CircRNA-0013747 induces mesangial cell proliferation in IgA nephropathy by targeting the Warburg effect via miR-330- 3p/PKM2 signaling

doi:10.21203/rs.3.rs-3996101/v1

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CircRNA-0013747 induces mesangial cell proliferation in IgA nephropathy by targeting the Warburg effect via miR-330- 3p/PKM2 signaling

https://doi.org/10.21203/rs.3.rs-3996101/v1

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Aberrant mesangial cell proliferation is a prevailing histopathological feature of immunoglobulin A nephropathy (IgAN) and is the primary driver of glomerular sclerosis and impaired renal function in IgAN patients. Prior research has revealed that PKM2-mediated aerobic glycolysis (the Warburg effect) frequently promotes mesangial cell growth and contributes to the development of various acute and chronic kidney diseases. However, the expression and functionality of PKM2 in IgA nephropathy, as well as the underlying molecular mechanisms governing its abnormal expression, remain elusive. Circular RNAs, a subset of noncoding RNAs, have garnered increasing attention due to mounting evidence of their pivotal roles in the initiation and progression of numerous disorders. The present study aimed to explore the effects of circRNA_0013747 on IgAN and the potential underlying mechanisms. The results indicated notable overexpression of circRNA_0013747 in lipopolysaccharide (LPS)-treated human mesangial cells (HMCs) and kidney biopsy samples from IgAN patients. CircRNA_0013747 was shown to facilitate mesangial cell proliferation and activate PKM2-mediated aerobic glycolysis, although these effects were mitigated by an increase in miR-330-3p. Mechanistically, circRNA_0013747 physically interacted with microRNA-330-3p (miR-330-3p) and hindered its function by directly binding to it. These findings imply that circRNA_0013747 can enhance glycolysis and proliferation in mesangial cells by modulating the miR-330-3p/PKM2 signaling pathway. In conclusion, the present results underscore the possibility of circRNA_0013747 serving as a promising therapeutic target for IgAN, suggesting new prospects for treating this disease.

IgA nephropathy

Mesangial cell

Warburg effect

PKM2

Circular RNA

Immunoglobulin A nephropathy (IgAN) is a primary glomerular disease characterized by glomerular mesangial deposition of IgA or IgA-based immunoglobulin[1]. Since its identification by the French scholar Berger in 1968, it has become the most prevalent primary glomerular disease worldwide[2]. Patients with moderate clinical disease exhibit mesangial cell proliferation and extracellular matrix growth, but 30–40% of patients develop end-stage renal disease as a result of glomerular and interstitial sclerosis approximately 20 years after diagnosis[3–4]. Mesangial cells are activated via the deposition of the galactose-deficient IgA1 (Gd-IgA1) immune complex to mediate particular intracellular signal transduction pathways, and Gd-IgA1 immune complex deposition also promotes mesangial cell growth and initiates kidney injury[5–6]. Additionally, a number of proinflammatory and fibroblastic cytokines are released by mesangial cells to mediate oxidative stress and complement activation, which in turn causes damage to podocytes and proximal tubular epithelial cells, glomerular sclerosis, and interstitial fibrosis, all of which contribute to the progression of IgAN[7–8]. The early pathogenic changes in IgAN are thought to be largely mediated by mesangial cells, according to the following findings.

Glucose is vital for cellular functions because it serves as the primary substrate for energy production. When glucose is fully oxidized within a cell in the presence of oxygen, 38 molecules of adenosine triphosphate (ATP) are generated, which serves as the cell's essential energy source[9]. However, when cells are under hypoxic conditions, pyruvic acid is not converted into acetyl-coenzyme A but rather into lactic acid, a process known as anaerobic cellular respiration[10]. In this scenario, the net energy balance is reduced to just two ATP molecules, making anaerobic glucose metabolism energetically inefficient[11]. In the early 1920s, Warburg, Posener, and Negelein made noteworthy discoveries regarding the behavior of tumor tissue in vitro. Their examination of respiration and glycolysis in different tissue sections revealed that tumors exhibit unusually high glycolytic activity and lactic acid production from glucose when compared to normal tissues. Surprisingly, glycolysis is "aerobic" and is not inhibited by oxygen in malignant cells, signifying that cancer cells lack the "Pasteur effect"[12–13]. This gave rise to the concept of aerobic glycolysis, which is the fermentation of glucose even in the presence of ample oxygen. Type M2 pyruvate kinase (PKM2) plays a key role in glycolysis by catalyzing the final and physiologically irreversible step of the process of converting phosphoenolpyruvate (PEP) to pyruvate through the transfer of a phosphate group to ADP[14]. The substantial upregulation of PKM2 expression in most human cancer types strongly suggests its importance in tumorigenesis. Transient transfection of tumor cell lines with PKM2 small interfering RNA (siRNA) resulted in reduced cell proliferation and increased apoptosis[15–17]. Metabolic changes have also been implicated in the development of several kidney diseases. Li et al. reported a shift toward aerobic glycolysis in the pathogenesis of chronic kidney disease (CKD)[18]. Furthermore, the inhibition of PKM2 by shikonin significantly ameliorated the histopathological symptoms of LPS-induced acute kidney injury (AKI)[19]. These findings underscore the potential role of PKM2-mediated aerobic glycolysis in various kidney diseases, although the precise regulatory mechanisms involved remain to be elucidated.

Circular RNAs (circRNAs) represent a distinct class of noncoding RNAs highly prevalent in mammalian cells and are involved in the regulation of gene expression[20]. They have been found in various human organs and exhibit unique spatial and temporal expression patterns[21]. Due to their closed-loop structure, circRNAs exhibit greater stability than linear RNAs, rendering them promising candidates as biomarkers for disease diagnosis[22]. Research suggests that circRNAs can act as 'sponges' for miRNAs, selectively binding to miRNAs and consequently leading to the upregulation of miRNA target genes[23]. For example, Niu et al. demonstrated that Circ_0008529 contributes to renal tubular cell dysfunction under high glucose stress through the miR-185-5p/SMAD2 pathway in diabetic nephropathy[24]. Furthermore, circRNA_0017076 modulates epithelial-to- mesenchy -mal transition during renal interstitial fibrosis by acting as a sponge for miR-185-5p[25]. These discoveries underscore the crucial role of circRNAs in kidney diseases.

In our investigation, we noted a considerable increase in the expression of hsa_circ_0013747 in human IgA nephropathy tissue compared to normal kidney tissue. Additionally, we found that circRNA_0013747 can enhance aerobic glycolysis and stimulate the proliferation of mesangial cells by modulating the miR-330-3p/PKM2 signaling axis. In an IgAN mouse model, the administration of adeno-associated virus with mmu_circ_0010297 knockdown led to a substantial reduction in kidney lactate levels, effectively ameliorating kidney damage. These findings indicate that the circRNA_0013747/miR-330-3p/PKM2 pathway could be a valuable target for the early diagnosis and treatment of IgA nephropathy.

2.1 Ethics Approval:

The research protocols involving human specimens were granted approval by the Institutional Ethics Committee of Guizhou Medical University, located in Guiyang, China (Approval Number: 2021-43). Patients who participated in the study were recruited from the Affiliated Hospital of Guizhou Medical University in Guiyang, China, and provided written informed consent. Animal experiments were conducted under the authorization of the Institutional Animal Ethics Committee of Guizhou Medical University (Approval Number: 2100031). These studies were carried out in strict adherence to the guidelines outlined in the "Guide for the Care and Use of Laboratory Animals" by the US National Institutes of Health.

2.2 Human tissue specimens

We collected fresh kidney tissues from a total of 32 patients from the Nephrology and Urology Departments of the hospital. Sixteen of these patients were pathologically diagnosed with primary IgA nephropathy. The kidney tissues of these patients exhibited significant mesangial proliferation and glomerulosclerosis. These tissues were obtained through percutaneous biopsy under the guidance of ultrasound. The control group included kidney samples from 16 patients who had undergone nephrectomy due to traumatic kidney injury. Tissue samples were taken approximately 5 cm from the injury site and were pathologically confirmed to be normal kidney tissues. After brief flash freezing in liquid nitrogen for 30 seconds, the tissues were stored at a temperature of -80°C.

2.3 Animal model

We obtained eighteen female BALB/c mice aged five weeks with weights ranging from 20 to 25 g. These mice were procured from the Laboratory Animal Center at Guizhou Medical University, located in Guiyang, China. They were kept in standard cages in a pathogen-free environment with controlled temperature and humidity conditions. Following a three-day acclimation period, the mice were randomly divided into three groups: "Blank control," "IgAN + AAV-shNC," and "IgAN + AAVsh-mmu_circ_0010297," with six mice in each group. Mice were intraperitoneally administered 2% pentobarbital at a dose of 4 ml/kg for anesthesia. Subsequently, under ultrasound guidance, a solution containing the RNAi adeno-associated virus (AAV) vector-9 was microinjected into the kidneys using a 31G needle. The AAV vector for silencing mmu_circ_0010297 and the negative control AAV vector were obtained from GeneChem in China. One week later, we induced IgAN through oral mucosal immunization. The mice were gavaged with acidified bovine serum albumin (BSA) every other day at a dose of 800 mg/kg. Additionally, they were administered a mixed solution of CCL4 and castor oil (at a 1:5 ratio) subcutaneously every week (0.1 ml) and intraperitoneally every two weeks (0.06–0.08 ml). At weeks 6 and 8, lipopolysaccharide (LPS, 50 µg) was injected into the tail vein. The successful establishment of the IgAN model was confirmed at the end of week 11 via glomerular IgA immunofluorescence. By week 17, the mice were humanely euthanized under anesthesia, and blood, urine, and kidney tissue samples were collected for further analysis.

2.4 Histological analyses

Kidney tissues were dehydrated, embedded, and subsequently sectioned at a thickness of 4 mm. To evaluate collagen deposition in the renal sections, Sirius red staining was performed. Immunohistochemistry (IHC) was used to quantify and locate specific proteins within these sections. Images were captured using a microscope and analyzed using ImageJ software. Further information about the antibodies utilized can be found in Table 1.

Table 1

Antibodies
Names	Manufacturer	Cat. No
PKM2	Abcam, UK	ab150377
collagen IV	Abcam, UK	ab6586
Fibronectin	Abcam, UK	ab2413
IgA	Abcam, UK	ab214003
Tubulin	Abcam, UK	ab8245

2.5 RNA fluorescence in situ hybridization (RNA FISH)

Cells or kidney sections were subjected to a 12-hour incubation with RNA probes in hybridization buffer. The circRNA_0013747 probe was labeled with Cyanine 3 (Cy3), while the miR-330-3p probe was labeled with fluorescein isothiocyanate (FITC). Both probes were obtained from RiboBio, China.

2.6 Immunofluorescence staining

Cells and tissues, which were subsequently grown on cover slips, were subjected to a series of steps. The cells were initially fixed using 4% formaldehyde, permeabilized using 0.25% Triton X-100, and blocked quickly with blocking solution. Subsequently, the sections were subjected to overnight incubation at 4°C with primary antibodies targeting IgA, PKM2, collagen IV, or fibronectin. The next day, the cells and tissues were thoroughly washed with PBS containing 0.05% Tween 20 before they were incubated with a fluorescent secondary antibody. DAPI staining was used to visualize the cell nuclei. The prepared samples were stored in a light-protected environment and subsequently examined using a laser confocal microscope at the earliest possible time points.

2.7 Cell culture

Human mesangial cells (HMCs) were procured from the American Type Culture Collection (ATCC, USA). The cells were transfected using Lipofectamine RNAiMAX (Invitrogen, USA) with either a circRNA_0013747-specific siRNA obtained from GeneSeed (China) or a negative control. Following transfection, the HMC cells were exposed to LPS. Additionally, cells were transfected with a pLCDH-ciR plasmid containing circRNA_0013747 (GeneSeed, China), a miR-330-3p mimic sourced from RiboBio, China, or a PKM2 siRNA provided by GenePharma, China. After these transfection procedures, the cells were incubated for 24 hours under standard growth conditions before they were transitioned to the experimental media. The harvested cells were used in the subsequent experiments. The specific siRNA sequences used are detailed in Table 2.

Table 2

siRNA sequences
siRNA targets	circRNA_0013747 siRNA1# circRNA_0013747 siRNA2#	GCTGAAACATTAAAGCTATAG AACATTAAAGCTATAGAAAAA
PKM2 siRNA	Sense Antisense	AGT ACC ATG CGG AGA CCA TC GCG TTA TCC AGC GTG ATT TT

2.8 RNA extraction and quantitative real-time PCR (RT‒qPCR) analysis

Total RNA was isolated from kidney tissues or cultured cells via the TRIzol method. Subsequently, the extracted RNA was subjected to reverse transcription into cDNA employing a kit sourced from Takara, Japan. RT‒qPCR was performed utilizing SYBR Green reagent (Takara, Japan). The relative gene expression was determined after normalization to the internal control, GAPDH, employing the 2^−ΔΔCt method. For the primer sequences used in the RT‒qPCR experiments, please refer to Supplementary Table 3.

Table 3

PCR primer sequences
	Forward primer (5'-3')	Reverse primer(5'-3')
CircRNA_0013747	GTCAAAGATGTATATTCCTCT	TGCTCTGGAACCATCTGCTCC
MiR-330-3p	GCGGCGGGCAAAGCACACGGCC	ATCCAGTGCAGGGTCCGAGG
PKM2	TCGCATGCAGCACCTGATT	CCTCGAATAGCTGCAAGTGGTA
GAPDH	TGTGGGCATCAATGGATTTGG	ACACCATGTATTCCGGGTCAAT

2.9 Western blot (WB) analysis

Proteins were extracted from tissues and cells using lysis buffer, and the total protein concentration was determined with a BCA kit (Beyotime Biotechnology, China). In brief, after the proteins were transferred to PVDF membranes, the membranes were blocked with 5% nonfat milk for one hour. The membranes were then incubated with the diluted primary antibody overnight at 4°C. On the following day, the secondary antibody was applied, and the samples were incubated at room temperature for an hour. The protein bands were visualized using the ECL method, and their intensity was quantified using a gel imaging system. Specific information about the antibodies utilized can be found in Table 1.

2.10 Cell viability assay

HMCs were seeded in 96-well plates and subjected to treatment with 10 µL of Cell Counting Kit-8 (CCK-8) reagent (Dojindo, Japan) per well. After incubation at 37°C in a controlled atmosphere, the optical density (OD450) was determined using a microplate reader (Bio-Rad, USA) on Days 1, 2, 3, 4, 5, and 6 following treatment. To evaluate the number of apoptotic cells, 1×10⁶ cells from each group were rinsed with PBS and subsequently stained with PE-conjugated Annexin V and 7AAD dyes. Flow cytometry was used to quantify apoptotic cells using a Becton Dickinson instrument located in Guiyang, China.

2.11 Lactate production, glucose uptake, and ATP levels

The glucose level was measured using a glucose assay kit from Merck KGaA, Germany. The lactate level was determined using a Lactate Assay Kit (Merck KGaA, Germany). ATP levels were assessed using the Luminescent ATP Detection Assay from Abcam, UK.

2.12 RNA pull‑down assay

Streptavidin-coated magnetic beads (Thermo Fisher Scientific, USA) were conjugated with biotin-labeled CircRNA_0013747 probes or control probes (RiboBio, China). Subsequently, these conjugates were mixed with cell lysates. After incubation, the bound RNAs were isolated and analyzed by RT–qPCR to determine the expression levels of circRNA_0013747 and miR-330-3p.

2.13 Dual‑luciferase reporter assay

Following transfection with either the pmiRGLO-CircRNA_0013747-wt plasmid or the pmiRGLO-CircRNA_0013747-Mut plasmid (GeneSeed, China), the cells were cotransfected with either a control or a miR-330-3p mimic (GeneSeed, China). After 48 hours of transfection, luciferase activity was assessed using a Dual-Luciferase Reporter Assay Kit (Promega, USA). The reporter activity was determined based on both firefly and Renilla luciferase activities.

2.14 Statistical analysis

Group differences were assessed via one-way ANOVA, followed by the least significant difference test for post hoc comparisons. The linear relationship between circRNA-0013747 and miR-330-3p expression in kidney tissues was analyzed using Spearman's correlation coefficient. The data are presented as the means ± SDs. All of the statistical analyses were performed using GraphPad Prism 7 software (GraphPad Software, USA), and the statistical significance was set at *P < 0.05.

3.1 Circ_0013747 expression is markedly elevated in human IgA nephropathy tissues

Dysregulated gene expression is a contributing factor in the pathogenesis of various diseases. In our research, we examined 16 pairs of human IgA nephropathy tissues and compared them to normal renal tissues to evaluate the expression of Circ_0013747. Our staining analysis revealed a substantial increase in Sirius red staining and IgA immunofluorescence in the IgA nephropathy group compared with the normal control group. This elevated staining indicated heightened deposition of IgA and collagen, suggesting more severe pathological damage (Fig. 1A). CircRNA_0013747 is generated from the exon of MAN1A2 through a process known as backsplicing, forming a 509-nucleotide circular structure, as illustrated in Supplementary Fig. S1. To specifically detect and quantify circRNA_0013747, we developed a tailored fluorescent probe. Notably, compared with that in control tissues, the fluorescence intensity in targeted IgA nephropathy tissues was significantly greater. Additionally, we designed specific PCR primers for circRNA_0013747 and applied RT‒qPCR to assess its expression levels in 16 paired IgAN and normal renal tissues. These results convincingly demonstrated elevated expression of circ_0013747 in IgAN tissues compared to normal tissues, as depicted in Fig. 1B. The circular structure of circRNA is more stable than the structure of linear RNA. While RNase R can effectively degrade linear RNA, it is incapable of cleaving circRNA. Our use of RNase R unequivocally confirmed the circular nature of circRNA_0013747; this circRNA exhibited remarkable resistance to RNase R digestion, notably outperforming the linear MAN1A2 transcript, as depicted in Fig. 1C. Furthermore, upon assessing RNA stability following treatment with the transcription inhibitor actinomycin D, it became evident that circRNA_0013747 displayed significantly enhanced stability when compared with the linear MAN1A2 mRNA transcript in human IgA nephropathy tissues, as illustrated in Fig. 1D. These findings underscore the upregulation of circRNA_0013747 in IgA nephropathy tissues.

3.2 Silencing of CircRNA_0013747 mitigates the proliferation of mesangial cells induced by lipopolysaccharide (LPS).

IgAN is fundamentally an inflammatory disorder in which persistent inflammation drives the proliferation of glomerular mesangial cells, ultimately leading to substantial collagen deposition and glomerular sclerosis. Lipopolysaccharide (LPS), a primary component of the outer membrane in gram-negative bacteria that is composed mainly of lipids and polysaccharides, is the gold standard for simulating acute inflammation due to its ease of control, reproducibility, and well-defined systemic effects. To determine the role of CircRNA_0013747 in inflammation-triggered mesangial cell proliferation, we downregulated its expression in LPS-stimulated human mesangial cells (HMCs). RT‒qPCR analysis revealed that LPS induced significant upregulation of circRNA_0017076 in HMCs, and this change was effectively reversed by siRNA treatment (Fig. 2A). Concurrently, the CCK-8 assay results illustrated that LPS amplified the proliferation of HMCs, whereas the inhibition of CircRNA_0013747 significantly suppressed this effect (Fig. 2B). Moreover, the flow cytometry results, which evaluated both apoptosis levels and cell cycle progression, were consistent with the CCK-8 assay results (Fig. 2C and 2D). Activated mesangial cells are known to produce notable quantities of collagen IV and fibronectin, both of which are major contributors to the development of glomerular sclerosis and the loss of renal function. Western blot and immunofluorescence analyses demonstrated a substantial increase in the levels of Collagen IV and fibronectin in LPS-exposed cells. However, the suppression of circRNA_0017076 effectively mitigated the LPS-induced upregulation of both proteins (Fig. 3E, F). Collectively, these findings strongly suggest the pivotal role of circRNA_0013747 in regulating HMC proliferation.

3.3 CircRNA_0013747 silencing results in downregulated PKM expression and a decrease in LPS-induced aerobic glycolysis in mesangial cells

Emerging evidence suggests that aerobic glycolysis may serve as a pivotal driver of pathological mesangial cell proliferation[26]. Many chronic kidney diseases characterized by mesangial hyperplasia commonly exhibit elevated glucose consumption and lactic acid accumulation. The results demonstrated a significant increase in glucose uptake and a concurrent increase in ATP and lactate production in HMCs when exposed to LPS. However, the effective silencing of circRNA_0013747 almost entirely reversed the stimulatory effect of LPS on HMCs (Fig. 3A, B, C). These findings strongly suggest that the activation of aerobic glycolysis might be one of the key factors contributing to the promotion of mesangial cell proliferation by circRNA_0013747. Key glycolysis enzymes, including PKM2, HK2, and PFK1, are known to be strongly involved in a wide range of cellular physiological functions and pathological processes. In our study, stimulation with LPS led to a notable increase in the expression of these three pivotal enzymes in HMCs. However, upon transfection with siRNA specifically targeting circRNA_0013747, we observed a significant reduction in PKM2 expression, with minimal effects on HK2 and PFK1 (as depicted in Fig. 3D). These findings strongly indicate that PKM2 is a crucial downstream regulatory signaling molecule influenced by circRNA_0013747. To further investigate the connection between PKM2 and circRNA_0013747, we assessed PKM2 mRNA levels in 16 paired kidney tissues. Intriguingly, compared with those in the normal tissue, PKM2 mRNA levels in the IgAN tissue were significantly greater (see Fig. 3H). Subsequent Spearman correlation analyses revealed a robust positive correlation between circRNA_0013747 and PKM2 mRNA levels (P < 0.001, r = 0.833; as depicted in Fig. 3I, J). Furthermore, the immunohistochemistry results provided additional support for these findings, as there was a conspicuous increase in PKM2 expression levels in IgA kidney biopsy samples compared to those in normal kidney tissue (refer to Fig. 3G). These combined results strongly suggest that PKM2 plays a pivotal role in the circRNA_0013747-induced process of aerobic glycolysis.

3.4 Silencing PKM2 attenuates circRNA-0013747-induced glycolysis and proliferation in mesangial cells

To investigate the influence of PKM2 on circRNA-0013747-induced glycolysis and proliferation, siRNA targeting PKM2 was cotransfected into HMC cells overexpressing circRNA-0013747. The mRNA and protein expression data revealed that the overexpression of circRNA-0013747 significantly upregulated PKM2, but this effect was subsequently suppressed by siRNA targeting PKM2 (as shown in Fig. 4A, B, C). Cell viability, as assessed by the CCK-8 assay, indicated that the increase in cell activity resulting from the overexpression of circRNA-0013747 was counteracted by PKM2 knockdown (Fig. 4D). The findings obtained from flow cytometry, which assessed both apoptosis levels and cell cycle progression, were in line with the outcomes of the CCK-8 assay (Fig. 4E, F). Furthermore, the increase in circRNA_0013747 expression led to a significant increase in glucose uptake and a concurrent increase in ATP and lactate production in HMC cells, and these effects were partially offset by PKM2 silencing (Fig. 4G, H, I). All of these findings collectively emphasize the critical importance of PKM2 in mediating the promotion of glycolysis and proliferation in mesangial cells via circRNA-0013747.

3.5 MiR-330-3p mediates the positive regulatory effect of circRNA-0013747 on PKM2

CircRNAs primarily act as miRNA sponges, exerting regulatory effects on genes. Therefore, we hypothesized that certain miRNAs might mediate the regulatory effect of circRNA-0013747 on PKM2. Using bioinformatics analysis via miRTarBase, we identified more than 100 miRNAs that potentially interact with PKM2 in HMC cells (Fig. 5A). The CircInteractome database lists several miRNAs as potential targets of circRNA-0013747 (Fig. 5B). Among these candidates, miR-184, miR-330, and miR-887 were found in both categories (Fig. 5C). Importantly, the upregulation of circRNA-0013747 in HMC cells significantly inhibited miR-330-3p expression, while miR-184 and miR-887 expression remained unchanged (Fig. 5D), indicating that miR-330-3p may be a crucial downstream target of circRNA-0013747.

To investigate the direct RNA interactions in this study, we conducted additional experiments. The FISH assay confirmed the colocalization of circRNA_0013747 and miR-330-3p in both mesangial cells and human renal glomerular tissues (Fig. 6A). Furthermore, a circRNA_0013747-specific probe showed enrichment of both circRNA_0013747 and miR-330-3p (Fig. 6B), indicating that circRNA_0013747 targets miR-330-3p in HMC cells. To validate this interaction, we generated wild-type and mutant dual-luciferase reporter vectors for circRNA_0013747 based on the potential binding sites of miR-330-3p and circRNA_0013747. We cotransfected a miR-330-3p mimic or a negative control (NC) mimic with the luciferase reporter into human embryonic kidney 293 (HEK293) cells. The overexpression of miR-330-3p reduced the luciferase activity of the wild-type reporters but had no effect on the luciferase activity of the mutant reporters (Fig. 6C). In a separate experiment using dual-luciferase reporter vectors for PKM2, miR-330-3p mimics decreased the luciferase activity of the reporter (Fig. 6D). These results confirmed that circRNA_0013747 functions as a miR-330-3p sponge and that miR-330-3p directly binds to the 3' UTR of PKM2 mRNA.

3.6 Knockdown of Mmu_circ_0010297 alleviates renal damage caused by IgA nephropathy in mice

Mmu_circ_0010297, which is the homologous circular RNA of hsa_circ_0013747, originates from the primary transcript of the MAN1A2 gene (gene symbol). In our study, we randomly assigned 18 BALB/c mice to three groups: "Blank Control", "IgAN + AAV-shNC", and "IgAN + AAVsh-mmu_circ_0010297". We utilized AAV9-sh-Mmu_circ_0010297 to suppress the expression of mmu_circ_0010297 in the kidneys of BALB/c mice. As a control, another group of mice received an injection of an empty AAV9 vector. Subsequently, both sets of mice were induced with IgA models and compared with untreated BALB/c mice. RT‒qPCR analysis revealed increased expression of Mmu_circ_0010297 in the kidneys of the IgAN + AAV-shNC group compared to that in the blank control group. This elevation was notably reduced following treatment with AAVsh-mmu_circ_0010297, as illustrated in Fig. 7A. Concurrent with the decrease in Mmu_circ_0010297, there was a significant reduction in lactate levels within the kidneys of the AAVsh-mmu_circ_0010297 group, as shown in Fig. 7B. Western blot analysis revealed that the increased expression of PKM2, fibronectin, and collagen IV in the IgAN model group was reversed in the IgAN + AAV-sh-mmu_circ_0010297 group, as depicted in Fig. 7C. Sirius Red staining clearly revealed that, compared with the AAV-sh-NC mice, the mmu_circ_0010297 knockdown mice showed notably less glomerulosclerosis. Furthermore, the FISH results revealed a significant decrease in miR-330-3p levels in the kidneys of the AAV-shNC group compared to those in the control group. This decrease was effectively counteracted following treatment with AAVsh-mmu_circ_0010297. The results of immunohistochemistry assays for collagen IV in kidney tissues from all three groups were consistent with the Western blot findings, as shown in Fig. 7D. IgA nephropathy typically presents with severe kidney dysfunction characterized by increasing levels of blood urea nitrogen (BUN), serum creatinine (Scr), and uric acid (UA) and 24-hour proteinuria (24 h-pro) as the disease progresses. Biochemical analysis demonstrated a reduction in the levels of these elevated markers in the IgAN + AAV-sh-mmu_circ_0010297 group compared to those in the IgAN model group. Additionally, positive correlations between mmu_circ_0010297 and BUN levels, Scr levels, UA levels, and 24 h-pro levels were detected using Spearman's correlation, as depicted in Fig. 7E and F. In summary, these findings collectively suggest that inhibiting Mmu_circ_0010297 mitigates the renal damage caused by IgA nephropathy in mice.

In mature organisms, quiescent cells typically exhibit heightened replication rates during tumorigenesis or as part of the tissue repair process following injury [27]. Within the renal glomerulus, various forms of injury can incite localized inflammatory reactions involving resident glomerular cells. These injuries may arise from immune-mediated responses, infections, toxins, mechanical stress, or other causative factors [28]. A prominent histopathological feature of numerous human and experimental glomerular inflammatory conditions is an increase in cellular density within the mesangium. This increase is attributed to the proliferation of mesangial cells (MCs) and the influx of leukocytes [29]. Irrespective of the specific injury mechanism, an early and pivotal factor in the development of progressive glomerular injury and glomerulosclerosis seems to be an imbalance in regulating MC proliferation. In experimental models of nephritis, MC proliferation frequently precedes and correlates with an increase in the accumulation of extracellular matrix (ECM) within the mesangium and the subsequent development of glomerulosclerosis [30–31]. Moreover, interventions that reduce cell proliferation in glomerular disease models, such as treatment with heparin, a low-protein diet, or the use of neutralizing antibodies against platelet-derived growth factor, have been demonstrated to mitigate ECM expansion and sclerotic changes[32]. Notably, a reduction in MC replication is correlated with a marked decrease in mesangial ECM accumulation and diminished deposition of collagen type IV, laminin, and fibronectin [33]. However, the specific molecular mechanisms underlying the abnormal proliferation of mesangial cells in various types of nephritis, including IgA nephropathy, have yet to be fully elucidated.

In the 1920s, Otto Warburg and his colleagues made an important observation that tumors exhibit an unusually high rate of glucose uptake in comparison to the surrounding tissue. Furthermore, they noted that glucose was metabolized to produce lactate even when oxygen was present, which led to the coining of the term "aerobic glycolysis."[34]. The Warburg effect has been postulated to be an adaptive mechanism aimed at fulfilling the biosynthetic demands associated with uncontrolled cell proliferation. In this context, increased glucose consumption serves as a carbon source for the anabolic processes required to facilitate cell proliferation. Excess carbon is allocated for the synthesis of new nucleotides, lipids, and proteins and can be channeled into various branching pathways that stem from glycolysis[35–36]. Furthermore, the Warburg effect may offer a growth advantage to cells within a multicellular environment[37]. However, the reason behind this relatively inefficient metabolic pathway in tumor cells remains unclear. However, current research points to mitochondrial dysfunction in cancer cells and changes in essential enzymes such as pyruvate kinase (PK), which are involved in glycolysis[38]. PK plays a crucial role in glycolysis by catalyzing the final and physiologically irreversible step, which involves converting phosphoenolpyruvate into pyruvate through the transfer of a phosphate group to adenosine diphosphate [39]. In mammals, there are four distinct PK isoforms encoded by two genes. The PKLR gene encodes PKL and PKR. The PKM gene encodes PKM1 and PKM2 through alternative splicing, utilizing mutually exclusive exons that are the same length but encode a 56-amino acid region differing at 22 residues[40]. PKM2 is universally expressed during embryogenesis, regeneration, and cancer development. This observation implies that the capacity to regulate pyruvate kinase enzymatic activity is a crucial factor in actively proliferating cells[41]. Numerous studies have indicated that elevated levels of PKM2 in circulation could serve as a diagnostic marker for various cancer types. Furthermore, the overexpression of PKM2 is positively correlated with tumor progression, primarily owing to its involvement in glycolysis, proliferation, and apoptosis[42]. A reduction in PKM2 expression has been demonstrated to lower the glycolytic rate and inhibit tumor growth in various types of cancer. Administering the PKM2 activator TEPP-46 to H1299 xenograft model mice resulted in a delay in tumor onset and the development of smaller tumors compared to those in control mice that received a vehicle[43]. Similarly, the deletion of PKM2 in a xenograft mouse model of NCI-N87 cells led to the formation of smaller tumors than those formed in the control counterparts[44]. Intriguingly, an expanding body of literature offers substantiating evidence regarding the potential of PKM2 as a biomarker for nephrotoxicity. This growing interest in revealing the possible involvement of PKM2 in renal diseases is well founded. In a recent study, the induction of nephrotoxicity in rats through cisplatin led to a notable increase in urinary PKM2 levels, which coincided with elevated lactate excretion and significant alterations in amino acids, glucose, and TCA intermediates within the urine. Correspondingly, renal tubular HK-2 cells exposed to cisplatin, as well as other nephrotoxic agents such as cyclosporine A, exhibited increased PKM2 secretion in conditioned media[45]. Furthermore, Chen et al. reported that PKM2 contributes to kidney fibrosis, particularly during the transition from acute kidney injury to chronic kidney disease[46]. These collective findings underscore PKM2 as a substantial contributor to renal function and a potential novel marker for nephrotoxicity. These discoveries suggest that targeting PKM2 could represent an innovative strategy for the prevention and treatment of renal diseases. In our study, we noted a significant increase in lactate and PKM2 expression in the kidneys of IgA model mice. Silencing PKM2 resulted in pronounced inhibition of LPS-induced aerobic glycolysis and proliferation in human mesangial cells. Notably, the expression of PKM2 gradually diminishes during the transition from embryonic development to mature tissue. However, PKM2 expression resurfaces during tissue repair and tumor growth, implying that its regulation in tissue cells is strictly controlled by molecular regulation. Research has demonstrated that miRNAs can bind to PKM2 mRNA, effectively suppressing its expression during gene translation. This suppression can decelerate cellular glycolysis and hinder tumor growth. Hence, there is a growing focus on the regulation of PKM2 by noncoding genes.

Unlike the majority of linear messenger RNAs and long noncoding RNAs, which are characterized by 5′ N7-methylguanosine caps at the beginning and 3′ polyadenylated tails at the end, circular RNAs represent a distinct class of RNA molecules[47]. Circular RNAs are characterized by their unique feature of being covalently closed single-stranded RNAs. These circular RNAs have recently gained recognition as a prevalent category of RNA species[48]. The circular nature of single-stranded RNA was initially observed in plant viroids, and subsequently, circular transcripts were discovered in eukaryotes through electron microscopic evidence, revealing a circular morphology despite their unknown functions[49]. In the 1980s, other examples of circular RNA genomes were identified, as was the case for the hepatitis δ virus. Over the past decade, the emergence of RNA sequencing technologies, enriched for nonpolyadenylated and circular transcriptomes, along with computational tools for circular RNA annotation, has revealed the widespread expression of circRNAs across various metazoan cell types and tissues[50]." A groundbreaking discovery in 2013 highlighted the ability of circRNAs to act as "molecular sponges" that bind and inhibit corresponding miRNAs and consequently enhance the expression of the miRNA target genes[51]. Liao et al. reported that circRNA_45478, which functions as a miR-190a-5p sponge, exacerbates ischemic acute kidney injury[52]. Furthermore, circPlekha7 inhibits the epithelial-to-mesenchymal transition of renal tubular epithelial cells by targeting miR-493-3p to derepress KLF4 expression[53]. Recent comparisons of urinary circRNA profiles between IgAN patients and healthy controls highlighted the upregulation of circRNA_0013747 in IgAN patients, with an associated expression pattern with PKM2[54]. These findings suggested that circRNA_0013747 may play an important role in the onset and progression of IgA nephropathy. In our study, we demonstrated that circRNA_0013747 was significantly upregulated in the kidney tissue of IgA nephropathy patients and promoted mesangial cell proliferation through the induction of PKM2-mediated aerobic glycolysis. Our results confirm the central role of the circRNA_0013747/miR-330-3p/PKM2 axis in driving aerobic glycolysis and mesangial cell proliferation in IgAN. Nevertheless, our study has certain limitations. We did not investigate the relationship between circRNA_0013747 expression and clinicopathological parameters such as mesangial IgA deposition and renal interstitial lymphocyte infiltration. The exact distribution of circRNA_0013747 in the kidney has not been determined, and its specific impact on mesangial cells has yet to be determined. Finally, whether this molecular target can be applied in clinical diagnosis and treatment requires further investigation.

To summarize, our research provides a novel perspective by suggesting that the Warburg effect may not only be central to tumor cell proliferation but also to the proliferation of mesangial cells. These findings indicated that circRNA_0013747 was elevated in IgAN tissues and induced mesangial cell proliferation and the Warburg effect by regulating the miR-330-3p/PKM2 signaling pathway. Therefore, our study suggested that circRNA_0013747 could serve as a promising new biological marker and therapeutic target for IgAN.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Author Contribution

Zou drafted the article, and Zhang revised the article.

Acknowledgement

The current work was supported by the National Natural Science Foundation of China (No. 82160137 and No. 82360147) and the Natural Science Foundation of Guizhou Province, China (QianKeHeJiChu-ZK[2022] General 409 and [2020] 1Y305).

Availability of data and materials

The datasets obtained and analyzed during the current study were made available from the corresponding authors through request.

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Supplementary Figure 1 and Supplementary Table 3 are not available with this version.

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CircRNA-0013747 induces mesangial cell proliferation in IgA nephropathy by targeting the Warburg effect via miR-330- 3p/PKM2 signaling

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Abstract

Figures

1. Introduction

2. Methods and materials

2.1 Ethics Approval:

2.2 Human tissue specimens

2.3 Animal model

2.4 Histological analyses

2.5 RNA fluorescence in situ hybridization (RNA FISH)

2.6 Immunofluorescence staining

2.7 Cell culture

2.8 RNA extraction and quantitative real-time PCR (RT‒qPCR) analysis

2.9 Western blot (WB) analysis

2.10 Cell viability assay

2.11 Lactate production, glucose uptake, and ATP levels

2.12 RNA pull‑down assay

2.13 Dual‑luciferase reporter assay

2.14 Statistical analysis

3. Results

3.1 Circ_0013747 expression is markedly elevated in human IgA nephropathy tissues

3.2 Silencing of CircRNA_0013747 mitigates the proliferation of mesangial cells induced by lipopolysaccharide (LPS).

3.4 Silencing PKM2 attenuates circRNA-0013747-induced glycolysis and proliferation in mesangial cells

3.5 MiR-330-3p mediates the positive regulatory effect of circRNA-0013747 on PKM2

3.6 Knockdown of Mmu_circ_0010297 alleviates renal damage caused by IgA nephropathy in mice

4. Discussion

Declarations

Conflicts of Interest

Author Contribution

Acknowledgement

Availability of data and materials

References

Supplementary Figure and Table

Additional Declarations

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