miR-205-5p ameliorates podocyte injury in diabetic nephropathy by targeting vascular endothelial growth factor A

Abstract

Objective

This work aims to elucidate the effect and the regulatory mechanisms of miR-205-5p on podocyte injury and oxidative stress in diabetic nephropathy.

Methods

A mouse model of diabetic nephropathy was established. Fasting blood glucose, 24 hours urinary albumin, serum creatinine and blood urea nitrogen of mice were detected. H&E and Tunel staining of mice renal tissues were executed to detect histological changes and apoptosis. A cell model of diabetic nephropathy was constructed by inducing mouse podocytes with high glucose. The function of miR-205-5p on viability, apoptosis, and levels of malondialdehyde, superoxide dismutase and glutathione in the diabetic nephropathy cell model was evaluated by CCK-8 assay, Tunel staining and enzyme-linked immunosorbent assay. Binding of miR-205-5p and vascular endothelial growth factor A was verified by dual luciferase reporter gene assay. Rescue experiment was implemented on the diabetic nephropathy cell model to research whether miR-205-5p regulated diabetic nephropathy development by targeting vascular endothelial growth factor A. Quantitative reverse transcription-polymerase chain reaction and Western blot were for the detection of gene expression.

Results

The increased fasting blood glucose, 24 hours urinary albumin, serum creatinine and blood urea nitrogen levels, the intensified apoptosis and injury, and the down-regulated miR-205-5p were observed in renal tissues. miR-205-5p relieved podocyte injury in diabetic nephropathy, as it increased cell viability, decreased cell apoptosis, reduced malondialdehyde, and elevated superoxide dismutase and glutathione in the diabetic nephropathy cell model. Vascular endothelial growth factor A was up-regulated in renal tissues of diabetic nephropathy mice, and directly suppressed by miR-205-5p. Vascular endothelial growth factor A up-regulation abolished the protection of miR-205-5p on the diabetic nephropathy cell model.

Conclusions

miR-205-5p might relieve podocyte injury in diabetic nephropathy by suppressing Vascular endothelial growth factor A. It might be a promising target for diabetic nephropathy treatment.

Keywords

DN miR-205-5p VEGFA apoptosis viability

Introduction

Diabetic nephropathy (DN) is a common, serious diabetes complication causing chronic and end-stage renal disease.¹ About 35%–40% of diabetics develop DN, impacting quality of life and posing a serious threat.² Current standard DN treatment focuses on controlling blood glucose and pressure, but only slows progression, not stopping or curing it.³ Podocytes are crucial cells in the glomerulus, and the integrity of their structure and function is essential for maintaining the glomerular filtration barrier. In DN, hyperglycemia-induced oxidative stress is one of the core pathogenesis mechanisms, where excessive accumulation of reactive oxygen species (ROS) can directly damage podocyte structure and function. As the end product of lipid peroxidation, malondialdehyde (MDA) reflects the degree of oxidative damage, while superoxide dismutase (SOD) and glutathione (GSH) serve as key indicators of the endogenous antioxidant defense system. Oxidative stress promotes podocyte epithelial-mesenchymal transition (EMT) through activation of signaling pathways such as Wnt/β-catenin, leading to foot process effacement and proteinuria. Meanwhile, depletion of GSH exacerbates mitochondrial dysfunction and accelerates podocyte apoptosis. Podocyte damage, manifesting as fusion, retraction, and disappearance of podocyte foot processes, which in turn disrupts the glomerular filtration barrier and leads to the occurrence of proteinuria.⁴ Podocyte injury is considered to be one of the important mechanisms underlying the development and progression of DN. Therefore, elucidating the mechanisms of podocyte injury will conducive to providing effective therapeutic strategies for the treatment of DN.

MicroRNAs (miRNAs) are a class of highly stable and conserved single-stranded non-coding RNAs with 20–22 nucleotides in length, and abnormal expression levels or functions of miRNAs may mediate pathophysiological changes such as damage to glomerular basement membrane and mesangial cells, renal tissue fibrosis, and podocyte apoptosis, thereby regulating the occurrence and development of DN.⁵ A lot of miRNAs (miR-192, miR-21, and miR-29) have been extensively studied and have been shown to be closely related to various pathophysiological processes in DN.^6–8 Recently, Yun et al.⁹ established a DN cell model by inducing human renal mesangial cells with high glucose (HG) and found the down-regulated miR-205-5p in these HG-induced renal mesangial cells; miR-205-5p was found to block the progression of DN by targeting high-mobility group AT-hook 2 (HMGA2). Interestingly, a recent study constructed a DN mouse model by streptozotocin (STZ) induction, and a DN cell model by inducing mouse podocyte with HG; an abnormal reduction in miR-205-5p expression was discovered in renal tissue of DN mouse and DN cell models; the restoration of miR-205-5p expression might attenuated the HG-induced mouse podocyte injury by targeting early growth response protein 1 (EGR1).¹⁰ Thus, miR-205-5p has initially shown its potential for use in DN target therapy. More studies remain to be done to support the clinical application of miR-205-5p in the target treatment of DN. Our present study further explored the influence of miR-205-5p on podocyte injury in DN.

To identify the downstream target gene of miR-205-5p, this work implemented bioinformatic analysis. Vascular endothelial growth factor A (VEGFA) was finally selected as the target of miR-205-5p, as it showed the highest relevance score with DN and exhibited the mutual binding site to miR-205-5p in its 3′-UTR region. VEGFA is a member of the vascular endothelial growth factor (VEGF) family, which has gained a reputation for promoting angiogenesis.¹¹ A previous study has been suggested that VEGFA might be involved in the pathogenesis and progression of DN.¹² Thus, this study scrutinized whether miR-205-5p regulated DN progression by targeting VEGFA. This article will provide definitive data support and molecular basis for the utilization of miR-205-5p as therapeutic targets for DN.

Materials and methods

Animals and construction of a mouse model of DN

A total of 12 C57BL/6J male mice (8 weeks old, n = 6/group) were purchased from Vital River Laboratory Animal Technology (Beijing, China). Mice were group-housed (3–4 per cage) in individually ventilated cages (IVCs; dimensions: 30 × 15 × 12 cm) with corn cob bedding (changed twice weekly). The facility maintained a 12-h light/dark cycle (lights on at 07:00), temperature of 22 ± 1 °C, and humidity of 50 ± 10%. Standard rodent chow (SPF-grade pellet diet) and autoclaved water were provided ad libitum. Environmental enrichment included nesting material (cotton squares) and PVC tunnels. Animal study was conducted after the approval of Shanghai General Hospital Clinical Center Laboratory Animal Welfare & Ethics Committee (No. JDB-SH 05A8). The animal experiment of this study conforms to ARRIVE 2.0 guidelines.¹³ The mice were under adequate care followed the Guide for the Care and Use of Laboratory Animals, 8th Edition.¹⁴ All experimental procedures were designed to adhere to the principles of the 3Rs (replacement, reduction, and refinement). We minimized animal use by employing statistical power analysis to determine the smallest sample size required for robust results.

Six mice were randomly selected for the induction of diabetes via intraperitoneal injection of STZ (Beyotime, Shanghai, China) at a dose of 50 mg/kg for 5 consecutive days¹⁵ (named the DN group). The other six mice served as the control, which were injected intraperitoneally with normal saline at the same dose (named the negative control (NC) group). All mice were free access to food and water throughout the course of the experiment. The DN mouse model was successfully constructed if the fasting blood glucose (FBG) of mouse exceeded 250 mg/dL for 2 consecutive days. After 12 weeks, all mice were deeply anaesthetized by inhalation of 5% isoflurane (Reward Life Sciences, Shenzhen, China) mixed with oxygen for inhalation induction via an induction chamber. During surgical procedures, the isoflurane concentration was adjusted to 1.5%–2% for maintenance of anesthesia, delivered continuously through a nose cone. The depth of anesthesia was monitored by the absence of toe pinch reflex and respiratory rate (maintained at 40–60 breaths per minute). This anesthesia protocol complies with the Guide for the Care and Use of Laboratory Animals (8th edition) and ensured no pain perception during tissue collection. All procedures were performed in a biosafety cabinet equipped with a gas scavenging system. The mice were then euthanized to collect the renal tissues, which was according to Guide for the Care and Use of Laboratory Animals (8th edition) issued by National Research Council. The renal tissues were kept at −80 °C for preservation.

Biochemical analysis

On the last morning of the experiment, fasting blood samples from mice were obtained through the abdominal aorta, and the FBG level was monitored by utilizing the Mouse blood glucose assay kit (Yaji Biotechnology, Shanghai, China). Besides, on the last day of the experiment, the 24 hours urine of each mouse was collected for the detection of urinary albumin (UAlb) based on the UAlb assay kit (Jingkang Biological Engineering, Shanghai, China). At the end of the experiment, the blood samples (800 μL per mouse) of mice were harvested via the abdominal aorta. Serum creatinine (SCr) and blood urea nitrogen (BUN) were, respectively, detected according to the mouse SCr assay kit (Huding Biotechnology, Shanghai, China) and the mouse BUN assay kit (Yaji Biotechnology, Shanghai, China).

Hematoxylin and eosin (H&E) staining

Renal tissues of mice were immobilized in 4% paraformaldehyde (Yubo Biotechnology, Shanghai, China) for 24 hours, and then sectioned to a thickness of 5 μm after dehydrated and paraffin-embedded. The sections were rehydrated in paraffin and graded alcohol, and then stained by hematoxylin staining solution (Yubo Biotechnology, Shanghai, China) for 5 minutes and eosin staining solution (Yubo Biotechnology, Shanghai, China) for 1 minutes. Residual staining solution was washed off with tap water. After dehydration by alcohol and xylene, the sections were disclosed in neutral resin, followed by being observed under a light microscope (BX51, Olympus, Tokyo, Japan).

Cell lines and treatment

Mouse podocytes (MPC5; Catalogue No.: LHY2125; RRID: CVCL_AS87) were purchased from Lianmai Bioengineering (Shanghai, China), and cultivated in Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 °C and 5% CO₂. To construct a cell model of DN, this study applied HG (30 mM) to treat MPC5 cells for 48 hours, 37 °C and 5% CO_2.^16,17 Meanwhile, MPC5 cells treated with normal glucose (NG) (5 mM) for 48 hours were regarded as the control.

Cell transfection

MPC5 cells in serum-free DMEM were inoculated into the 6-well plates with 1 × 10⁶ cells/well. miR-205 mimic, mimic negative control (NC), and pcDNA-VEGFA vectors (GeneChem, Shanghai, China) were individually transfected into MPC5 cells by Lipofectamine 3000 (Thermo Fisher Scientific, San Jose, CA, USA) in line with the manufacturer's protocol. The sequences were miR-205-5p mimic, 5′-UCCUUCAUUCCACCGGAGUCUG-3′; miR-NC mimic, 5′-UCGCUUGGUGCAGGUCGGGAA-3′; miR-205-5p inhibitor, 5′-CAGACUCCGGUGGAAUGAAGGA-3′; and miR-NC inhibitor, 5′-CAGUACUUUUGUGUAGUACAA-3′. Then cells were treated with HG or NG for 48 hours at 37 °C and 5% CO₂. Depending on the transfection and treatment conditions, cells were named the NG + miR-NC group, the HG + miR-NC group, the HG + miR-205 group, and the HG + VEGFA group, respectively.

Besides, MPC5 cells were divided into (i) HG + miR-NC + oe-NC group (co-transfected with non-targeting miRNA mimic + pcDNA3.1 empty vector), and (ii) HG + miR-205 + VEGFA group (co-transfected with miR-205 mimic + pcDNA3.1-VEGFA plasmid).

Cell counting kit-8 (CCK-8) assay

MPC5 cells were cultivated in the relevant conditions for 48 hours in the 96-well plates with an inoculation density of 5 × 10³ cells/well. Five duplicate wells were set in each group. CCK-8 solution (10 μL, Beyotime, Shanghai, China) was added into each well to treat cells for 2 hours at 37 °C. The absorbance values of the wells were measured under a microplate reader (Biotek, Winooski, VT, USA). Cell viability was calculated by the percent absorbance of the control (the untreated cells).

Tunel staining

After rehydration, the sections (5 μm) of renal tissues were permeabilized by 0.25% Triton-X 100 (Beyotime, Shanghai, China) for 20 minutes. Tunel Apoptosis Detection Kit (Beyotime, Shanghai, China) was applied for the Tunel staining of the section in line with the manufacturer's protocol. After washed by phosphate buffered saline (PBS), the sections were stained for 1 minutes by 4′, 6-diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China). The apoptotic cells (green fluorescence) were observed under a fluorescent microscope (IX-71, Olympus, Tokyo, Japan). Apoptotic cells were counted in 10 random fields (400 ×) per sample using ImageJ software (v1.53), expressed as apoptotic cells/mm².

A coverslip was pre-positioned in each well of the 6-well plates. MPC5 cells were grown in the relevant conditions for 48 hours in these 6-well plates with an inoculation density of 1 × 10⁵ cells/well. Then cells were immobilized for 30 minutes in 4% paraformaldehyde. After permeabilized for 15 minutes by 0.25% Triton-X 100, MPC5 cells were subjected to the Tunel staining by recruiting the Tunel Apoptosis Detection Kit (Zeye Biotechnology, Shanghai, China) according to the manufacturer's directions. DAPI staining was implemented on these cells for 1 minutes at room temperature. The apoptotic cells (red fluorescence) under the fluorescent microscope were apoptotic cells. Three independent replicates were analyzed, with apoptosis rate calculated as (TUNEL + cells/DAPI + cells) × 100% per field) (200 cells/field minimum).

Enzyme-linked immunosorbent assay (ELISA)

After cultured for 48 hours in the relevant conditions, MPC5 cells of each group were harvested and lysed by lysis solution (Zeye Biotechnology, Shanghai, China) for 30 minutes on ice. The supernatant of the lysate was collected after centrifugation (10,000 r/min, 15 minutes, at 4 °C). The levels of MDA, SOD, and GSH in the supernatant was tested by employing the mouse MDA ELISA kit (Xinyu Biotechnology, Shanghai, China), the mouse SOD ELISA kit (Jingkang Biological Engineering, Shanghai, China), and the mouse GSH ELISA kit (Yiyan Biotechnology, Shanghai, China).^18–20 The testing procedure was carried out based on the manufacturer's directions.²¹

Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

This work utilized the Total RNA Extraction kit (Tiangen Biochemical Technology, Beijing, China) to extract the total RNA in renal tissues and MPC5 cells by following the manufacturer's protocol. Reverse transcription reaction was implemented with the PrimeScript RT kit (Takara, Tokyo, Japan) the miRNA First-Strand Synthesis kit (Takara, Tokyo, Japan). PCR was executed to detect the expression of miR-205-5p and VEGFA mRNA by utilizing the SYBR Premix Ex Taq II (TaKaRa, Shiga, Japan) on the 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), according to the following conditions: 95 °C for 3 minutes, followed by 40 cycle of 95 °C for 15 seconds and 58 °C for 30 seconds. U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were served as the reference for miR-205-5p and VEGFA, respectively. Primers were VEGFA (forward: 5′-CGAAACCATGAAGTTTACTGCAACTG-3′, reverse : 5′-TTGTGTCCTGGAAGCTCAGGAT-3′); U6 (forward : 5′-CTCGCTTCGGCAGCACATATACTA-3′, reverse : 5′-ACGCTTCACGAATTTGCGTGTCAT -3′); GAPDH (forward: 5′-AACTTTGGCATTGTGGAAGGGCTC-3′, reverse: 5′-TGGAAGAGTGGGAGTTGCTGTTGA-3′); and miR-205-5p (forward: 5′-GCGGCGGTGTAGTGTTTCCTA-3, reverse: 5′-GTGCAGGGTCCGAGGT-3′). The relative expression of miR-205-5p and VEGFA was determined by the 2^−ΔΔCt method.

Western blot

MPC5 cells cultured for 48 hours in the relevant conditions were harvested and lysed in radio-immunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) for total protein extraction. The total protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to the polyvinylidene fluoride (PVDF) (Lianshuo Biotechnology, Shanghai, China) membranes, and then blocked in 5% skimmed milk (Beyotime, Shanghai, China) for 1 hours. In the following, rabbit anti-primary antibodies (1:1000) were added to the PVDF membranes to probe the proteins overnight at 4 °C. All the primary antibodies were purchased from Abcam (Shanghai, China), including anti-cleaved-caspase-3 (ab2302), anti-Bax (ab262929), anti-Bcl-2 (ab59348), anti-VEGFA (ab185238), and anti-GAPDH (ab70699). Thereafter, goat anti-rabbit secondary antibody (1:5000, ab6721, Abcam, Shanghai, China) was employed to react with the proteins for 2 hours at room temperature. To develop the protein blots, the enhanced chemiluminescent reagent (Beyotime, Shanghai, China) was utilized to treat the PVDF membranes. Image J software (Version 1.46r, ImageJ, NIH, Bethesda, MD, USA) was responsible for the qualification of the blot grey values. Three independent biological replicates were quantified for each condition. Normalized ratios were calculated as (target protein intensity/GAPDH intensity).

Bioinformatic analysis

This study selected 4277 genes associated with “Diabetes nephropathy” from GeneCard (Version: V4.14, https://www.genecards.org/), with gene-disease relevance score > 1 as the selection criteria. Besides, 251 downstream genes of miR-205-5p were selected from the TargetScan7.2 (Version 7.2, http://www.targetscan.org/vert_72/), with the binding site type of 8 mer as the screening criterion. In the next, the 4277 genes associated with “Diabetes nephropathy” and the 251 downstream genes of miR-205-5p were intersected. Then genes with gene-disease relevance score in the top 10 were selected, which was shown in Table 1.

Table 1.

Genes with gene-disease relevance score in the top 10.

Gene	Relevance score
VEGFA	56.78420258
SLC19A2	35.99349213
GATA3	12.68791676
ADAMTS9	11.8816433
ONECUT1	10.69738102
INPPL1	10.06921577
CREB1	9.905687332
CSF1	9.267751694
LRP1	8.744279861
SMAD4	8.653975487

VEGFA: vascular endothelial growth factor A.

Dual luciferase reporter gene assay

The binding site of VEGFA and miR-205-5p was obtained from TargetScan7.2. Then the wild type (Wt) 3′-UTR sequences of VEGFA containing the miR-205-5p targeting sequence, as well as the mutant type (Mut) 3′-UTR sequences of VEGFA, were synthesized, cloned, and inserted into the pGL3 luciferase reporters by GeneChem (Shanghai, China). Then miR-205 mimic combined with the pGL3 luciferase reporters (Wt or Mut) were cotransfected into 293 T cells by Lipofectamine 3000. Meanwhile, 293 T cells were subjected to the cotransfection by mimic NC and the pGL3 luciferase reporters (Wt or Mut). After transfection, 293 T cells were cultivated for 48 hours in DMEM containing 10% FBS. The luciferase activity was then detected by the Dual-Luciferase Reporter Assay system (Promega, Madison, WI, USA).

Statistical analysis

All experiments were carried out in triplicate. Data were presented into mean ± standard deviation and GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA) was recruited for statistical analysis. Two tailed paired Student's t-test was employed to show the difference between two groups, and one-way analysis of variance combined with post-hoc Tukey's test was applied to exhibit the difference in more than two groups. p < 0.05 was considered the threshold for a statistically significant difference.

Results

The expression of miR-205-5p was declined in renal tissues of DN mice

This study established a mouse model of DN by STZ induction. As presented in Figure 1(a), DN mice had higher relative levels of FBG, 24 hours UAlb, SCr, and BUN content than NC mice (p < 0.01). By H&E staining of renal tissues, obvious DN signs were found in DN mice, such as the thickened mesangial matrix and glomerular hypertrophy, in comparison to NC mice (Figure 1(b)). Besides, relative to NC mice, Tunel staining showed more apoptotic cells in renal glomerulus of DN mice (Figure 1(c)). qRT-PCR displayed the reduced miR-205-5p expression in renal tissues of DN mice, as compared to the NC mice (p < 0.01) (Figure 1(d)). Therefore, miR-205-5p was down-regulated in renal tissues of DN mice.

Figure 1.

miR-205-5p was down-regulated in renal tissues of DN mice. (a) Fasting abdominal aortic blood samples were collected from mice. An increased FBG content was found in DN mice (n = 6). (b) The 24 hours urine samples were acquired from mice. DN mice showed a higher 24 hours UAlb content (n = 6). (C and D) Blood samples were collected from the abdominal aorta of mice (n = 6). DN mice had higher levels of SCr and BUN content. Data was shown as mean ± SD. ** p < 0.01 versus NC mice.

miR-205-5p overexpression protected mouse podocytes against the HG-induced injury and dysfunction

A cell model of DN was constructed by inducing mouse podocytes (MPC5 cells) with HG (30 mM). As exhibited in Figure 2(a), MPC5 cells induced with HG had lower miR-205-5p expression and cell viability than those treated with NG (5 mM) (p < 0.05 or p < 0.01). This suggested that the cell model of DN was successfully constructed and miR-205-5p was down-regulated in the cell model of DN.

Figure 2.

miR-205-5p overexpression protected mouse podocytes against HG-induced injury. (a) A cell model of DN was constructed by inducing mouse podocytes (MPC5 cells) with HG. qRT-PCR and CCK-8 assay suggested the down-regulated miR-205-5p and the attenuated cell viability in the DN cell model (n = 6). (b) qRT-PCR implied that miR-205-5p was effectively up-regulated in the HG-induced MPC5 cells by miR-205 mimic transfection (n = 6). (c) CCK-8 assay illustrated the promotion role of miR-205-5p on the viability of the HG-induced MPC5 cells (n = 6). (d) Tunel staining indicated the suppression effect of miR-205-5p on the apoptosis of the HG-induced MPC5 cells. (e) ELISA revealed that miR-205-5p could down-regulate MDA and up-regulated SOD and GSH in the HG-induced MPC5 cells (n = 6). (f) By Western blot, miR-205-5p could suppress the expression of cleaved-caspase-3 and Bax proteins but increase the expression of Bcl-2 protein in the HG-induced MPC5 cells (n = 3). Data was shown as mean ± SD. * p < 0.05 or ** p < 0.01 versus the NG group or the NG + miR-NC group. ^^ p < 0.01 versus the HG + miR-NC group.

Subsequently, this study explored the function of miR-205-5p in the HG-induced injury of mouse podocytes. A lower miR-205-5p level could be noticed in MPC5 cells of the HG + miR-NC group, in comparison to the NG + miR-NC group (p < 0.01); in contrast to the HG + miR-NC group, MPC5 cells of the HG + miR-205 group expressed higher miR-205-5p (p < 0.01) (Figure 2(b)). Thus, miR-205-5p was effectively up-regulated in the HG-induced MPC5 cells. CCK-8 assay and Tunel staining showed the decreased cell viability but the intensified apoptosis in MPC5 cells of the HG + miR-NC group, when matched to the NG + miR-NC group (p < 0.01); however, relative to MPC5 cells of the HG + miR-NC group, those of the HG + miR-205 group displayed the increased cell viability but the attenuated apoptosis (p < 0.01) (Figure 2(c) and (d)). By ELISA, a higher MDA level but lower levels of SOD and GSH occurred in MPC5 cells of the HG + miR-NC group, compared to the NG + miR-NC group (p < 0.01); conversely, MPC5 cells of the HG + miR-205 group had lower MDA level but higher SOD and GSH levels than the HG + miR-NC group (p < 0.01) (Figure 2(e)). Next, this research executed Western blot to monitor the expression of apoptotic proteins. MPC5 cells of the HG + miR-NC group expressed higher cleaved-caspase-3 and Bax proteins but lower Bcl-2 protein than the NG + miR-NC group (p < 0.01); oppositely, a lower cleaved-caspase-3 and Bax proteins but a higher Bcl-2 protein was observed in MPC5 cells of the HG + miR-205 group, when matched to HG + miR-NC group (p < 0.01) (Figure 2(f)). All these findings implied that miR-205-5p up-regulation could protect mouse podocytes against the HG-induced injury and dysfunction.

miR-205-5p could directly suppress the expression of VEGFA

Through bioinformatic analysis, this study screened the top 10 miR-205-5p downstream genes that were closely relevant to DN, including VEGFA, SLC19A2, GATA3, ADAMTS9, ONECUT1, INPPL1, CREB1, CSF1, LRP1, and SMAD4. Among them, VEGFA had the highest relevance score with DN (Table 1). Then PCR was implemented on renal tissues of DN and NC mice, and Heatmap was produced. Compared with NC mice, VEGFA, GATA3, CREB1, and SMAD4 were up-regulated in renal tissues of DN mice; besides, among the four up-regulated genes, VEGFA was the most significantly up-regulated gene in renal tissues of DN mice (Figure 3(a)). Thus, VEGFA was selected as the subject in the next research. As presented in Figure 3(b), MPC5 cells of the HG + miR-NC group expressed higher VEGFA mRNA than the NG + miR-NC group (p < 0.01); however, a much reduction in the expression of VEGFA mRNA was observed in MPC5 cells of the HG + miR-205 group, when matched to the HG + miR-NC group (p < 0.01). By Western blot, DN mice expressed a distinctly higher VEGFA protein than NC mice (p < 0.01) (Figure 3(c)). Additionally, the increased VEGFA protein was displayed in MPC5 cells of the HG + miR-NC group, in contrast to the NG + miR-NC group (p < 0.01); intriguingly, relative to the expression of VEGFA protein in MPC5 cells of the HG + miR-NC group, it was dramatically reduced in those of the HG + miR-205 group (p < 0.01) (Figure 3(d)). TargetScan showed the binding site of VEGFA to miR-205-5p; according to dual luciferase reporter gene assay, miR-205 mimic did not influence the luciferase activity in MPC5 cells that cotransfected with the plasmid expressing the VEGFA Mut sequence; however, it pronouncedly reduced the luciferase activity in MPC5 cells that cotransfected by the plasmid expressing the VEGFA Wt sequence (p < 0.01) (Figure 3(e)). Thus, as a target of miR-205-5p, VEGFA could be directly suppressed by miR-205-5p.

Figure 3.

miR-205-5p could directly suppress the expression of VEGFA. (a) By qRT-PCR, VEGFA was the most significantly up-regulated gene in renal tissues of DN mice among the four up-regulated genes (including VEGFA, GATA3, CREB1, and SMAD4) (n = 6). (b) qRT-PCR indicated that miR-205-5p reduced the expression of VEGFA mRNA in the HG-induced MPC5 cells (n = 6). ** p < 0.01 versus the NG + miR-NC group. ^^ p < 0.01 versus the HG + miR-NC group. (c) Western blot implied the up-regulated VEGFA protein in renal tissues of DN mice (n = 3). ** p < 0.01 versus NG mice. (d) Western blot revealed the suppression role of miR-205-5p on the e expression of VEGFA protein in the HG-induced MPC5 cells. ** p < 0.01 versus the NG + miR-NC group (n = 3). ^^ p < 0.01 versus the HG + miR-NC group. (e) TargetScan showed the binding site of VEGFA to miR-205-5p. Dual luciferase reporter gene assay demonstrated that VEGFA could be directly suppressed by miR-205-5p (n = 6). Data was shown as mean ± SD. ** p < 0.01 versus the miR-NC group.

miR-205-5p relieved the HG-induced injury of mouse podocytes by blocking the expression of VEGFA

In this part, rescue experiment was executed by cotransfecting the HG-induced mouse podocytes with miR-205 mimic and plasmid expressing VEGFA. A much higher miR-205-5p level was observed in MPC5 cells of the HG + miR-205 group, in contrast to the HG + miR-NC + oeNC group (p < 0.01); MPC5 cells of the HG + miR-205 + VEGFA group exhibited a higher miR-205-5p expression than those of the HG + VEGFA group; besides, matched to MPC5 cells of the HG + miR-NC + oeNC group, a lower VEGFA mRNA expression in the HG + miR-205 group but a higher VEGFA mRNA expression in the HG + VEGFA group were discovered (p < 0.01); MPC5 cells of the HG + miR-205 + VEGFA group expressed lower VEGFA mRNA than the HG + VEGFA group (p < 0.01); additionally, in comparison to the viability of MPC5 cells in the HG + miR-NC + oeNC group, it was much enhanced in the HG + miR-205 group but significantly attenuated in the HG + VEGFA group (p < 0.05 or p < 0.01); a markedly intensified viability was observed in MPC5 cells of the HG + miR-205 + VEGFA group, in comparison to the HG + VEGFA group (p < 0.05) (Figure 4(a)). By Western blot, a decreased VEGFA protein in the HG + miR-205 group but an increased VEGFA protein in the HG + VEGFA group was found, when matched to that in MPC5 cells of the HG + miR-NC + oeNC group (p < 0.01); however, MPC5 cells of HG + miR-205 + VEGFA group showed a much reduction in the expression of VEGFA protein, compared to the HG + VEGFA group (p < 0.01) (Figure 4(b)).

Figure 4.

miR-205-5p relieved the HG-induced injury of mouse podocytes by blocking the expression of VEGFA. (a) The HG-induced MPC5 cells were successfully cotransfected by miR-205 mimic and plasmid expressing VEGFA. VEGFA counteracted the enhancement of miR-205-5p on the viability of the HG-induced MPC5 cells (n = 6). (b) Western blot proved that the expression of VEGFA was effectively regulated by transfection (n = 3). (c) Tunel staining revealed that VEGFA reversed the suppression of miR-205-5p on the apoptosis of the HG-induced MPC5 cells (n = 6). (d) By ELISA, VEGFA abrogated the suppression of miR-205-5p on MDA expression and the promotion of it on SOD and GSH expression in the HG-induced MPC5 cells (n = 6). (e) Western blot illustrated that VEGFA counteracted the inhibition of miR-205-5p on cleaved-caspase-3 and Bax protein expression and the promotion of it on Bcl-2 protein expression in the HG-induced MPC5 cells (n = 3). Data was shown as mean ± SD. ** p < 0.01 versus the HG + miR-NC + oeNC group. ^ p < 0.05 or ^^ p < 0.01 versus the HG + VEGFA group.

Tunel staining of MPC5 cells exhibited the weakened apoptosis in the HG + miR-205 group and the intensified apoptosis in the HG + VEGFA group, when relative to the HG + miR-NC + oeNC group; interestingly, matched to the HG + VEGFA group, the apoptosis was suppressed in MPC5 cells of the HG + miR-205 + VEGFA group (Figure 4(c)). ELISA was implemented to test the levels of MDA, SOD and GSH. Compared to the HG + miR-NC + oeNC group, MPC5 cells of the HG + miR-205 group had lower MDA level but higher SOD and GSH levels (p < 0.01); meanwhile, MPC5 cells of the HG + VEGFA group exhibited higher MDA level but lower SOD and GSH levels than the HG + miR-NC + oeNC group (p < 0.01); when relative to MPC5 cells of the HG + VEGFA group, the decreased MDA level and increased SOD and GSH levels occurred in the HG + miR-205 + VEGFA group (p < 0.05 or p < 0.01) (Figure 4(d)).

The expression of apoptosis-related proteins in MPC5 cells of each group was determined by Western blot. MPC5 cells of the HG + miR-205 group expressed lower cleaved-caspase-3 and Bax proteins but higher Bcl-2 protein than the HG + miR-NC + oeNC group (p < 0.01); on the contrary, higher cleaved-caspase-3 and Bax proteins and lower Bcl-2 protein was discovered in MPC5 cells of the HG + VEGFA group, in contrast to the HG + miR-NC + oeNC group (p < 0.01); as compared to the HG + VEGFA group, lower cleaved-caspase-3 and Bax proteins as well as higher Bcl-2 protein were shown in MPC5 cells of the HG + miR-205 + VEGFA group (p < 0.01) (Figure 4(e)). According to these data, VEGFA abrogated the protection of miR-205-5p on the HG-induced injury of mouse podocytes. Hence, miR-205-5p was suggested to protect mouse podocytes against the HG-induced injury by suppressing VEGFA.

Discussion

Since their initial discovery in 1993, miRNAs have been recognized as critical regulators of gene expression, with dysregulated expression closely linked to diverse pathologies including cancers, cardiovascular diseases, and viral infections. Several miRNA-targeted therapeutics, such as the anti-hepatocellular carcinoma agent miR-34 (MRX34) and the anti-hepatitis C drug miR-122 (Miravirsen), have entered clinical trials worldwide and demonstrated promising therapeutic potential. However, challenges persist in achieving efficient systemic delivery, minimizing off-target effects, and ensuring in vivo stability of miRNA-based therapies.^22–24 Recent advancements in chemical modifications, novel nanocarriers, and precision-targeted delivery systems have progressively addressed these technological limitations.²⁵ The emergence of innovative approaches such as CRISPR-miRNA editing technologies is now driving the evolution of miRNA therapeutics from single-target interventions toward precision medicine frameworks, offering novel strategic avenues for treating complex diseases.

The present study indicated that miR-205-5p was abnormally down-regulated in the renal tissues of DN mice. Glomerular podocyte injury is regarded as a major reason leading to DN.²⁶ Thus, in vitro study in this article constructed the DN cell model by inducing mouse podocytes with HG. It was suggested that the restoration of miR-205-5p expression could alleviate the injury of the HG-induced mouse podocytes. Regarding the mechanism, it was implied that miR-205-5p overexpression might protect mouse podocytes from the HG-induced injury via directly repressing the expression of VEGFA.

Accumulating evidence has been suggested the involvement of miRNAs in regulating the progression of DN, such as miR-1187,⁴ which has been revealed to induce podocyte injury, renal dysfunction, glomerular apoptosis and proteinuria in DN mouse. Conversely, miR-30a-5p has been indicated to ameliorate DN by reducing podocyte injury and suppressing its apoptosis.²⁷ In this paper, miR-205-5p was detected to be down-regulated in renal tissues of DN mice, which was consistent with the previous study.¹⁰ This work established a DN cell model by inducing mouse podocytes with HG. It was suggested that miR-205-5p up-regulation enhanced viability but weakened apoptosis of the HG-induced mouse podocytes. Besides, miR-205-5p overexpression decreased cleaved-caspase-3 and Bax proteins and reduced Bcl-2 protein in the HG-induced mouse podocytes. Bcl-2 is a well-known anti-apoptotic protein,²⁸ and cleaved-caspase-3 and Bax are pro-apoptotic markers.²⁹ Thus, in this study, miR-205-5p could attenuate apoptosis of the HG-induced mouse podocytes by down-regulating cleaved-caspase-3 and Bax proteins and up-regulating Bcl-2 protein. Due to technical limitations, the area of the glomerulus could not be assessed, and subsequent research will focus on this aspect. Moreover, Nephrin (NPHS1), one of the most critical structural and functional markers of podocytes, will be systematically incorporated into our subsequent investigations to validate podocyte-specific injury mechanisms.

In diabetes, excessive oxidative stress induced by metabolic abnormalities is a major cause of diabetic kidney injury.³⁰ Hyperglycemia-induced oxidative stress can lead to the excessive production of reactive oxygen species; however, the excessive production of reactive oxygen species can induce kidney cell injury by further inducing the production of MDA, a marker of oxidative damage.³¹ SOD and GSH are considered to be important antioxidant enzymes in the antioxidant defense system in renal injury, as SOD is able to catalyze superoxide radicals into hydrogen peroxide and GSH is capable of catalyzing the hydrogen peroxide into water.³¹ It has been discovered the increased MDA as well as the decreased antioxidant enzymes such as SOD and GSH in DN mouse.³⁰ This research investigated that the restoration of miR-205-5p expression reduced MDA level but elevated the levels of SOD and GSH in the HG-induced mouse podocytes. Thus, miR-205-5p could relieve the oxidative stress damage of podocytes in DN. While miR-205-5p demonstrates antioxidative efficacy in murine models, clinical translation requires solutions for renal-targeted delivery (e.g. ligand-modified nanoparticles). Combining miR-205-5p with Nrf2 activators could synergistically amplify GSH production, addressing both ROS generation and clearance. Further validation in human podocyte cultures is warranted to assess species-specific responses.

In this study, VEGFA was identified to be a target of miR-205-5p, and it could be directly suppressed by VEGFA. Besides, we revealed the aberrantly overexpressed VEGFA in renal tissues of DN mice. As we know, DN is a common microvascular complication of diabetes.³² VEGFA has been proposed to be a major mediator of vascular dysfunction in DN, because both deletion and overexpression of VEGFA can exacerbate DN by inducing microvascular abnormality.³³ ROS activates HIF-1α and NF-κB signaling pathways to induce VEGFA secretion, as observed in glomerular podocytes.^34,35 VEGFA is produced by podocytes, which is necessary for the survival and morphological maintenance of podocytes; hyperglycemia-induced excessive production of VEGFA in podocytes drives the onset and progression of DN.³⁶ It has been researched that the greatly enhanced VEGFA signal worsens DN in mouse and VEGFA level can determine the severity of DN.³⁷ Emerging evidence also revealed that under hyperglycemic conditions, substantial ROS accumulation occurs, which directly upregulates VEGFA gene transcription by activating HIF-1α while simultaneously stimulating the NF-κB inflammatory pathway to synergistically enhance VEGFA expression.³⁸ The overexpression of VEGFA promotes pathological angiogenesis in glomeruli, accelerating proteinuria formation. Concurrently, VEGFA binding to its receptor VEGFR2 activates downstream PI3K signaling, further stimulating NOX enzymatic activity and thereby promoting continuous ROS generation.³⁹ This positive feedback loop exacerbates local oxidative damage in renal tissues, directly impairing tubular cells and basement membrane structure. Consistently, this work implied the role of VEGFA in exacerbating the HG-induced mouse podocyte injury. More interestingly, VEGFA abolished the protection of miR-205-5p on the HG-induced injury of mouse podocytes. This suggested that miR-205-5p could protect mouse podocytes against the HG-induced injury by suppressing VEGFA. Despite providing valuable insights into the role of miR-205-5p in DN, this study still has several limitations. The study employed high-glucose-induced mouse podocytes and mouse models to simulate diabetic nephropathy. However, these animal models may not fully replicate all the complexities and diversities of human DN. The research focuses on miR-205-5p and its impact on VEGFA, whereas DN is a multifactorial disease involving multiple signaling pathways and molecular mechanisms. Further investigation into the underlying molecular mechanisms is needed. Future research efforts will focus on addressing these limitations and further validating the findings of the current study. Moreover, the rescue experiments showed partial but not complete reversal of VEGFA effects, suggesting involvement of additional pathways. miR-205-5p may alleviate podocyte epithelial-mesenchymal transition (EMT) by suppressing the TGF-β1/Smad3 signaling pathway, and this mechanism may involve cross-regulatory interactions with the VEGFA pathway.⁴⁰

Our study demonstrated decreased miR-205-5p expression in both DN mice and HG-stimulated podocytes. The observed protective effects of miR-205-5p against HG-induced podocyte injury, potentially mediated through VEGFA inhibition, suggest its possible therapeutic relevance for DN. While these preclinical findings require further validation, targeted modulation of miR-205-5p may represent a novel therapeutic strategy worthy of future clinical exploration.

Footnotes

ORCID iD

Zhihuan Tang

Ethics approval and consent to participate

This study was conducted after obtaining the Shanghai General Hospital Clinical Center Laboratory Animal Welfare & Ethics Committee (No. JDB-SH 05A8). All methods were performed in accordance with the relevant guidelines and regulations.

Authors’ contributions

Yingdan Zhao and Yunhai Tang planned the study. Qingqing Wang, Xia Wu, Zifan He, and Yayun He conducted a survey. Yingdan Zhao, Yunhai Tang, Qingqing Wang, Xia Wu, and Zifan He performed the data analyses and wrote the manuscript. Yayun He and Zhihuan Tang performed the data analyses and revised the manuscript. All the authors read and agreed to submit the manuscript. Zhihuan Tang submitted the study.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Nature Cultivation Project (grant number 202140B).

Declaration of conflicting interests

The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: The animal experiment of this study conforms to ARRIVE 2.0 guidelines.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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