WITHDRAWN: Long noncoding RNA Glis2 regulates podocyte apoptosis by mediating mitochondrial function in diabetic nephropathy

doi:10.21203/rs.3.rs-2912492/v1

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WITHDRAWN: Long noncoding RNA Glis2 regulates podocyte apoptosis by mediating mitochondrial function in diabetic nephropathy

https://doi.org/10.21203/rs.3.rs-2912492/v1

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Objectives

Diabetic nephropathy (DN) is one of the most serious microvascular complications of diabetes and the main cause of end-stage kidney disease. Podocyte injury or apoptosis exerts a crucial role in the pathogenesis of DN. Recently, long noncoding RNAs (lncRNAs) have been gradually identified to be functional in a variety of different mechanisms associating with DN. However, the relationship between lncRNAs and podocyte apoptosis in DN is still in its infancy. This study aimed to investigate whether lncRNA Glis2 could regulate podocyte injury via miR-328-5p in DN and uncover the underlying mechanism.

Methods

Normal-glucose or high-glucose cultured podocytes and diabetic db/db mice were used to investigate the exact role and regulatory mechanism of lncRNA Glis2 on podocyte apoptosis in DN. Apoptosis rate of podocyte was detected by flow cytometry. Cell viability was measured using the Cell Counting Kit-8 colorimetric assay (CCK-8). The expressions of lncRNA Glis2 and miR-328-5p were measured by qRT-PCR. The relationship between lncRNA Glis2 and miR-328-5p was examined by dual luciferase reporter assay. Mitochondrial membrane potential (ΔΨM) was measured using JC-1 staining. Mitochondrial morphology was detected by MitoTracker Deep Red staining. Then, the histopathological and ultrastructure changes of renal tissues in diabetic mice were observed using periodic acid-Schiff (PAS) staining and transmission electron microscopy. Finally, the effect of lncRNA Glis2 on podocyte mitochondrial dysfunction and apoptosis through miR-328-5p/Sirt1 was detected by western blot.

Results

We found that lncRNA Glis2 was significantly downregulated in high-glucose cultured podocytes and renal tissues of db/db mice. Furthermore, lncRNA Glis2 overexpression or knockdown was found to regulate podocyte mitochondrial dysfunction and apoptosis. The direct interaction between lncRNA Glis2 and miR-328-5p was confirmed by dual luciferase reporter assay. LncRNA Glis2 overexpression alleviated podocyte mitochondrial dysfunction and apoptosis via miR-328-5p/Sirt1 pathway in podocytes and diabetic mice.

Conclusion

Taken together, this study demonstrated that lncRNA Glis2, acting as a competing endogenous RNA (ceRNA) of miRNA-328-5p, regulated Sirt1 mediated mitochondrial dysfunction and podocyte apoptosis in DN.

long noncoding RNAs (lncRNAs)

diabetic nephropathy (DN)

podocyte apoptosis

mitochondrial dysfunction

competing endogenous RNA (ceRNA)

Diabetic nephropathy (DN) is one of the most serious microvascular complications of diabetes and the main leading cause of chronic kidney disease (CKD) and end-stage renal disease in the world [1]. Clinically, DN is characterized by progressive proteinuria with a subsequent glomerular filtration rate reduction, resulting in irreversible renal dysfunction and uremic symptoms. The histopathological features of DN involve several complicated mechanisms, including glomerular hypertrophy, thickening of the glomerular basement membrane, massive accumulation of the extracellular matrix, podocyte reduction, and ultimately the development of interstitial fibrosis and glomerular sclerosis. Podocytes, which located outside the glomerular capillaries, are a key component of the kidney filtration barrier. The podocytes possess a unique complex architecture consisting of cell body, major processes, and foot processes, covering the outer side of glomerular basement membrane (GBM) [2]. Major processes that extend outward from their cell body formed interdigitated foot processes. The neighboring foot processes are connected by the slit diaphragms, which are a key structure maintaining the permeability of the glomerular filtration barrier to prevent proteinuria [3]. In healthy podocytes, slit diaphragms are composed by various proteins, such as podocin, nephrin and zonula occludens-1 (ZO-1) [4, 5]. When podocytes suffer from various external stimuli including high glucose, they undergo foot process effacement and loss of slit diaphragms, leading to podocyte injury or apoptosis [6]. Podocyte injury or apoptosis could disrupt the glomerular filtration barrier and induce the appearance of albuminuria, resulting in progressive loss of kidney function [7, 8], which has been widely considered as a crucial pathological mechanism in the progress of DN. Thus, exploring the therapies of preventing podocyte injury or promoting podocyte repair may help to seek for efficient targets and prospective strategies for DN.

Long noncoding RNAs (lncRNAs) are a class of RNA transcripts with no protein-coding ability longer than 200 nucleotides in length [9]. More and more research related to lncRNAs have been carried out extensively in recent years, leading to the discovery of their significant roles in gene modulation through epigenetic, transcriptional and post transcriptional regulatory mechanisms [10]. Growing evidences have indicated that lncRNAs played vital roles on various physiological and pathological processes of human diseases [11–13]. LncRNAs also participated in the progression of DN, including inflammation [14], mesangial cells proliferation and fibrosis [15], podocyte apoptosis [16] and so on. However, the expression profile and potential mechanisms of lncRNAs in podocyte apoptosis in DN remain largely unexplored.

To further investigate the role of lncRNAs in podocyte injury and apoptosis, high-throughput RNA-sequencing (RNA-seq) technologies and bioinformatics analytical were used to examine differentially expressed lncRNAs in cultured mouse podocytes under normal-glucose (5.5mM) and high-glucose (25mM) conditions. In this present study, we identified a number of significant differentially expressed lncRNAs in high-glucose cultured mouse podocytes. Among these lncRNAs, ENSMUST00000122896 (lncRNA Glis2 for short) stood out and was chosen for further research. The function of lncRNA Glis2 and its molecular mechanism in podocyte apoptosis have not been reported until now.

The present study aimed to investigate the function and underlying molecular mechanism of lncRNA Glis2 in podocyte apoptosis, providing a theoretical basis for lncRNA treatment in DN.

2.1 Cell culture

The conditionally immortalized mouse podocytes were purchased from the Cell Culture Center (PUMC, CAMS, Beijing, China). The podocytes were cultured in Dulbecco’s modified eagle medium (DMEM) with 10% fetal bovine serum, 100U/ml penicillin and 100µg/ml streptomycin at 37℃ under 5% CO2 atmosphere incubator. Podocytes were subsequently sub-cultured at 80–90% confluence for further experiments. The culture medium was changed every daily or two days. To mimic high-glucose environment or normal growth status, the podocytes were incubated in DMEM with high glucose (HG, 25mmol/L glucose) or normal glucose (NG, 5.5 mmol/L glucose plus 19.5mmol/L mannitol), respectively.

2.2 RNA sequencing (RNA-Seq)

Total RNA from the podocytes of NG and HG group were extracted using TRIzol® Reagent (Life Technologies) in accordance with the manufacturer’s instructions. RNA concentration was measured using Qubit® RNA Assay Kit in Qubit®2.0 Flurometer (Life Technologies, CA, USA). The total RNA was purified by depleting ribosomal RNA using Ribo-off rRNA Depletion kit (Vazyme Biotech Co., Ltd, Nanjing, China). Then, these samples were used for constructing a cDNA library using VAHTS™ Stranded mRNA-seq Library Prep Kit for Illumina® (Vazyme Biotech Co., Ltd, Nanjing, China). cDNA libraries were sequenced using an Illumina Novaseq6000 sequencer (Illumina Inc., San Diego, CA, USA), which was conducted by Sangon Biotech (Shanghai, China). Differentially expressed lncRNAs with statistical significance between NG and HG group were identified through P-value/false discovery rate (FDR) filtering. A volcano plot filtering approach (|log2(fold change)|≥1.0; q value ≤ 0.05) was considered as differentially expressed lncRNAs between two groups.

2.3 Cell transfection

For transfection, the podocytes from each group were seeded in 6 wells and incubated for 24h. After the cell fusion degree was approximately 60–80%, the transfection was conducted using opti-MEM and lipofectamine 2000 reagents (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. To overexpress lncRNA Glis2, pcDNA3.1-lncRNA Glis2 was constructed by inserting lncRNA Glis2 coding sequence into plasmid pcDNA3.1, and pcDNA3.1 empty vector was used as a negative control. For lncRNA Glis2 knockdown, small interference RNA targeting lncRNA Glis2 (siRNA-lncRNA Glis2) was synthesized by Hunan Fenghui Biotechnology (Changsha, China). siRNA-NC was used as a negative control. For miR-328-5p overexpression and knockdown, miR-328-5p mimics, miR-328-5p inhibitors and a matched miRNA negative control were synthesized and purified by Hunan Fenghui Biotechnology (Changsha, China), respectively. After 48h of transfection, the podocytes were collected and used for further analysis.

2.4 Animal experiments

All experimental procedures were in accordance with the institutional animal care and use committee and approved by the Animal Care and Ethics Committee of Second Hospital of Hebei Medical University (approval ID: 2022-AE051). Eight-week-old male C57BLKS/J db/db mice and their normal littermates (db/m) were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China). During the experiments, all mice were fed in a standard animal house with 12-hour light/12-hour dark cycle and given ad libitum access to food and water. After one week of adaptive feeding, db/db mice were randomly divided into three groups: db/db group, db/db + pcDNA3.1-NC group (db/db mice injected with empty vector) and db/db + pcDNA3.1-lncRNA Glis2 group (db/db mice injected with 50µl 1×10⁶ infective units lncRNA Glis2 overexpression lentivirus every week). Meanwhile, db/m mice served as control group. At the end of 12 weeks after treatment, mice were kept in individual metabolic cages for urine collection to measure urinary albumin-to-creatinine ratio (UACR). Blood samples were obtained for blood glucose, blood urea nitrogen (BUN), and serum creatinine (Scr). Then, the mice were sacrificed and the kidneys were harvested immediately for follow-up experiments.

2.5 Flow cytometry

After 48h transfection, cell apoptosis from each group was detected with an Annexin V-FITC and propidium iodide (PI) double staining kit (MultiSciences Biotechnology Corporate Limited, China) by flow cytometry (BD Biosciences, USA). Cells were collected and single cell suspensions (1×10⁶ cells/ml) were prepared. Then the cells were resuspended in binding buffer. After that, the cells were dual stained with annexin-V FITC/propidium iodide (PI) at room temperature for 5 min. Finally, apoptotic cells were detected by flow cytometry.

2.6 Measurement of cell viability

Cell viability was assessed with Cell Counting Kit-8 (CCK-8) assay. The podocytes were seeded in a 96-well plate and cultured overnight at 37℃. After that, each well was mixed with 10µl CCK-8 agent (Sigma-Aldrich, St Louis MO, USA). After 1h incubation, the light absorbance value was measured using microplate reader at 450 nm.

2.7 Immunofluorescence

For immunofluorescence, the coverslips were fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Next, the coverslips were blocked in 5% bovine serum albumin for 1h. Subsequently, the coverslips were incubated with the following primary antibodies (anti-podocin; anti-nephrin) at 37℃ overnight. The coverslips were washed and incubated with the corresponding secondary antibodies at room temperature for 1 h. After tinted with DAPI, the images were observed with a florescent microscope (Olympus FV10-ASW, Tokyo, Japan).

2.8 Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA from cultured cells or renal tissues was isolated using TRIzol reagent following the manufacturer’s instructions. The quality and concentration of RNA was detected using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The RNA quality was examined based on the absorbance ratio at 280/260 nm, and the RNA concentration was examined based on absorbance at 260 nm. Reverse transcription experiment was performed by the Revert Aid First Strand cDNA Synthesis kit (Thermo Fisher Scientific). Then, qRT-PCR was performed using SYBR®Premix Ex Taq™ (Takara, Dalian, China). The relative quantitative result was calculated using 2^-△△Ct method. The expression of GAPDH was set as the internal normalization control.

2.9 Fluorescence in situ hybridization (FISH)

The subcellular distribution of lncRNA Glis2 was detected using FISH kit (Boster Biological Technology co.ltd, Wuhan, China) according to the manufacturer’s instructions. The lncRNA Glis2 FISH probe was designed and synthesized by Servicebio Technology (Wuhan, China). The slides were fixed in 4% paraformaldehyde at room temperature for 30min. Next, the cells were permeabilized with cold PBS containing 0.5% Triton X-100. Subsequently, the cells were incubated with the fluorescence probe at 37℃ overnight. Then, the slides were stained with DAPI. The images were visualized with a florescent microscope (Olympus FV10-ASW, Tokyo, Japan).

2.10 Dual luciferase reporter assay

The dual luciferase reporter assay was constructed to determine the direct interaction of lncRNA Glis2 and miR-328-5p, as well as miR-328-5p and Sirtuin1 (Sirt1). To construct plasmids used in dual luciferase reporter assay, the predicted 3’-UTR sequence of lncRNA Glis2 (or Sirt1) interacting with miR-328-5p and an artificially mutated sequences within the predicted target sites were synthesized and inserted into pmirGLO vector (Cosmo Bio, Tianjin, China), respectively. Then, podocytes were co-transfected with wide type (wt) or mutated (mut) luciferase reporter plasmid and miR-328-5p mimics or NC mimics using lipofectamine 2000 reagents (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After 48h of transfection, luciferase activity was detected using dual luciferase assay system (Promega, Madison, WI, USA). The relative luciferase activity was normalized to Renilla luciferase activity.

2.11 Mitochondrial membrane potential (ΔΨM)

The ΔΨM of podocytes was detected using mitochondrial membrane potential assay kit with JC-1 (Beyotime Biotechnology, Shanghai, China) in accordance with the manufacturer’s instructions. The cultured podocytes were washed with PBS, and then incubated with JC-1 working solution for 30min at 37°C in darkness. The podocytes were rinsed with JC-1 staining buffer. After that, the images were observed with a florescent microscope (Olympus FV10-ASW, Tokyo, Japan). The values were expressed as the ratio of red relative to green fluorescence levels.

2.12 Mitochondrial morphology

The mitochondrial morphology was observed by MitoTracker™ Deep Red FM staining. The cultured podocytes were incubated with MitoTracker Deep Red (Yeasen Biotechnology, Shanghai, China) for 30min at 37℃ in the dark. Fluorescence images were obtained using a florescent microscope (Olympus FV10-ASW, Tokyo, Japan).

2.13 Adenosine triphosphate (ATP)

ATP levels were assessed using an ATP Assay Kit (Beyotime Biotechnology, China). Briefly, the cultured cells were harvested and lysed with lysis reagent and then centrifuged at 12000×g for 10min. The supernatant was collected and mixed with ATP detection solution in a 96-well plate afterwards. The light absorbance value was detected using microplate reader.

2.14 Western blot

Total protein from cultured podocytes or renal tissues was extracted using RIPA lysis buffer. The protein concentration was detected by Bradford protein content assay kit (Boster Biological Technology co.ltd, Wuhan, China) following its standard protocol. Equal amount of proteins was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene difluoride (PVDF) membrane. Then, the membranes were blocked with 5% skim milk or bovine serum albumin for 1h and incubated with primary antibodies overnight at 4°C. GAPDH was used as an internal control. After washing with phosphate-buffered saline (PBS), the membranes were incubated with the corresponding secondary antibodies at room temperature. After that, the membranes were incubated in Western Lightning™ Chemiluminescence Reagent (PerkinElmer, USA) for 5 min and visualized by LabWorks™ imaging system. The gray value of the target band was analyzed with ImageJ software (National Institutes of Health, Bethesda, MD).

2.15 Periodic acid-Schiff (PAS) stain

Mice were perfused with PBS, and then the renal tissues were fixed in 10% neutral buffered formalin overnight and dehydrated through graded alcohol series prior to processing for histology. The renal tissues were embedded in paraffin and stained with PAS reagent.

2.16 Transmission electron microscopy

Renal tissues were quickly cut into small pieces and fixed in 2.5% glutaraldehyde overnight. After embedding in epoxy resin, the ultrathin tissue sections were stained with uranyl acetate and lead citrate, and then examined with transmission electron microscope (HITACHI, Japan) and photographed.

2.17 Statistical analysis

All data were expressed as mean ± SD. SPSS version 18.0 (SPSS, Chicago, IL) was performed for statistical analysis. The statistical difference between two groups was measured using Student’s t-test. Multiple groups were compared using one-way analysis of variance (ANOVA). The P < 0.05 was considered to be statistically significant.

3.1 LncRNA Glis2 was down-expressed in high-glucose cultured mouse podocytes

To explore the effect of hyperglycemia on podocytes, mouse podocytes were cultured under normal-glucose or high-glucose concentration for 48 hours. Podocyte apoptosis rate and cell viability were respectively evaluated with flow cytometry and CCK-8 assay. Compared with NG group, flow cytometry indicated that hyperglycemia exposure significantly increased the number of apoptotic cells (Fig. 1A-B). The CCK-8 assay showed that hyperglycemia exposure significantly reduced cell viability (Fig. 1C). As the markers of podocyte injury, the expressions of nephrin and podocin were further performed by immunofluorescence. The expressions of nephrin and podocin were significantly decreased after hyperglycemia exposure (Fig. 1D-E). All these data confirmed that hyperglycemia could induce podocyte apoptosis.

Then, RNA-seq analysis showed that a total of 51 lncRNAs were differentially expressed between NG and HG group using the following criteria: p value < 0.001, q value < 0.01 and |log2 (fold change)| > 1, including 20 up-regulated and 31 down-regulated genes (Fig. 1F). Among them, the expressions of 11 lncRNAs were successfully validated using qRT-PCR in NG and HG cultured podocytes. Data showed that the expressions of these candidate lncRNAs were consistent with the result of RNA-seq (Fig. 1G). Among these, the expression of ENSMUST00000122896 was markedly lower in HG group compared with that in NG group. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed to identify the biological role of lncRNAs. Bioinformatics analysis showed that ENSMUST00000122896 was associated with kidney development (GO:0060994, GO:0061005, GO:0001822). Therefore, ENSMUST00000122896 (lncRNA Glis2 for short) was focused on for further study to reveal the potential function and mechanism.

3.2 Silencing lncRNA Glis2 induced podocyte apoptosis and mitochondrial dysfunction

To research the functional role of lncRNA Glis2 in podocyte injury, we first successfully used pcDNA3.1 vector or chemically modified siRNA to transfect into normal-glucose cultured podocytes. The results of qRT-PCR showed that podocytes transfected with pcDNA3.1-lncRNA Glis2 or siRNA-lncRNA Glis2 could effectively up-regulate or down-regulate lncRNA Glis2. Flow cytometry showed that the apoptosis rate was decreased in pcDNA3.1-lncRNA Glis2 group compared with that in pcDNA3.1-NC group, whereas silencing lncRNA Glis2 significantly increased podocyte apoptosis in siRNA-lncRNA Glis2 group than that in siRNA-NC group (Fig. 2A-B). Next, CCK-8 assay revealed that lncRNA Glis2 overexpression increased cell viability in pcDNA3.1-lncRNA Glis2 group compared with that in pcDNA3.1-NC group, while the cell viability was differentially reduced in siRNA-lncRNA Glis2 group compared with siRNA-NC group (Fig. 2C). Immunofluorescence assay revealed that lncRNA Glis2 overexpression remarkably up-regulated the expressions of nephrin and podocin, while lncRNA Glis2 silencing showed the opposite effect in siRNA-lncRNA Glis2 group (Fig. 2D-E). Consistent with these results, pro-apoptotic caspase3 and caspase9 were significantly decreased in pcDNA3.1-lncRNA Glis2 group compared with that in pcDNA3.1-NC group. On the other hand, the expressions of caspase3 and caspase9 were reversed respectively once lncRNA Glis2 was knockdown in siRNA-lncRNA Glis2 group compared with siRNA-NC group (Fig. 2F-G). Therefore, these results indicated that lncRNA Glis2 down-regulation induced by hyperglycemia might be associated with podocyte apoptosis.

Podocytes, which are terminally differentiated visceral epithelial cells, have a limited capacity for renewal and can’t regenerate when they suffer from damage. Thus, podocytes rely on their ability to maintain their complex structure via regulation and organization of the actin cytoskeleton and extracellular matrix. This imposes a high-energy requirement and requires large amounts of mitochondria to supply energy [17, 18]. To explore whether lncRNA Glis2 involve in podocyte mitochondrial dysfunction, we assessed mitochondrial function with lncRNA Glis2 overexpression or inhibition in podocytes. The change of ΔΨM was detected using the fluorescent probe JC-1. As shown in Fig. 3A-B, silencing lncRNA Glis2 caused a large accumulation of JC-1 monomers, indicating the occurrence of decreased ΔΨM. A decline in the ΔΨm is a distinctive feature of change in mitochondrial activity during early-stage apoptosis of podocytes. ATP concentration was also decreased in siRNA-lncRNA Glis2 group than siRNA-NC group (Fig. 3C). The change of mitochondrial morphology was observed using confocal microscopy (Fig. 3D). Under physiological condition, mitochondria appeared rod-shaped in podocytes. Silencing lncRNA Glis2 markedly affected mitochondrial shape. Highly fragmented and discrete mitochondria were observed in siRNA-lncRNA Glis2 group compared with siRNA-NC group. The mitochondrial fusion and fission protein were detected by western blot (Fig. 3E-F). Mitofusin 1 (Mfn1) was significantly decreased in siRNA-lncRNA Glis2 group compared with siRNA-NC group. Dynamin related protein 1 (Drp1) was increased in siRNA-lncRNA Glis2 group than that in siRNA-NC group. However, up-regulation of lncRNA Glis2 had the adverse effect in mitochondrial function. Taken together, these results provided evidences that lncRNA Glis2 down-regulation induced by hyperglycemia is an important reason for podocyte mitochondrial dysfunction and apoptosis.

3.3 LncRNA Glis2 overexpression alleviated podocyte apoptosis and mitochondrial dysfunction induced by hyperglycemia

To further confirm whether lncRNA Glis2 overexpression alleviated podocyte apoptosis induced by hyperglycemia, pcDNA3.1-lncRNA Glis2 was used to transfect into high-glucose cultured podocytes. Flow cytometry showed that lncRNA Glis2 overexpression could alleviate podocyte apoptosis induced by hyperglycemia (Fig. 4A-B). CCK-8 assay revealed that lncRNA Glis2 overexpression could increase cell viability in HG + pcDNA3.1-lncRNA Glis2 group compared with that in HG + pcDNA3.1-NC group (Fig. 4C). Immunofluorescence assay revealed that the down-regulation of nephrin and podocin induced by hyperglycemia was significantly reversed by transfected with pcDNA3.1-lncRNA Glis2 compared with that transfected with pcDNA3.1-NC (Fig. 4D-E). In addition, lncRNA Glis2 overexpression significantly reversed the protein expressions associated with apoptosis in HG + pcDNA3.1-lncRNA Glis2 group, including decreasing caspase3 and caspase9 (Fig. 4F-G). Therefore, these data indicated that lncRNA Glis2 overexpression played a significant role in alleviating podocyte injury induced by hyperglycemia.

Then, we investigated whether lncRNA Glis2 overexpression by transfecting pcDNA3.1-lncRNA Glis2 alleviated mitochondrial dysfunction induced by hyperglycemia. JC-1 staining showed more accumulation of JC-1 aggregates and less diffuse JC-1 monomers in HG + pcDNA3.1-lncRNA Glis2 group, which indicated that the decrease of ΔΨM caused by hyperglycemia significantly reversed by lncRNA Glis2 overexpression (Fig. 5A-B). MitoTracker Deep Red staining showed that lncRNA Glis2 overexpression significantly prevented hyperglycemia induced changes in mitochondrial morphology (Fig. 5D). As shown, the down-regulation of ATP (Fig. 5C) and the changes in mitochondrial fusion or fission protein (Fig. 5E-F) induced by hyperglycemia were reversed in HG + pcDNA3.1-lncRNA Glis2 group compared with that in HG + pcDNA3.1-NC group. These results suggested that lncRNA Glis2 overexpression relieved mitochondrial dysfunction induced by hyperglycemia in podocytes.

3.4 LncRNA Glis2 played biological function by serving as a competing endogenous RNA (ceRNA) of miR-328-5p

To further explore the potential mechanism of lncRNA Glis2 in podocyte apoptosis, we detected the subcellular location of lncRNA Glis2 in NG and HG group by FISH. The results showed that lncRNA Glis2 was predominantly located in the cytoplasm of cells (Fig. 6A). As known, identified lncRNAs perform various functions based on their subcellular location. If the lncRNAs distribute in the nucleus, they are principally involved in the transcription process and chromatin remodeling. If the lncRNAs are located in the cytoplasm, they mainly participate in post-transcriptional gene regulation by acting as a ceRNA or forming complexes with specific protein [19, 20]. Thus, we speculated that lncRNA Glis2 could regulate the target genes by competitively binding miRNA as a ceRNA in the progression of podocyte apoptosis. Many lncRNAs could compete with other RNA transcripts for miRNA binding sites, resulting in affecting the binding of miRNAs to downstream target genes [21, 22]. MiRNAs are endogenous noncoding RNAs with 19–25 nucleotides in length. MiRNAs regulate gene expression through binding to complementary sequences of their target mRNA, resulting in translational repression or mRNA degradation [23].

To further elucidate the mechanism of lncRNA Glis2 in podocyte apoptosis, we used microarray chip and bioinformatics to interrogate the potential miRNAs interacting with lncRNA Glis2. Nine candidates of putative miRNA for lncRNA Glis2 were predicted online databases (StarBase, miRbase, USCS, and BiBiserv2 software). Among these, miR-328-5p was chosen for predicted candidate because not only it was the highest after transfecting with siRNA-lncRNA Glis2 (Fig. 6B) but also it was reported to be correlated with mitochondrial activity [24]. Data from qRT-PCR also revealed that the expression level of miR-328-5p is negatively correlated with lncRNA Glis2 (Fig. 6C). Figure 6D illustrated the binding sequence prediction of lncRNA Glis2 and miR-328-5p. To validate the bioinformatics results, the direct interaction between lncRNA Glis2 and miR-328-5p was confirmed by dual luciferase reporter assay (Fig. 6E). We constructed wild type and mutant 3’-UTR lncRNA Glis2 luciferase reporter vectors. A wild type plasmid lncRNA Glis2-wt or mutant plasmid lncRNA Glis2-mut were co-transfected with miR-328-5p mimics or NC mimics in podocytes and the luciferase activity was examined. When lncRNA Glis2-wt was used for co-transfecting, the luciferase activity was striking inhibited in miR-328-5p mimics group compared with NC mimics group. Whereas, when lncRNA Glis2-mut was used for co-transfecting, there was no significant difference between miR-328-5p mimics and NC mimics group. Taken together, these findings confirmed that there was a direct interaction between lncRNA Glis2 and miR-328-5p.

3.5 Inhibiting of miR-328-5p alleviated podocyte apoptosis and mitochondrial dysfunction

In order to investigate the function of miR-328-5p in podocyte injury, we used miR-328-5p mimics or miR-328-5p inhibitors to transfect into a panel of normal-glucose cultured podocytes. Flow cytometry revealed that miR-328-5p mimics significantly induced podocyte apoptosis, whereas miR-328-5p inhibitors ameliorated podocyte apoptosis (Fig. 7A-B). Next, the results of CCK-8 assay showed that miR-328-5p mimics decreased cell viability compared with NC mimics group, while inhibiting the expression of miR-328-5p increased cell viability compared with NC inhibitors group (Fig. 7C). Moreover, immunofluorescence assay revealed that miR-328-5p mimics obviously reduced the protein expression of nephrin and podocin, whereas miR-328-5p inhibitors up-regulated the expression of nephrin and podocin (Fig. 7D-E). Western blot displayed that pro-apoptotic caspase3 and caspase9 were significantly increased in miR-328-5p mimics group compared with that in NC mimics group. On the other hand, the expressions of caspase3 and caspase9 were reversed respectively once miR-328-5p was knockdown in miR-328-5p inhibitors group (Fig. 7F-G). Therefore, these results confirmed that up-regulation of miR-328-5p induced podocyte injury, whereas inhibition of miR-328-5p alleviated podocyte injury.

Furthermore, we assessed the role of miR-328-5p in mitochondrial dysfunction by transfecting with miR-328-5p mimics or miR-328-5p inhibitors. In contrast to NC mimics group, overexpression of miR-328-5p caused a large accumulation of JC-1 monomers, indicating the decline of ΔΨM in miR-328-5p mimics group (Fig. 8A-B). Overexpression of miR-328-5p markedly reduced ATP concentration in miR-328-5p mimics group than NC mimics group (Fig. 8C). Meanwhile, overexpression of miR-328-5p significantly affected mitochondrial morphology, including more fragmented and discrete mitochondria (Fig. 8D). Western blot displayed that up-regulation of miR-328-5p decreased the expression of Mfn1 and increased the expression of Drp1 (Fig. 8E-F). However, inhibition of miR-328-5p had the adverse effect in mitochondrial function. These data suggested that overexpression of miR-328-5p induced mitochondrial dysfunction, while inhibition of miR-328-5p alleviated mitochondrial dysfunction.

3.6 LncRNA Glis2 targets miR-328-5p in podocyte apoptosis

To further validate whether lncRNA Glis2 could interact with miR-328-5p to affect podocyte apoptosis, rescue experiments were performed by co-transfecting pcDNA3.1-lncRNA Glis2 and miR-328-5p mimics. As shown, up-regulation of miR-328-5p could apparently induce podocyte apoptosis, but co-transfection with pcDNA3.1-lncRNA Glis2 could partially reverse the effect (Fig. 9). The ability of inhibiting podocyte apoptosis which was caused by lncRNA Glis2 overexpression was partly countervailed by co-transfection with miR-328-5p mimics (Fig. 9). Taken together, the above results elucidated that lncRNA Glis2 regulated podocyte apoptosis through interacting with miR-328-5p.

3.7 MiR-328-5p targets Sirt1 in podocyte apoptosis

In order to further decipher the mechanism of miR-328-5p in podocyte apoptosis, we predicted the potential target genes that miR-328-5p may regulate by bioinformatics analysis (TargetScan, http://www.targetscan.org). Among these, Sirt1 was chosen for predicted candidate because it was reported to be related to mitochondrial function and podocyte injury [25–27]. We found that there was a direct binding site between miR-328-5p and Sirt1 (Fig. 10A). In addition, up-regulation of miR-328-5p reduced the expression of Sirt1. However, the expression of Sirt1 was increased when miR-328-5p was inhibited (Fig. 10B). Importantly, dual luciferase reporter assay revealed that miR-328-5p mimics co-transfection with a wild-type Sirt1 reporter gene (Sirt1-wt) could reduce the relative luciferase activities. Nevertheless, the relative luciferase activities were not significantly changed when miR-328-5p mimics co-transfected with a mutant Sirt1 reporter gene (Sirt1-mut) (Fig. 10C). These data indicated that miR-328-5p negatively regulated the expression of Sirt1 in a direct manner.

3.8 Sirt1 overexpression alleviated podocyte apoptosis and mitochondrial dysfunction induced by hyperglycemia

To identify the biological function of Sirt1, we investigated the effect of Sirt1 overexpression by transfecting pcDNA3.1-Sirt1 in podocyte apoptosis induced by hyperglycemia. Strikingly, Sirt1 overexpression reduced high glucose-induced podocyte apoptosis (Fig. 11A-B) and increased cell viability (Fig. 11C) in HG + pcDNA3.1-Sirt1 group. Sirt1 overexpression significantly prevented the down-regulation of nephrin and podocin induced by hyperglycemia compared with HG + pcDNA3.1-NC group (Fig. 11D-E). Up-regulation of Sirt1 significantly reversed the protein expressions associated with apoptosis, including decreasing caspase3 and caspase9 (Fig. 11F-G). Therefore, these results confirmed that up-regulation of Sirt1 alleviated podocyte apoptosis induced by hyperglycemia.

To further evaluate whether Sirt1 overexpression alleviated mitochondrial dysfunction induced by hyperglycemia, pcDNA3.1-Sirt1 was transfected into high-glucose cultured podocytes. The decrease of ΔΨM (Fig. 12A-B), the down-regulation of ATP (Fig. 12C) and abnormal mitochondrial morphology (Fig. 12D) caused by hyperglycemia were significantly reversed by transfection with pcDNA3.1-Sirt1. Furthermore, western blot showed that these abnormal changes in mitochondrial fusion or fission protein induced by hyperglycemia were impeded by Sirt1 overexpression in HG + pcDNA3.1-Sirt1 group (Fig. 12E-F). Collectively, these results elucidated that up-regulation of Sirt1 alleviated mitochondrial dysfunction induced by hyperglycemia in podocytes.

3.9 Up-regulation of lncRNA Glis2 attenuated podocyte apoptosis in diabetic mice

The above findings demonstrated that up-regulation of lncRNA Glis2 might be a valuable therapeutic approach in DN. To verify whether lncRNA Glis2 also attenuated podocyte apoptosis in the same way in diabetic mice, lncRNA Glis2 overexpression lentivirus was transfected into db/db mice by tail vein injection. Compared with those in db/db + pcDNA3.1-NC group, analyses of biochemical parameters, including blood glucose (Fig. 13A), body weight (Fig. 13B), kidney weight (Fig. 13C), UACR (Fig. 13D), BUN (Fig. 13E) and Scr (Fig. 13F), were evidently lower in db/db + pcDNA3.1-lncRNA Glis2 group. Compared with that in db/db + pcDNA3.1-NC group, a significant increase in the expression of lncRNA Glis2 (Fig. 13G) and a significant decrease in the expression of miR-328-5p (Fig. 13H) were detected in db/db + pcDNA3.1-lncRNA Glis2 group.

PAS staining revealed the stromal hyperplasia and thickened glomerular basement membrane in db/db mice. LncRNA Glis2 overexpression lentivirus treatment produced dramatic reductions in these pathological changes in db/db + pcDNA3.1-lncRNA Glis2 group (Fig. 13I). TEM confirmed the alleviation of podocyte foot processes effacement and glomerular basement membrane thickening in db/db + pcDNA3.1-lncRNA Glis2 group compared with db/db + pcDNA3.1-NC group (Fig. 13I).

Our findings demonstrated that lncRNA Glis2 was markedly down-regulated and associated with podocyte apoptosis in high-glucose cultured podocytes and diabetic db/db mice. More importantly, silencing of lncRNA Glis2 triggered mitochondrial dysfunction and podocyte apoptosis. Further, lncRNA Glis2 exerted biological function by serving as a ceRNA of miR-328-5p, which negatively regulated the expression of its target Sirt1 and downstream proteins. Overall, lncRNA Glis2, acting as a ceRNA of miR-328-5p, regulated Sirt1 mediated mitochondrial function in podocyte apoptosis in DN. Targeting lncRNA Glis2 may be a therapeutic approach for podocyte injury in DN.

The rising and emerging evidences have demonstrated that lncRNA effectively participated in the pathophysiological process of DN. A recent study found that LINC01619 was down-regulated in DN patients’ renal tissues, diabetic rats and high-glucose cultured podocytes, which is associated with proteinuria and declined renal function [28]. In diabetic rats and high-glucose cultured podocytes, LINC01619 down-regulation promoted podocyte injury and disease progression by promoting the inhibitory effect of miR-27a on FOXO1 [28]. In high-glucose cultured podocytes, SPAG5-AS1 level was up-regulated. Further study indicated that lncRNA SPAG5-AS1 inhibited autophagy and aggravated podocyte apoptosis via acting as a ceRNA of miR-769-5p [29]. However, the exact mechanism of lncRNAs in DN remained mysterious and need to be further explored. An in depth understanding of the function of lncRNA Glis2 in podocyte apoptosis was performed in the current study. In this study, we found that the expression of lncRNA Glis2 was down-regulated in high-glucose cultured podocytes, which negatively correlated with nephrin and podocin expressions. Overexpression of lncRNA Glis2 rescued podocyte apoptosis and reduced nephrin and podocin expressions in podocytes under high-glucose condition. Furthermore, lncRNA Glis2 overexpression attenuated podocyte apoptosis in diabetic mice. These results provided evidences that lncRNA Glis2 down-regulation induced by hyperglycemia was an important reason for podocyte apoptosis. LncRNA Glis2 played a renoprotection role in DN.

Mitochondria are highly plastic organelles that constantly change their number, shape and intracellular localization through fusion and fission processes in response to energy demands [30]. Mitochondrial dynamics is governed by the balance between these two opposite processes of fusion and fission [31]. Mitochondrial fusion promotes the generation of interconnected mitochondria and is regulated by fusion proteins (mitofusins 1 and 2 (Mfn1, Mfn2) and optical atrophy (OPA1)). On the contrary, mitochondrial fission produces numerous mitochondrial fragments in order to remove the impaired mitochondria and is regulated by fission proteins (dynamin related protein 1 (Drp1) and mitochondrial fission 1 (Fis1)) [32]. Mitochondrial dysfunction leads to impaired ATP generation, increases oxidative stress and alters mitochondrial membrane permeability, resulting in cell dysfunction or cell apoptosis [33]. Mitochondria are a major source of intracellular reactive oxygen species (ROS) generation in the majority of cell types. ROS can increase the release of mitochondrial intermembrane space proteins to the cytoplasm[34], which leads to activation of the caspase cascade (apoptotic markers) and mitochondria-mediated apoptosis [35]. Caspases are the central components in the execution of apoptosis, which are divided into the initiator and executioner caspases. Caspase-9 is an essential initiator caspase in the mitochondrial pathway [36]. Caspase-3, which is downstream of caspase-9, is the key executioner caspase in apoptosis [37]. Accumulating studies have indicated that improving mitochondrial function attenuated high-glucose induced podocyte injury. In diabetic mice, treatment with recombinant human progranulin (rPGRN) attenuated high-glucose induced podocyte injury through maintaining mitochondrial homeostasis via Sirt1-PGC-1α/FoxO1 signaling [38]. In vitro studies showed that Sirt6 overexpression alleviated high-glucose induced podocyte apoptosis through protecting mitochondrial function [39]. The current study revealed that silencing of lncRNA Glis2 effectively increased mitochondrial fission and promoted mitochondrial dysfunction in podocytes under normal-glucose condition, whereas overexpression of lncRNA Glis2 attenuated mitochondrial dysfunction in podocytes under high-glucose condition. The relationship between lncRNA Glis2 and mitochondrial function in podocytes indicated that lncRNA Glis2 overexpression reduced podocytes apoptosis through ameliorating mitochondrial dysfunction.

LncRNA Glis2 (Ensembl ID: ENSMUST00000122896), which is a 3512bp lncRNA, locates in chromosome 16 (Chr16: 4426491–4433806). A large amount of evidence had demonstrated that lncRNAs could participate in diabetes and diabetic complications via acting as a ceRNA. Chen W et al. identified the DN-related ceRNA network and explored that lncRNA H2k2 promoted mesangial cell proliferation by acting as a ceRNA of miR449a/b [40]. Another study demonstrated that overexpression of lncRNA LEGLTBC could function as a ceRNA of miR-34a and improve glucolipotoxicity-induced oxidative stress and apoptosis in INS-1 cells [41]. To elucidate the mechanism of lncRNA Glis2 in regulating podocyte mitochondria dysfunction and apoptosis, we detected the subcellular distribution of lncRNA Glis2 by FISH and found that lncRNA Glis2 was mainly located in the cytoplasm of podocytes. According to the results, we speculated that lncRNA Glis2 maybe participated in podocyte mitochondria dysfunction through acting as a ceRNA. Next, we predicated the target gene of lncRNA Glis2 by bioinformatics analysis and found that miR-328-5p is its potential binding target. QRT-PCR data showed that the expression of miR-328-5p was opposite to lncRNA Glis2. The direct relationship between lncRNA Glis2 and miR-328-5p was further confirmed using dual luciferase reporter gene assay. These results indicated that lncRNA Glis2 participate in podocyte apoptosis maybe through directly binding to miR-328-5p.

Additionally, the role of miRNAs in mitochondrial function has been elucidated in previous research. A previous study showed that miR-328 regulated cell resistance to complement-dependent cytotoxicity (CDC) by modifying the expression of membrane complement regulators CD46 and CD59 and the response of the mitochondria to complement lytic attack [24]. Inhibition of miR-27a-3p ameliorated renal fibrosis and improved renal function in the kidney of diabetic mice through relieving mitochondrial dysfunction, including up-regulation of mitochondrial membrane potential, adenosine triphosphate, and mitochondrial cytochrome C [42]. In mouse models of CKD induced by obstruction, ischemia/reperfusion, and albumin overload, miR-214 was reported to play a pathogenic role through the disruption of mitochondrial oxidative phosphorylation [43]. In the current study, a negative feedback regulatory mechanism between lncRNA Glis2 and miR-328-5p was demonstrated. Furthermore, miR-328-5p overexpression aggravated mitochondrial dysfunction and podocyte apoptosis. Based on these views, this study illustrated that lncRNA Glis2 overexpression reduced mitochondrial dysfunction and podocyte apoptosis maybe through acting as a ceRNA of miR-328-5p.

Subsequently, this study confirmed that miR-328-5p effectively inhibited Sirt1 expression by directly binding to the 3’UTRs of its mRNAs. The Sirt1 expression and its effect in podocyte apoptosis was opposite to miR-328-5p. The direct relationship between miR-328-5p and Sirt1 was also verified using dual luciferase reporter assay. Sirt1, a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, provided protective effects by deacetylating many target proteins against a variety of metabolic diseases, including DN in mammals [44]. It has been reported that the protective role of Sirt1 in both podocytes and renal tubular cells in multiple kidney disease settings, including DN [45]. Both podocyte-specific Sirt1 overexpression and Sirt1 agonist treatment could attenuate diabetes-induced podocyte injury and effectively alleviate the progression of diabetic kidney disease in diabetic mice [26]. Ligustilide treatment alleviated the podocyte injury under a hyperglycemia environment and reduced the symptoms of renal dysfunction in DN rats. Increasing Sirt1 expression by ligustilide might be involved in its molecular mechanism [46]. Growing evidences described that Sirt1 activation might play a renoprotective role through regulating mitochondrial function in DN [47]. In vitro study, grape seed procyanidin B2 (GSPB2) protected against high-glucose induced mitochondrial dysfunction and podocyte injury via the activation of the AMPK-SIRT1-PGC-1α axis [48]. Salidroside played a beneficial role against DN as evidenced by decreased urinary albumin, blood urea nitrogen and serum creatinine via Sirt1/PGC-1α mediated mitochondrial biogenesis in mice [49]. A recent study further revealed that phosphorylation regulation of Sirt1 play a crucial role in its therapeutic effects on DN. Phosphorylation of Sirt1 was significantly up-regulated and lead to mitochondrial dysfunction in high-glucose treated podocyte and in diabetic mice [27]. Consistent with previous studies, this present study reconfirmed the protective effect of Sirt1 in mitochondrial function and podocyte apoptosis in DN. Based on the above findings, this study demonstrated that miR-328-5p regulated mitochondrial function and podocyte apoptosis by directly targeting Sirt1.

In summary, these results together revealed that lncRNA Glis2 overexpression ameliorated Sirt1-mediated mitochondrial dysfunction and podocyte injury by severing as a ceRNA of miR-328-5p.

Our investigation unraveled that lncRNA Glis2 down-regulation is an important reason for podocyte mitochondrial dysfunction and apoptosis in DN. LncRNA Glis2 down-regulation promoted the inhibitory effect of miR-328-5p on Sirt1, thus triggering mitochondrial dysfunction and podocyte apoptosis in DN (Fig. 14). This study provided new evidences into the mechanisms of renoprotection associated with lncRNA Glis2. The interaction between lncRNA Glis2 and miR-328-5p may provide a novel and specific therapeutic target for tackling podocyte injury in DN.

Acknowledgements

The authors thank the patients who participate in this study and the members who involved in the data collection and analysis.

Funding

This study was funded by the Natural Science Funds for Young Scholar of Hebei, China (grant numbers: 2020206108), and the subject of Health Commission of Hebei, China (grant numbers: 20210151).

Ethics approval

This study was conducted in accordance with the institutional animal care and use committee and approved by the Animal Care and Ethics Committee of Second Hospital of Hebei Medical University (approval ID: 2022-AE051).

Conflict of interest

The authors declare no competing interest.

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WITHDRAWN: Long noncoding RNA Glis2 regulates podocyte apoptosis by mediating mitochondrial function in diabetic nephropathy

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Editorial Note

Abstract

Objectives

Methods

Results

Conclusion

Figures

1. Introduction

2. Material and methods

2.1 Cell culture

2.2 RNA sequencing (RNA-Seq)

2.3 Cell transfection

2.4 Animal experiments

2.5 Flow cytometry

2.6 Measurement of cell viability

2.7 Immunofluorescence

2.8 Quantitative real-time polymerase chain reaction (qRT-PCR)

2.9 Fluorescence in situ hybridization (FISH)

2.10 Dual luciferase reporter assay

2.11 Mitochondrial membrane potential (ΔΨM)

2.12 Mitochondrial morphology

2.13 Adenosine triphosphate (ATP)

2.14 Western blot

2.15 Periodic acid-Schiff (PAS) stain

2.16 Transmission electron microscopy

2.17 Statistical analysis

3. Results

3.1 LncRNA Glis2 was down-expressed in high-glucose cultured mouse podocytes

3.2 Silencing lncRNA Glis2 induced podocyte apoptosis and mitochondrial dysfunction

3.3 LncRNA Glis2 overexpression alleviated podocyte apoptosis and mitochondrial dysfunction induced by hyperglycemia

3.4 LncRNA Glis2 played biological function by serving as a competing endogenous RNA (ceRNA) of miR-328-5p

3.5 Inhibiting of miR-328-5p alleviated podocyte apoptosis and mitochondrial dysfunction

3.6 LncRNA Glis2 targets miR-328-5p in podocyte apoptosis

3.7 MiR-328-5p targets Sirt1 in podocyte apoptosis

3.8 Sirt1 overexpression alleviated podocyte apoptosis and mitochondrial dysfunction induced by hyperglycemia

3.9 Up-regulation of lncRNA Glis2 attenuated podocyte apoptosis in diabetic mice

4. Discussion

5. Conclusion

Declarations

References

Supplementary Files

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