lncRNA Meg3 in podocytes ameliorated albuminuria and renal dysfunction of STZ-induced diabetic miceTo determine the effects of podocyte-specific Meg3 deficiency under diabetic conditions, we generated a diabetic state by intraperitoneal injection of STZ into Meg3+/+ and Cre+Meg3fl/fl mice. Diabetic mice exhibited lower body weights and higher blood glucose, but no significant differences were observed between the two groups (Fig. 3a, b). Higher UACR, kidney/body weight ratio, and increased levels of urine albumin excretion (UAE) and serum blood urea nitrogen were detected in diabetic mice; all of these parameters in diabetic Cre+Meg3fl/fl mice were significantly lower than in diabetic Meg3+/+ mice (Fig. 3c–f). These data suggest that Meg3-specific knockdown in podocytes can improve physiological parameters independent of blood glucose in diabetic mice.Fig. 3: Knockdown of lncRNA Meg3 in podocytes ameliorated albuminuria and renal dysfunction in STZ-induced diabetic mice.a Changes of body weight measured every 2 weeks during the 12-week follow-up period. b Changes of blood glucose measured every 2 weeks during the 12-week follow-up period. c Changes of urinary urine albumin/creatinine ratio (UACR) in each group during the 12-week follow-up period. d Changes of kidney weight/body weight ratio in each group. e Changes of urine albumin excretion at the time of 12 weeks after the establishment of hyperglycemia. f Changes of blood urea nitrogen (BUN) in each group. Data were shown as mean ± SD in all statistical graphs (n = 6 mice per group). *P < 0.05 and ****P < 0.0001.Knockdown of Meg3 in podocytes improved glomerular injury and podocyte mitochondrial fission in STZ-induced diabetic miceTo ascertain the influence of Meg3 knockdown in podocytes on renal pathology under diabetic conditions, histopathological and morphometric analyses were performed. Diabetic mice exhibited more severe mesangial matrix expansion and glomerulosclerosis, increased glomerular basement membrane thickness, and aggravated podocyte foot process effacement, and these pathological features were improved in diabetic Cre+Meg3fl/fl mice (Fig. 4a, c–f). Using transmission electron microscopy (TEM), more fragmented mitochondria were observed in podocytes from diabetic states, demonstrated by the lower aspect ratio (AR) and form factor of podocyte mitochondria, while more elongated mitochondria with a higher AR and form factor were found in podocytes from diabetic Cre+Meg3fl/fl mice relative to diabetic Meg3+/+ mice (Fig. 4b, g–l). These data indicate that Meg3-specific knockdown in podocytes can improve renal pathology and mitochondrial fission in diabetic mice.Fig. 4: Glomerular injury and podocyte excessive mitochondria fusion were attenuated in STZ-induced diabetic mice with podocyte-specific lncRNA Meg3 knockdown.a Representative images of HE, periodic Acid-Schiff (PAS), PASM, and Masson staining in kidney sections (original magnification, ×400; scale bar, 20 μm). b Representative images of transmission electron microscopy (TEM) visualizing the glomerular ultrastructure (top panel, original magnification, ×12,000). Representative TEM images in lower panels display mitochondrial shape in podocytes (original magnification, ×30,000). The red dotted lines indicated the mitochondria, with a double membrane. c Comparison of mesangial matrix index in each group. d Comparison of Masson Trichrome staining area in each group. e Comparison of glomerular basement membrane (GBM) thickness in each group. f Degree of podocyte foot process effacement was quantified by assessing foot process width using TEM. g Comparison of mitochondria aspect ratio (AR) in podocytes of different group. h Comparison of mitochondria form factor in podocytes of different group. i Comparison of mitochondrial length in podocytes of a different group. j Comparison of the mitochondrial area in podocytes of a different group. k Comparison of mitochondrial circularity in podocytes of a different group. l Comparison of the mitochondrial perimeter in podocytes of a different group. Two hundred mitochondria were randomly selected in each group when mitochondrial morphology analyses were done. Normally distributed data were shown as mean ± SD, and non-normally distributed data were shown as median (interquartile range) in all statistical graphs (n = 6 mice per group). **P < 0.01, ***P < 0.001, and ****P < 0.0001.Knockout of Meg3 attenuated HG-induced mitochondrial fission and cell injury in human podocytesTo determine the role of Meg3 in mitochondrial fission and cell injury in podocytes induced by HG, we generated a stable human podocyte cell line for knockout (KO) of Meg3 (Supplementary Fig. 2A–D and Fig. 5a, b). Consistent with our previous report19,20, cultured human podocytes were injured by HG, as demonstrated by lower expression of Nephrin and Synaptopodin (Fig. 5c, e). At the same time, highly fragmented and discrete mitochondria were observed in podocytes incubated with HG (Fig. 5d, f), while podocyte injury was attenuated in the absence of Meg3. Moreover, less fragmented, more elongated, and interconnected mitochondria were observed in Meg3 KO podocytes. The trend was the same in the detection of mitochondrial membrane potential and ATP (Supplementary Fig. 2E–G). These data indicate that downregulation of Meg3 might protect against excessive mitochondrial fission and podocyte injury in HG conditions.Fig. 5: Knockout of lncRNA Meg3 attenuates high glucose-induced mitochondrial fission and cell injury in human podocytes.a Schematic diagram of target strategy of Meg3 knockout in human podocytes using the double-nickase/CRISPR-Cas9 System. Two pairs guide RNAs targeted to the promoter region and exon 3 of lncRNA Meg3, and the target sequence is approximately 3500 bp. b Expression of lncRNA Meg3 in human podocytes of different groups. c Expression of Nephrin and Synaptopodin in human podocytes of different groups. d Representative structured of mitochondria of human podocytes were stained by MitoTracker Deep Red and imaged by laser scanning confocal microscopy (original magnification, ×630; scale bar, 10 μm). e Quantitative analyses of expression of Nephrin and Synaptopodin in human podocytes of different groups. f Quantitative analyses of mitochondrial organization in human podocytes of different groups (mean branch length, mean number of branches per network, and mitochondrial footprint, n = 10 cells per group). Data were shown as mean ± SD in all statistical graphs. *P < 0.05, **P < 0.01, and ***P < 0.001 (n = 6 per group).Overexpression of Meg3 aggravated HG-induced mitochondrial fission and cell injury in human podocytesTo further confirm the role of Meg3 in mitochondrial fission and podocyte damage, we generated Meg3-overexpressing (OE) podocytes by infection with lentivirus-expressing Meg3 (Supplementary Fig. 3A–C and Fig. 6a). lncRNA Meg3 OE cells were damaged more severely compared with control cells under HG or normal glucose (NG) conditions, as demonstrated by decreased expression of Nephrin and Synaptopodin (Fig. 6b, d). In addition, more highly fragmented and discrete mitochondria were found in Meg3 OE cells, including significantly lower branch lengths, network sizes, and footprints (Fig. 6c, e). The generation of ATP was also dramatically reduced in lncRNA Meg3 OE cells compared with WT podocytes incubated in HG (Supplementary Fig. 3D). Collectively, these results suggest the possible roles of higher lncRNA Meg3 expression in the induction of mitochondrial fission and podocyte injury in HG.Fig. 6: Overexpression of lncRNA Meg3 aggravated high glucose-induced mitochondrial fission and cell injuries in human podocytes.a Expression of lncRNA Meg3 in Meg3 overexpression podocytes compared with the control. b Expression of Nephrin and Synaptopodin in human podocytes of different groups. c Representative structured of mitochondria of human podocytes were stained by MitoTracker Deep Red and imaged by laser scanning confocal microscopy (original magnification, ×630; scale bar, 10 μm). d Quantitative analyses of expression of Nephrin and Synaptopodin in human podocytes of different groups. e Quantitative analyses of mitochondrial organization on human podocytes from different groups (mean branch length, mean number of branches per network and mitochondrial footprint, n = 10 cells per group). Data were shown as mean ± SD in all statistical graphs. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (n = 6 per group).lncRNA Meg3 promotes mitochondrial fission in podocytes by regulation of Drp1 and its translocation to mitochondriaWe then assessed the role of Meg3 in mitochondrial fission in podocytes in diabetic states. First, we explored the changes in expression of proteins involved in fission and fusion of mitochondria in wild-type (WT) and Meg3 KO human podocytes cultured under NG or HG conditions. The expression of Opa1, Mfn1, and Mfn2 in podocytes cultured with HG was reduced relative to NG; however, no differences in the expression levels of these proteins were found in lncRNA Meg3 KO cells relative to WT podocytes (Fig. 7a, b). Drp1 expression in WT podocytes cultured with HG was higher than in podocytes treated with NG, while the expression of Drp1 in Meg3 KO cells cultured in HG was greatly reduced compared with WT podocytes cultured with HG. IF staining showed that Drp1 translocated to mitochondria in podocytes cultured with HG; this mitochondrial translocation was blocked in Meg3 KO podocytes (Fig. 7j). These data suggest that Drp1 might be related to Meg3-mediated mitochondrial fission and podocyte injury.Fig. 7: LncRNA Meg3 promotes fission of the mitochondria in podocytes by regulation of Drp1 and its translocation to mitochondria.a, b Western blot analysis of proteins (Opa1, Mfn1, Mfn2 and Drp1) involved in the regulation of mitochondrial fission and fusion in podocytes from different groups. c–f Western blot analysis of pDrp1 (Ser637) in Meg3 KO and OE podocytes. g–i Western blot analysis of proteins in podocytes treated with HG or HG + Mdivi1 in Meg3 OE cell line. j Representative confocal images of mitochondria in human podocytes were stained by MitoTracker Deep Red and incubated with anti-Drp1 (original magnification, ×630; scale bar, 10 μm). k Quantitative analyses of mitochondrial organization on human podocytes from different groups (mean branch length, mean number of branches per network, and mitochondrial footprint, n = 10 cells per group). Data were shown as mean ± SD in all statistical graphs. *P < 0.05, **P < 0.01, ***P < 0.001 (n = 6 per group), and n.s., not significant.Several kinases can influence the subcellular localization of Drp1 by phosphorylating two conserved serine residues of Drp121. Increased phosphorylation of Drp1 at Ser600 (Ser637 in humans) is essential for HG-mediated mitochondrial fission22. Thus, we detected phosphorylation of Ser637 in Meg3 KO and OE podocytes. Increased phosphorylation of Ser637 of Drp1 was observed in human podocytes cultured in HG relative to podocytes incubated in NG, while the phosphorylation induced by HG was reduced in Meg3 KO podocytes. By contrast, phosphorylation was increased in Meg3 OE podocytes (Fig. 7c–f). Mdivi1, a pharmacological inhibitor of Drp1, blocks this phosphorylation and subsequent mitochondrial translocation22. Human podocyte injury was attenuated following treatment with Mdivi1, as demonstrated by the increased expression of Nephrin and Synaptopodin in podocytes treated with Mdivi1 relative to those untreated in HG conditions (Fig. 7g–i). In addition, Mdivi1 treatment blocked Drp1 translocation to mitochondria and improved mitochondrial morphology: less fragmented, more elongated, and interconnected mitochondria were observed compared with lncRNA Meg3 OE podocytes cultured in HG (Fig. 7j, k). These data suggest that Meg3 can promote mitochondrial fission and induce podocyte injury by regulating Drp1 and its translocation to mitochondria.Specific knockdown of LncRNA Meg3 in podocytes alleviated Drp1 and its phosphorylation of Ser600 in podocyte from in STZ-induced diabetic micePrimary mice podocytes were isolated for RNA extraction. Meg3 expression in podocytes from Cre+Meg3fl/fl mice was lower than those isolated from Meg3+/+ mice (Fig. 8a). mRNA expression of Nephrin and Synaptopodin was also decreased in podocytes from STZ-induced diabetic Meg3+/+ mice compared with control Meg3+/+ mice, while expression of Nephrin and Synaptopodin in diabetic Cre+Meg3fl/fl mice was higher than in diabetic Meg3+/+ mice (Fig. 8b, c). We also found that the expression of Drp1 mRNA was increased in the diabetic group, and downregulated with Meg3 knockdown (Fig. 8d). Higher expression of pDrp1 (Ser600, corresponding to Ser637 in human Drp1) colocalized with Synaptopodin was observed in glomeruli of diabetic Meg3+/+ mice compared with control Meg3+/+ mice, while these two colocalized expression in diabetic Cre+Meg3fl/fl mice were lower than diabetic Meg3+/+ mice (Fig. 8e). These data suggest that knockdown of Meg3 in podocytes from STZ-induced diabetic mice can alleviate podocyte injury and reduce Drp1 expression and phosphorylation of Ser600 in podocytes in vivo.Fig. 8: Specific knockdown of lncRNA Meg3 in podocytes alleviated Drp1 and its phosphorylation of Ser600 in podocyte from in STZ-induced diabetic mice.a LncRNA Meg3 expression in primary podocytes isolated from mice kidneys. b Synaptopodin mRNA expression in primary podocytes isolated from mice kidneys. c Nephrin mRNA expression in primary podocytes isolated from mice kidneys. d Drp1 mRNA expression in primary podocytes isolated from mice kidneys. e Immunofluorescence staining for pDrp1 (Ser600) (green) and Synaptopodin (red) in the paraffin-embedded kidney sections was performed (original magnification, ×630, scale bar, 20 μm). Data were shown as mean ± SD in all statistical graphs (n = 6 mice per group). **P < 0.01, ***P < 0.001, and ****P < 0.0001.

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