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, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C under a 5% CO₂ atmosphere incubator. Podocytes were subsequently sub-cultured at 80%–90% confluence for further experiments. The culture medium was changed every day or 2 days. To mimic a high-glucose environment or normal growth status, the podocytes were incubated in DMEM with high glucose (HG, 25 mmol/L glucose) or normal glucose (NG, 5.5 mmol/L glucose plus 19.5 mmol/L mannitol), respectively. Mannitol was added to the cell incubation medium to ensure osmolar equivalence in the NG group.

2.2 RNA sequencing (RNA-Seq)

Total RNA from the podocytes of the NG and HG groups 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 Fluorometer (Life Technologies, CA, USA). The total RNA was purified by depleting ribosomal RNA using the 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 groups 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 NG and HG groups.

2.3 Cell transfection

For transfection, the podocytes from each group were seeded in six wells and incubated for 24 h. 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.

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 the 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). All mice were fed in a standard animal house with a 12-h light/12-h dark cycle and given ad libitum access to food and water. After 1 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 50 μL 1 × 10⁶ infective units lncRNA Glis2 overexpression lentivirus every week). Meanwhile, db/m mice served as a control group. At the end of 12 weeks after treatment, mice were kept in individual metabolic cages for urine collection to measure the 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 48 h 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 a 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 a Cell Counting Kit-8 (CCK-8) assay. The podocytes were seeded in a 96-well plate and cultured overnight at 37°C. After that, each well was mixed with 10 μL CCK-8 agent (Sigma-Aldrich, St Louis MO, USA). After 1 h incubation, the light absorbance value was measured using a 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 1 h. Subsequently, the coverslips were incubated with the following primary antibodies (anti-podocin; anti-nephrin) at 37°C overnight. The coverslips were washed and incubated with the corresponding secondary antibodies at room temperature for 1 h. After being tinted with DAPI, the images were observed with a fluorescent 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 were detected using a 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. A 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 the 2^−△△Ct method. The expression of GAPDH or U6 was set as the internal normalization control for lncRNA or miRNA.

2.9 Fluorescence in situ hybridization (FISH)

The subcellular distribution of lncRNA Glis2 was detected using a 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 30 min. 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°C overnight. Then, the slides were stained with DAPI. The images were visualized with a fluorescent 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 the 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 48 h of transfection, luciferase activity was detected using a 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 30 min at 37°C in darkness. The podocytes were rinsed with JC-1 staining buffer. After that, the images were observed with a fluorescent 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 30 min at 37°C in the dark. Fluorescence images were obtained using a fluorescent 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 12,000 × g for 10 min. The supernatant was collected and mixed with ATP detection solution in a 96-well plate afterwards. The light absorbance value was detected using a 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. An equal amount of protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane. Then, the membranes were blocked with 5% skim milk or bovine serum albumin for 1 h 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 grey value of the target band was analysed 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 the two groups was measured using Student's t-test. Multiple groups were compared using a 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 h. We confirmed that hyperglycemia could induce podocyte apoptosis (Figure S1A–E). In our study, RNA-seq analysis showed that a total of 51 lncRNAs were differentially expressed between NG and HG groups 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 (Figure 1A). 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 (Figure 1B). Among these, the expression of ENSMUST00000122896 (lncRNA Glis2 for short) was markedly altered in the HG group compared with that in the NG group. There was about a 7.76-fold change reduction of lncRNA Glis2 in the HG group compared with that in the NG group. Therefore, lncRNA Glis2 was focused on for further study to reveal the potential function and mechanism.

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

To research the functional role of lncRNA Glis2 in podocyte apoptosis, we first successfully used pcDNA3.1 vector or chemically modified siRNA to transfect into normal-glucose cultured podocytes. The results indicated that lncRNA Glis2 down-regulation induced by hyperglycemia might be associated with podocyte apoptosis (Figure S2A–G). 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 (Figure 2A,B). CCK-8 assay revealed that lncRNA Glis2 overexpression could increase cell viability in HG + pcDNA3.1-lncRNA Glis2 group (Figure 2C). Immunofluorescence assay revealed that the down-regulation of nephrin and podocin induced by hyperglycemia was significantly reversed by transfected with pcDNA3.1-lncRNA Glis2 (Figure 2D,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 (Figure 2F,G). Therefore, these data indicated that lncRNA Glis2 overexpression played a significant role in alleviating podocyte apoptosis induced by hyperglycemia.

Podocytes, which are terminally differentiated visceral epithelial cells, have a limited capacity for renewal and cannot 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.^{9, 10} To explore whether lncRNA Glis2 is involved in podocyte mitochondrial function, we first successfully used pcDNA3.1 vector or chemically modified siRNA to transfect into normal-glucose cultured podocytes. The results indicated that lncRNA Glis2 might be associated with mitochondrial dysfunction in podocytes (Figure S2H–M). Next, we assessed whether lncRNA Glis2 overexpression alleviated mitochondrial dysfunction in high-glucose cultured podocytes. Hyperglycemia exposure 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. More accumulation of JC-1 aggregates and less diffuse JC-1 monomers were observed in HG + pcDNA3.1-lncRNA Glis2 group, which indicated that the decrease of ΔΨM caused by hyperglycemia significantly reversed by lncRNA Glis2 overexpression (Figure 2H,I). ATP concentration was decreased in the HG group than NG group. LncRNA Glis2 overexpression could increase ATP concentration in the HG + pcDNA3.1-lncRNA Glis2 group compared with the HG group (Figure 2J). The change in mitochondrial morphology was observed using MitoTracker Deep Red staining. Under high-glucose condition, mitochondria appeared rod-shaped in podocytes. Highly fragmented and discrete mitochondria were observed in the HG group. LncRNA Glis2 overexpression significantly prevented hyperglycemia-induced changes in mitochondrial morphology (Figure 2K). Mitofusin 1 (Mfn1) was significantly decreased and dynamin-related protein 1 (Drp1) was increased in the HG group compared with the NG group. The changes in mitochondrial fusion and fission protein induced by hyperglycemia were reversed in HG + pcDNA3.1-lncRNA Glis2 group (Figure 2L,M). These results suggested that lncRNA Glis2 overexpression alleviated podocyte apoptosis induced by hyperglycemia by restoring mitochondrial dysfunction in podocytes.

3.3 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 by FISH. The results showed that lncRNA Glis2 was predominantly located in the cytoplasm of cells (Figure 3A). As known, 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. Thus, we speculated that lncRNA Glis2 could regulate the target genes by competitively binding miRNA as a ceRNA.

To further elucidate the mechanism of lncRNA Glis2 in podocyte apoptosis, we used a 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 as the predicted candidate because it was the highest after transfecting with siRNA-lncRNA Glis2 (Figure 3B). Data from qRT-PCR also revealed that the expression of miR-328-5p is negatively correlated with lncRNA Glis2 (Figure 3C). Figure 3D illustrates 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 (Figure 3E). 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 the miR-328-5p mimics group compared with the NC mimics group. Whereas, when lncRNA Glis2-mut was used for co-transfecting, there was no significant difference between miR-328-5p mimics and the NC mimics group. Taken together, these findings confirmed that there was a direct interaction between lncRNA Glis2 and miR-328-5p.

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. The ability of inhibiting podocyte apoptosis which was caused by lncRNA Glis2 overexpression was partly countervailed by co-transfection with miR-328-5p mimics (Figure 3F–L). Taken together, the above results elucidated that lncRNA Glis2 regulated podocyte apoptosis by interacting with miR-328-5p.

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

In order to investigate the function of miR-328-5p in podocyte apoptosis, 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 (Figure 4A,B). Next, the results of the CCK-8 assay showed that miR-328-5p mimics decreased cell viability, while inhibiting the expression of miR-328-5p increased cell viability (Figure 4C). 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 (Figure 4D,E). Western blot displayed that pro-apoptotic caspase3 and caspase9 were significantly increased in the miR-328-5p mimics group. On the other hand, the expressions of caspase3 and caspase9 were reversed respectively once miR-328-5p was knock down in the miR-328-5p inhibitors group (Figure 4F,G). Therefore, these results confirmed that up-regulation of miR-328-5p induced podocyte apoptosis, whereas inhibition of miR-328-5p alleviated podocyte apoptosis.

Furthermore, we assessed the role of miR-328-5p in mitochondrial dysfunction. In contrast to the 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 (Figure 4H,I). Overexpression of miR-328-5p markedly reduced ATP concentration in the miR-328-5p mimics group than the NC mimics group (Figure 4J). Meanwhile, overexpression of miR-328-5p significantly affected mitochondrial morphology, including more fragmented and discrete mitochondria (Figure 4K). Western blot displayed that up-regulation of miR-328-5p decreased the expression of Mfn1 and increased the expression of Drp1 (Figure 4L,M). However, inhibition of miR-328-5p had an adverse effect on mitochondrial function. These data suggested that overexpression of miR-328-5p induced mitochondrial dysfunction, while inhibition of miR-328-5p alleviated mitochondrial dysfunction.

3.5 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 as the predicted candidate because it was reported to be related to mitochondrial function and podocyte apoptosis.^11-13 We found that there was a direct binding site between miR-328-5p and Sirt1 (Figure 5A). 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 (Figure 5B). 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 were co-transfected with a mutant Sirt1 reporter gene (Sirt1-mut) (Figure 5C). These data indicated that miR-328-5p negatively regulated the expression of Sirt1 in a direct manner.

3.6 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 podocyte apoptosis (Figure 6A,B) and increased cell viability (Figure 6C). Sirt1 overexpression significantly prevented the down-regulation of nephrin and podocin induced by hyperglycemia (Figure 6D,E). Up-regulation of Sirt1 significantly reversed the protein expressions associated with apoptosis, including decreasing caspase3 and caspase9 (Figure 6F,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 (Figure 6H,I), the down-regulation of ATP (Figure 6J) and abnormal mitochondrial morphology (Figure 6K) 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 (Figure 6L,M). Collectively, these results elucidated that up-regulation of Sirt1 alleviated mitochondrial dysfunction induced by hyperglycemia in podocytes.

3.7 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 (Figure 7A–F), including blood glucose, body weight, kidney weight, UACR, BUN and Scr, 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 (Figure 7G) and a significant decrease in the expression of miR-328-5p (Figure 7H) in kidney tissue 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 the db/db + pcDNA3.1-lncRNA Glis2 group. TEM confirmed the alleviation of podocyte foot process effacement and glomerular basement membrane thickening in the db/db + pcDNA3.1-lncRNA Glis2 group compared with the db/db + pcDNA3.1-NC group (Figure 7I).