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FTO mediates the diabetic kidney disease progression through regulating the m6A modification of NLRP3

BMC Nephrology volume 25, Article number: 345 (2024) Cite this article

Abstract

Background

The objective of our research was to investigate the specific mechanism of FTO in diabetic kidney disease (DKD) progression.

Methods

The DKD model was established with renal tubular epithelial HK-2 cells and mice in vitro and in vivo. The N6-methyladenosine (m6A) content in cells was detected using dot plot assay and the m6A levels of NLRP3 was detected with the MeRIP assay. The mRNA and protein levels were tested with real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) and western blot. The IL-1β and IL-18 levels were assessed with enzyme-linked immunosorbent assay (ELISA). The cell viability was measured by cell counting kit (CCK)-8 assay and cell pyroptosis was determined with Annexin V and propidium iodide (PI) double staining followed by flow cytometry analysis. RNA-binding protein immunoprecipitation (RIP) and dual luciferase reporter assays were conducted to detect the interaction between FTO and NLRP3. m6A levels were detected by Me-RIP assay. The renal injury was measured by observing the renal morphology and urine and blood levels of relevant indicators.

Results

The results indicated that high glucose treatment induced HK-2 cell pyroptosis. m6A levels were prominently elevated in high glucose treated HK-2 cells while FTO expression were significantly down-regulated. FTO over-expression promoted cell viability but inhibited pyroptosis of HK-2 cells under high glucose (HG) treatment. Moreover, FTO could inhibit NLRP3 expression. RIP and Me-RIP assays indicated that FTO could bind with NLRP3 and regulate its m6A modification level. Further luciferase assay confirmed that FTO binds with the 233–237 bp region of NLRP3. NLRP3 neutralized the function of FTO in the HG stimulated HK-2 cells. In vivo, the H&E staining showed that FTO over-expression alleviated the kidney injury and suppressed the pyroptosis induced by DKD.

Conclusion

We found that FTO could inhibit the DKD progression in vivo and in vitro by regulated the m6A modification of NLRP3.

Peer Review reports

Introduction

Diabetic kidney disease (DKD) is a microvascular complication of diabetes [1]. DKD is characterized by the increase formation of extracellular matrix and disappearance of foot process, which eventually leads to increased urinary protein and decreased estimated glomerular filtration rate [2, 3]. Previous studies have shown that DKD is the result of the interaction of hemodynamic disorders and metabolic abnormalities [4]. However, increasing evidences have determined that the local inflammatory state of the kidney leads to the occurrence and progression of DKD [5]. In DKD patients, the driving effect of hyperglycemia, the increase of toxic metabolites (such as advanced glycation end), and the vascular mechanical changes caused by the disturbance of hemodynamics will lead to the inflammatory response in vivo, which further affect the proliferation, hypertrophy, aging and apoptosis of renal cells [6, 7].

In recent years, epigenetics, a branch of genetics, has developed rapidly. Epigenetics mainly regulates gene expression through gene modification, without changing the nucleotide sequence of the gene [8]. RNA modification widely affects the structure, function and stability of RNA, which plays a critical role in the regulation of various cell process [9]. Among all RNA modifications, N6 methyladenosine (m6A) is the most common modification form in eukaryotic mRNA, which is also one of the most studied RNA modifications at present [10]. Fat-mass and obesity-associated gene (FTO) is a central enzyme in the process of methylation modification, which has been confirmed to be closely related to many diseases progression [11]. Additionally, abnormal FTO levels participate in the regulation of inflammation and cell death, which are critical mechanisms in the malignant development of DKD [12]. However, whether FTO modulates the development of DKD and its potential mechanism are largely unknown.

Pyroptosis is a newly identified programmed cell death characterized by cell swelling and information reaction. Recent researches demonstrate that the pyroptosis of renal tubular epithelial cell has been found in the process of DKD [13,14,15]. The NLRP3 inflammasome is known to be the most important initiator of pyroptosis [16]. Thus, controlling the activation of the NLRP3 inflammasome and podocyte pyroptosis play important roles in the progression of DKD. Here in this study, we aimed to investigate the effect and mechanism of FTO in DKD development. We hypothesized that FTO involves in the DKD progression through m6A modification of NLRP3 and regulating cell pyroptosis.

Materials and methods

Cell culture and treatment

Human renal tubular epithelial cell (HK-2) was provided by Shanghai Mingjin Biotechnology Co., Ltd (Shanghai, China). The cells were maintained in the endothelial culture medium supplemented with 5.6 mmol/L glucose and 10% fetal bovine serum (FBS; Gibco). In order to induce the DKD model, HK-2 cells were treated with 30 mmol/L d-glucose for 48 h, named high glucose (HG) group. In addition, HK-2 cells were treated with 5.6 mmol/L d-glucose in the normal glucose (NG) group, while cells cultured in 25 mM mannitol were used as the osmotic control (M group).

Cell transfection

FTO over-expressing vector (oe-FTO), NLRP3 over-expressing vector (oe-NLRP3) and empty vector (oe-NC) were provided by GenePharma (Shanghai, China). In brief, pUC57-FTO and pLVX-mCMV-ZsGreen-IRES-Puro were digested by EcoRI and SpeI, and the products were connected by T4 DNA Ligase. Then, an Agarose Gel Extraction Kit was used to retrieve fragments. JM109, which was transfected with the ligation product, was inoculated and amplificated, and the sequencing primer CMV-F was used to verify the bacterial fluid. rLV-FTO/NLRP3 was obtained in 293T cells co-transfected with recombinant plasmid or control plasmid using Lipofectamine 3000 (Invitrogen, USA). HK-2 cells were transfected with rLV-FTO/NLRP3 on the basis of MOI = 20. Two days after transfection, lentivirus-infected HK-2 cells were cultured normally.

Real-time reverse transcriptase-polymerase chain reaction (RT-qPCR)

The total RNA was extracted with Trizol Kits (Beyotime, Shanghai, China), and the total RNA was reverse transcribed into cDNA according to the instructions of the PrimeScript RT reagent kit (Takara, Japan). The synthetic cDNA was used as the templates for the RT-qPCR reaction with a Fast SYBRGREEN PCR kit (Takara), and in a ABIPRISM 7300 RT-PCR system (Applied Biosystems). The amplification program was set as follows: initial denaturation, 95℃ for 15 min; denaturation, 95℃ for 15 s, 45 cycles; annealing, 55℃ for 15 s; extension, 72℃ for 30 s. In this experiment, β-actin was used as the internal control of genes. Relative quantification method (2−ΔΔCt method) was used to calculate the relative levels of related genes. The primer sequences were as follows: FTO, 5ʹ-GCTGCTTATTTCGGGACCTG-3ʹ and 5ʹ-AGCCTGGATTACCAATGAGGA-3ʹ; NLRP3, 5ʹ-CCACAAGATCGTGAGAAAACCC-3ʹ and 5ʹ-CGGTCCTATGTGCTCGTCA-3ʹ; CASP1, 5ʹ-TTTCCGCAAGGTTCGATTTTCA-3ʹ and 5ʹ-GGCATCTGCGCTCTACCATC-3ʹ; ASC, 5ʹ-CCCAAGCAAGTCAAGCGACA-3ʹ and 5ʹ-AAGCCGCTGAAGTTGAGCC-3ʹ; GSDMD, 5ʹ-GGACAGGCAAAGATCGCAG-3ʹ and 5ʹ-CACTCAGCGAGTACACATTCATT-3ʹ; and GAPDH, 5ʹ-GTGTTCCTACCCCCAATGTGT-3ʹ and 5ʹ-ATTGTCATACCAGGAAATGAGCTT-3ʹ.

RNA stability detection

For the determination of mRNA stability of NLRP3, the cells were treated with Actinomycin D for 2, 4, 6, 8 h. After the treatment of different times, the mRNA levels of NLRP3 were detected using RT-qPCR as mentioned above.

Cell counting kit (CCK)-8 assay

CCK-8 detection kit (Shanghai Liji Biotechnology Co., Ltd., Shanghai, China) was used to detect the cell viability. 100 μL of cell suspension was seeded in a 96-well plate at a density of 5 × 103/well. On the 24th, 48th, and 72th h after seeding, 10 μL of CCK-8 solution was added to each well, and mixed gently without bubbles. After incubated in a 5% CO2 incubator at 37 ℃ for 4 h, the absorbance of each well at 450 nm was measured with a microplate reader.

Annexin V and propidium iodide (PI) double staining

Pyroptosis of HK-2 cells was detected by using an Annexin V-FITC Detection Kit (Beyotime) according to the manufacturer’s instructions. Briefly, HK-2 cells at the concentration of 2 × 105 were centrifuged at 300 × g for 5 min, and the supernatant was discarded. The cells were re-suspended with 500 μL diluted 1 × Annexin V Binding Buffer. The cell suspension was added with 5 μL Annexin V-FITC staining solution and 5 μL PI staining solution (50 μg/mL). Cells ere incubated at room temperature for 15 ∼ 20 min away from light, and then analyzed by flow cytometry (Becton Dickinson, Germany). HK-2 cells in Q1 (Annexinv-/PI+) are necrotic cells [17], and were deemed as positive pyroptosis cells.

Western blot

The cultured cells were collected and the supernatant was discarded. Then the cells were lysed with enhanced RIPA lysate (Beyotime), and the protein concentration was determined with BCA protein quantitative Kit (Beyotime). Proteins (20 μg) were separated with 10% SDS-PAGE and transferred to PVDF membrane (Millipore). Membranes were blocked in 5% skimmed milk at room temperature for 1 h. Then the blots were treated with primary antibodies against (NLRP3, 27458-1-AP, Proteintech, China; ASC, ab307560, Abcam, USA; caspase-1, ab286125, Abcam, USA; GSDMD-N, EPR20829-408, Abcam, USA; IL-6, ab290735, Abcam, USA; caspase-3, ab32351, Abcam, USA) at 4℃ overnight. Next day, after washing, the membranes were treated with HRP labeled secondary antibody for 1 h. Thereafter, the membranes were visualized with ECL solution (Biomiga, USA) for 1 min at room temperature. ImageJ software was used to quantify the gray scale of each band. β-actin was used as the internal control.

m6A dot blot assay

Total RNA was extracted from the blood, cells and tissues by Trizol (Beyotime). The m6A content relative to the total mRNA level was measured by EpiQuik m6A RNA Methylation Quantification Kit (Colorimetric) (AmyJet Scientific, Wuhan, China) according to the instructions. Briefly, 4 μg of total RNA was and purified using the RNeasy Mini Kit (Qiagen). Then the RNA was separated on 1.2% formaldehyde-agarose gels and transferred onto Hybond N + membranes (Amersham). Then the membranes were incubated with m6A antibody overnight and and then treated with horseradish peroxidase conjugate anti-rabbit immunoglobulin G. Finally, after washing, the blots were developed on PhosphorImager screens and quantited with ImageQuant.

Me-RIP qPCR experiment

Magna RIPTM RNA binding protein immunoprediction kit was purchased from Millipore (MA, USA) and the experiment was carried out according to the instructions. After the cells were treated with Rip lysate, anti-m6A antibody was used to incubate the cell lysate. Then the mixture was incubated with magnetic beads. After that, protease K was used to remove proteins. Finally, the RNA was purified and reverse transcribed into cDNA, and the expression of target genes was detected by RT-qPCR.

RNA-binding protein immunoprecipitation (RIP) experiment

The Rip Kit (Millipore, USA) was used to detect the binding of FTO protein with NLRP3 RNA. HK-2 cells were washed twice with cold PBS and collected. The cells were then resuspended with RIPA lysate (Beyotime) and centrifuged (14 000 r/min, 4 ℃) for 10 min to collect the supernatant. The experimental procedures were as follows: 50 μL of magnetic beads were washed and then resuspended in 100 μL of RIP Wash Buffer, and then incubated with 5 μg of anti-FTO or IgG. The magnetic bead-antibody complexes were washed and resuspended in 900 μL RIP Wash Buffer, and then incubated with 100 μL cell extract at 4℃ overnight. The sample was then placed on a magnetic holder to collect the magnetic bead-protein complexes. The complex pulled down were digested with proteinase K to extract RNA, and the expression level of NLRP3 was detected by RT-qPCR as mentioned above.

Double luciferase reporter assay

The wild-type and mutant fragments of NLRP3 were constructed and inserted into the pmirGLO reporter vector using endonuclease sites Spe I and Hind III, named as NLRP3-WT and NLRP3-MUT, respectively. These reporter plasmids along with oe-nc or oe-FTO were co-transfected into HK-2 cells using the LipofectamineTM3000 reagent. After 48 h, the cells were collected and fully lysed. The firefly luciferase activity was finally measured and normalized to the renin luciferase activity.

Animal experiment

The experimental procedures followed the recommendations in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and were approved by Laboratory Animal Ethics Committee of the Beijing Medconor Biotechnology Co., LTD. (MDKN-2022-70). Five weeks old male C57bl/6 mice (20 ± 2 g) were divided into DKD model group and control group. The fasting blood glucose of all mice was measured before the experiment, which was required to be less than 7 mmol/L. The mice in DKD model group were fed with high-fat and high-glucose diet for 8 weeks. Then, 1 day after restoring the high-fat and high-glucose diet, the mice in the DKD model group were fasted for 16 h and injected with streptozotocin (STZ) at 50 mg/kg per day for 5 days. The fasting blood glucose was measured 2 weeks after the last injection. If the fasting blood glucose was greater than 16 mmol/L, the medium-term diabetes model was established successfully. The mice in the control group were received standard chow and injected with the same amount of normal saline. Subsequently, all DKD mice were randomly divided into lv-NC group and lv-FTO group. After DKD model establishment, the lentiviruses carrying empty vector (NC) or FTO over-expressing vector (FTO) (MOI = 50) were injected into caudal vein at the dose of 1 μg/g according to the weight of mice, once a week for 8 weeks.

H&E staining

Mice were sacrificed with 160 mg/kg pentobarbital sodium injection to cause respiratory arrest. The kidneys of the mice were collected and fixed in 4% paraformaldehyde. After the fixation, dehydration and paraffin embedding, the paraffin sections were cut into sections with a thickness of 2 μm. H&E staining kit were purchased from Shanghai GEFAN Biotechnolog Co., Ltd. (Shanghai, China). The sections were stained according to the instructions of the kits. The pathological changes of renal glomerulus, basement membrane and mesangial matrix were observed under the microscope. The renal tubule injury was evaluated as described previously [18].

Detection of renal tubule injury

The IL-1β, IL-18 and LDH levels in the kidneys of the mice and the HK-2 cells were tested by corresponding ELISA kits purchased from Jiancheng Bio (Nanjing, China). All operations shall be carried out according to the operation instructions of the kits. Urine samples were collected from the metabolic cage 2 days before sacrifice, and the urinary albumin level and urinary albumin excretion rate (mg/24 h) were measured according to the instructions of enzyme-linked immunosorbent assay (ELISA) kit (Ethos Biosciences). Femoral vein blood was collected with heparin anti-coagulation capillary tubes for measurement of blood urea nitrogen, serum creatinine and glomerular filtration rate using biochemical analyzer (Mindray). To measure urinary N-acetyl-beta-D-glucosidase (NAG) and retinol-binding protein-4 (RBP-4) levels, particle-enhanced turbidimetric immunoassay (PETIA) kits (Diazyme, USA) were implemented.

Statistical analysis

SPSS 22.0 software was applied for data analysis. The results were expressed by mean ± SD. The independent sample T-test was used for the comparison between the two groups, and the one-way ANOVA was used for the comparison between multiple groups. The m6A methylation sites of NLRP3 were predicted using the SRAMP database (http://www.cuilab.cn/sramp). P < 0.05 was statistically significant.

Results

HG induced pyroptosis and elevated the m6A level of renal tubular epithelial cells

After HG treatment, cell viability of HK-2 cells was suppressed compared with NG and M groups (Fig. 1A). The LDH release in the culture medium of HK-2 cells was also significantly increased (Fig. 1B). Flow cytometry results indicated that HG notably induced pyroptosis of HK-2 cells. ELISA results showed that HG promoted the release of IL-1β and IL-18 of HK-2 cells (Fig. 1C, D, E). Western blot was used to assess the protein expression. It was showed that HG promoted the expression of NLRP3, ASC, cleaved caspase-1, caspase-3 and GSDMD-N (Fig. 1F, Fig. S1A). These results demonstrated that HG could induce pyroptosis of HK-2 cells in vitro. Subsequently, we investigated whether HG could influence the m6A level. Dot plot staining showed that HG did elevate the m6A level of HK-2 cells (Fig. 1G). The performance of HK-2 cells treated with mannitol was not statistically different from that of the NG group. Then, we tried to figure out which m6A related regulators lead to the abnormal m6A level of DKD. The results suggested that the FTO level was prominently decreased in the HG stimulated HK-2 cells (Fig. 1H). Thus, we selected FTO for the further study.

Fig. 1
figure 1

(A) Cell viability was assessed by CCK-8 after HG treatment. (B) LDH release in HK-2 cell culture medium was determined by commercial kits. (C) The pyroptosis of HK-2 cells was detected by flow cytometry. (D) The release of IL-1β and IL-18 in HK-2 cells was assessed. (F) HG promotes protein expression of NLRP3, ASC, cleared caspase-1 and GSDMD-N. (G) m6A dot plot staining was performed to evaluate the level of m6A in HK-2 cells. (H) The expressions of m6A related regulatory factors were detected by qPCR in HK2 cells after HG treatment. The one-way ANOVA was used for the comparison between multiple groups, and data were expressed by mean ± SD

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Over-expression of FTO alleviated the injury in HK-2 cells induced by HG treatment

Then, we constructed FTO over-expressing vectors. The qPCR results confirmed that oe-FTO successfully elevated the FTO level in HK-2 cells (Fig. 2A). HG treatment dramatically suppressed the cell viability of the HK-2 cells, while oe-FTO significantly enhanced it (Fig. 2B). The promoted release of LDH was reversed by FTO, either (Fig. 2C). Besides, HG treatment dramatically elevated the pyroptosis rate of HK-2 cells while FTO reduced it (Fig. 2D). In addition, FTO inhibited the HG promoted release of IL-1β and IL-18 (Fig. 2E, F) and the expression of NLRP3, ASC, cleaved caspase-1, caspase-3 and GSDMD-N (Fig. 2G, Fig. S1B).

Fig. 2
figure 2

FTO over-expression moderated the injury in the HK-2 cells induced by HG treatment. (A) The expression of FTO after transfection was evaluated by RT-qPCR. oe-FTO successfully increased the level of FTO in HK-2 cells. (B) Cell viability after treatment and transfection was assessed by CCK-8. (C) Promoted release of LDH. (D) Flow cytometry was used to evaluate the pyroptosis of HK-2 cells. (E) FTO inhibited the HG promoted release of IL-1β (F) and IL-18. (G) The expression of NLRP3, ASC, cleaved caspase-1 and GSDMD-N. The independent sample T-test was used for the comparison between the two groups, and the one-way ANOVA was used for the comparison between multiple groups. The data were expressed by mean ± SD

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FTO regulated m6A modification of NLRP3

As FTO participates in the regulation of pyroptosis, we are wondering whether FTO could regulate the m6A modification of pyroptosis related genes. The Me-RIP assay indicated that FTO could significantly reduce the m6A level of NLRP3 but not that of caspase-1, ASC or GSDMD (Fig. 3A). Furthermore, the RIP assay showed that the FTO antibody could enriched the NLRP3 mRNA in HK-2 cells (Fig. 3B). After FTO over-expression, the m6A levels of NLRP3 were prominently decreased (Fig. 3C). Then, SRAMP, a sequence-based m6A modification site predictor, was applied to predict the potential theoretical binding sites of NLRP3, and there are 3 site for m6A modification located at 233–237, 866–870 and 1404–1408 bp (Fig. 3D). Afterwards, whether the site can play the m6A modification function is verified by the base mutation of the binding site (Fig. 3E). The luciferase assay indicated that FTO prominently reduced the luciferase activity of WT-NLRP3 carrying 233–237 bp region but not MUT-NLRP3 (Fig. 3F). qPCR results indicated that FTO over-expression reduced the stability of NLRP3 mRNA (Fig. 3G).

Fig. 3
figure 3

FTO over-expression moderated the DKD progression in vivo. (A) m6A level of NLRP3, caspase-1, ASC and GSDMD. (B) RIP assay was performed to detect the binding of FTO with NLRP3 mRNA. (C) m6A level of NLRP3 after FTO knockdown was detected. (D) Prediction of potential m6A modification sites of NLRP3. (E) The wild type (WT) and mutant (mut) sequence was showed. (F) Luciferase activity of WT-NLRP3 and MUT-NLRP3 after FTO over-expression was assessed. (G) The half life of FTO mRNA after FTO over-expression was detected by qPCR. The independent sample T-test was used for the comparison between the two groups, and the one-way ANOVA was used for the comparison between multiple groups. The data were expressed by mean ± SD

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NLRP3 over-expression neutralized the function of FTO in the HG stimulated HK-2 cells

Finally, to confirm the interaction between FTO and NLRP3, the rescue experiments were conducted. We established the NLRP3 over-expressing vector and found that it prominently elevated the mRNA expression of NLRP3 in HK-2 cells (Fig. 4A). NLRP3 over-expression prominently decreased the cell viability (Fig. 4B), but elevated the LDH release (Fig. 4C). Pyroptosis rate and IL-1β and IL-18 amount were elevated by NLRP3 over-expression (Fig. 4D-F). Additionally, after NLRP3 over-expression, the protein expressions of NLRP3, ASC, cleaved caspase-1, caspase-3 and GSDMD-N were promoted (Fig. 4G, Fig. S1C). These results further confirmed the interaction between FTO and NLRP3.

Fig. 4
figure 4

FTO regulated m6A modification of NLRP3. (A) The NLRP3 levels were measured by RT-qPCR in HK-2 cells. Over-expression of NLRP3 reversed the effect of FTO on the (B) proliferation, (C) LDH release, (D) pyroptosis rate, (E, F) IL-18 and IL-1β release of HK-2 cells. (G) The effect of FTO on expression of NLRP3, ASC, cleared caspase-1 and GSDMD-N protein was reversed by NLRP3 over-expression. The independent sample T-test was used for the comparison between the two groups, and the one-way ANOVA was used for the comparison between multiple groups. The data were expressed by mean ± SD

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Over-expression of Fto inhibited the DKD progression in vivo

Next, we focused our interest on renal tubular function and evaluated the potential protective roles of over-expression of Fto against DKD. The ratio of kidney weight to body weight was increased with exposure to STZ, and this trend was altered by over-expression of Fto. Meanwhile, STZ treatment resulted in a remarkable renal tubular injury as evidenced by increased urinary albumin, blood urea nitrogen, serum creatinine, glomerular filtration, 24 h urinary albumin excretion rate, NAG and RBP-4 levels. However, the application of elevated Fto greatly reduced these metabolites (Table 1). Then the pathologic changes of renal tissues were observed by H&E staining. Remarkable renal damage with many dilated and atrophic tubules were observed in DKD group, and over-expression of Fto significantly alleviated the above renal damages (Fig. 5A). The increased abundance of renal IL-6 was also down-regulated by elevated Fto (Fig. 5A). Meanwhile, the kidney damage injury was evaluated based on the results of H&E staining, and the data demonstrated that elevation of Fto significantly down-regulated the scores induced by DKD (Fig. 5B). Then, to check if Fto over-expression influence the pyroptosis, we evaluated the expression of pyroptosis related proteins. The results indicated that Fto over-expression inhibited the amount of IL-1β, IL-18 and the expression of NLRP3, ASC, cleaved caspase-1, caspase-3 and GSDMD-N (Fig. 5C-E, Fig. S1D).

Table 1 Effects of overexpression of FTO on renal function in DKD model mice
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Fig. 5
figure 5

Over-expression of FTO inhibited the DKD progression in vivo. (A) H&E staining and renal IL-6 levels evaluated by immunoblotting were performed to evaluate the effect of FTO on the kidney injury induced by high-fat and high-glucose diet. (B-D) ELISA was performed to detect the amount of IL-1β and IL-18 in the kidney tissue. (E) Expression of pyroptosis related proteins including NLRP3, ASC, cleared caspase-1 and GSDMD-N was evaluated by western blot. The one-way ANOVA was used for the comparison between multiple groups. The data were expressed by mean ± SD

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Discussion

In the present research, we demonstrated that FTO inhibited the NLRP3 expression via m6A modification. Additionally, we illustrated that FTO was prominently lowly expressed in HK-2 cells treated by HG. FTO over-expression relieved the injury of HK-2 cells induced by HG treatment and repressed the kidney injury in the DKD mice. This research is the first to investigate the function of FTO on the NLRP3 signaling mediated pyroptosis in DKD progression.

Identifying targets for therapeutic interventions is critical to developing strategies to combat the development and progression of DKD disease [19]. Accumulating evidence suggested that the mainstay treatments of DKD including renin-angiotensin system inhibitors, sodium-glucose co-transporter 2 inhibitors, incretin-based therapeutic agents, and non-steroidal mineralocorticoid receptor antagonists have been reported to treat DKD with different targets [20, 21]. Despite the many advances in the clinical management of DKD, the number of cases of diabetic kidney disease continues to increase, in part, owing to a pandemic increase of people with diabetes [19]. It is of great significance to find new therapeutic targets for DKD, and the combination of new targets and existing therapeutic methods may be more helpful for the treatment of DKD.

m6A methylation is confirmed to be the most common RNA modification in mammals and is involved in the RNA metabolism processes including, such as RNA biosynthesis and decay [22, 23]. Demethylase FTO, as an important m6A regulator, has been highlighted to participate in various diabetic complications, including microvascular complication [24], diabetic cataract [25], diabetic foot ulcers [26]. Due to the different modification sites and m6A binding readers, the function of m6A modification is complex. For instance, Xu et al. [27] found that methylase METTL14 levels were prominently depleted in the HK-2 cells after HG treatment, which would further inhibited the histone deacetylase 5 and TGF-β1 expression levels. Eventually affecting the EMT of renal tubular cells in DKD. However, Li et al. [28] suggested that METTL14 levels were dramatically up-regulated in kidneys of DKD, which was further demonstrated to elevate the ROS, TNF-α and IL-6 levels in the regulation of m6A levels. Here, we confirmed that FTO was lowly expressed in DKD. FTO over-expression promoted the cell growth while inhibited the cell pyroptosis in vivo and in vitro. These findings are similar to the previous report of Jiang et al. [12], who also confirmed FTO silencing effectively relieved the podocyte injury in DKD mice. All these results implied that FTO-mediated m6A modification might be a critical factor in DKD progression. We found that FTO was dysregulated in the serum of DKD patients. In this study, we evaluated the protective effect of FTO on the kidneys in vivo by detecting changes in interleukin markers and pyroptosis related protein levels throughout the kidney tissue. However, whether this protective effect is accurately located in the renal tubules and glomeruli deserves further study. Moreover, we did not evaluate whether FTO could be the biomarker for the diagnose of DKD. It will be also studied in our further research.

Autophagy is a protective mechanism for maintaining cellular homeostasis, and impairment of autophagy can aggravate renal cell dysfunction and apoptosis [29]. In the microenvironment of diabetes, autophagy deficiency can induce the pathological changes of different renal cells and further promote the progression of renal disease [30,31,32]. Mitochondrial autophagy termed as mitophagy was discovered to decline the release of ROS, promote apoptotic substances and reduce apoptosis [33], and the increases of inflammatory cytokines and ROS have also been shown in DKD [34]. Recently, mitophagy mediated by NLRP3/Parkin signaling pathway has been proved to participate in various diseases and is the main pathway for the removal of damaged mitochondria [35, 36]. In this study, the western blot results revealed that in the HG-stimulated HK-2 cells, the ratio of LC3-II/LC3-I was prominently down-regulated, while p62 was up-regulated. FTO silencing reversed the effect of HG treatment. Additionally, we found that NLRP3/Parkin signaling pathway was inhibited in the HG-stimulated HK-2 cells. After FTO silencing, it was activated. Therefore, we speculate whether FTO regulates the signaling pathway by regulating the m6A methylation modification of NLRP3.

Interestingly, through the RIP and dual luciferase report assay, we confirmed the interaction between FTO and NLRP3. And FTO silencing prominently decreased the m6A levels of NLRP3 and increased the production of NLRP3 mature mRNA levels. Furthermore, NLRP3 silencing neutralized the functions of sh-FTO in the HG-stimulated HK-2 cells. These results indicated that FTO participated in the DKD progression through modulating the NLRP3/Parkin signaling pathway.

To sum up, our study determined that FTO mediates the DKD progression through regulating the pyroptosis of renal tubular epithelial cells. Mechanistically, FTO regulates the m6A modification and expression of NLRP3 (Fig. 6). We hope these findings could provide novel targets for the treatment of DKD disease.

Fig. 6
figure 6

Mechanism diagram

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Data availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

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  1. Department of Nephrology, Guang’anmen Hospital South Campus, China Academy of Chinese Medical Sciences, No.138, Xingfeng Street, Huangcun Village, DaXing District, Beijing, 102600, China

    Qiang Li & Shujuan Mu

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Q L drafted the work and revised it critically for important intellectual content and was responsible for the acquisition, analysis and interpretation of data for the work; S M made substantial contributions to the conception or design of the work. All authors read and approved the final manuscript.

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Correspondence to Shujuan Mu.

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All experimental protocols were approved by approved by Laboratory Animal Ethics Committee of the Beijing Medconor Biotechnology Co., LTD. (MDKN-2022-70). All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org) for the reporting of animal experiments. All methods were performed in accordance with the Basel Declaration.

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Li, Q., Mu, S. FTO mediates the diabetic kidney disease progression through regulating the m6A modification of NLRP3. BMC Nephrol 25, 345 (2024). https://doi.org/10.1186/s12882-024-03741-5

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Keywords

BMC Nephrology

ISSN: 1471-2369

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