Acute kidney injury (AKI), also known as acute renal failure, is a clinical syndrome with a rapid decline of renal function caused by multiple factors. The kidney is rich in microvascular network, and the insufficient blood supply is the most crucial factor that leads to renal circulatory disorder and tissue injury. Furthermore, renal tissue can be damaged again during blood recovery and reperfusion by triggering inflammation or cell apoptosis
[1-2].
Long noncoding RNAs (lncRNAs) represent a class of functional RNA molecules with a length of more than 200 nucleotide (nt) without the ability to encode proteins
[3]. Recent studies have shown that lncRNAs can regulate cell biology by competing with microRNA (miRNAs) or directly interacting with proteins
[4]. MiRNAs are endogenous, single stranded, noncoding and small regulatory RNAs, with a length of <22 nt. MiRNAs are considered to be important regulators in a variety of biological processes such as cell fate determination, stress response, cell proliferation or death
[5-6]. It is reported that miRNAs can regulate gene expression by inhibiting the translation of target messenger RNAs (mRNA) or promoting mRNA degradation through binding to the 3'-untranslated region (3'-UTR) of target mRNA
[5]. LncRNAs can function as the competing endogenous RNA (ceRNA) or “sponge” to bind with miRNAs through the same miRNA target sequence during RNA transcription to regulate the expression of downstream target genes
[4]. The role of lncRNAs in AKI was widely studied over the past 5 years. Lu, et al
[7] found that knockdown of lncRNA MALAT1 could ameliorate AKI progression and inflammation by targeting miR-204 through apolipoprotein L1/nuclear factor kappa (APOL1/NF-κB) signaling pathway. LncRNA TCONS_00016406 could attenuate sepsis-associated AKI by modulating the miR-687/phosphatase and tensin homolog (PTEN) axis
[8].
Based on systematic screening of differentially expressed RNAs in renal tissues after ischemia-reperfusion, this study aims to investigate the role of lncRNA AK154753 (AK154753) in acute renal ischemia reperfusion injury (IRI) and to elucidate the molecular mechanism of the AK154753 via miR-345-3p/Bcl-2 homologous antagonist/killer (Bak) and miR-708-5p/Bcl-2 interacting mediator of cell death (Bim) axis.
1 Materials and methods
1.1 Ethics statement
The experimental protocol and animal use plan in this study were approved by the Animal Ethics Committee of Second Xiangya Hospital, Central South University (Approval No. 20241078).
1.2 AKI mouse model
The kidney IRI mouse model was established according to a previous study
[9]. Male C57BL/6 mice aged 8 weeks were weighed and anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg). All the mice were controlled the anal temperature within the range of 36.5 to 36.7 ℃ during the entire operation. After skin preparation, a longitudinal incision was taken on the back of the mouse, and the bilateral kidneys and renal arteries were exposed and separated. The back incision of the mice in the sham group was directly sutured without subsequent treatment. In the IRI group, after the bilateral kidneys and renal arteries were separated, the bilateral renal arteries were clamped with arterial clamps. After ischemia for 30 minutes followed by reperfusion for 24 hours (IRI-24 h) and 48 hours (IRI-48 h), the mice were sacrificed. The kidneys were dissected, and blood was collected and sent to the clinical laboratory of our hospital for the measurement of serum creatinine (Scr) and blood urea nitrogen (BUN) to evaluate renal function. Each group was 3 mice.
To further explore the function of AK154753 in kidney injury, we used the adeno-associated virus (AAV)-shRNA AK154753 silencing AK154753 to investigate if targeting AK154753 in vivo can alleviate the development of IRI. The AAV-shRNA AK154753 was purchased from Hanbio (Shanghai, China). AAV (at the titer of 6×10
10 viral genomes/mL) was injected into the tail vein when mouse was 4 weeks old, IRI model was performed at 8 weeks old
[10-11].
1.3 Oxygen and glucose deprivation/reperfusion model
To explore the potential impact of AK154753/miR-345-3p/miR-708-5p interaction in IRI. An oxygen and glucose deprivation/reoxygenation (OGD/R) model was established in BUMPT cells (mouse renal proximal tubular cell line). The BUMPT cell line was derived from Dr. Wilfred Lieberthal and John Shwartz of Boston University, which was kindly given by Professor Dong Zheng. The OGD/R cell model was established according to a previous study
[12]. When the cells adhered to the wall and grow to a density of about 80%, then discard the entire medium. Washing 3 times with phosphate buffered saline (PBS), replacing the dosing medium containing 5 μmol/L Antimycin A and Oligomycin (Abcam, Cambridge, UK), and then incubating for 2 hours. After 2 hours, discarding the dosing medium, and washing it gently with PBS. After changing the culture medium, incubating for another 2 hours. The cells of control group were consistently cultured in complete medium.
1.4 Fluorescence in situ hybridization (FISH)
The fluorescence probe of AK154753 was synthesized by Ribobio (Guangzhou, China). To detect in BUMPT cells, nuclei was stained with 40,6-diamidino-2-phenylindole (DAPI), U6 (nucleus positive), and 18S rRNA (cytoplasmic positive), and AK154753 was labeled by CY3. Briefly, the slides of BUMPT cells were hybridized overnight with the probe and subsequently stained with DAPI. A fluorescence microscope (Leica, Weltzlar, Germany) was used for fluorescence imaging analysis.
1.5 Transfection
The lentivirus vector (LV)-shRNA AK154753 used in this study was purchased from genechem (Shanghai, China). Sequence for sh-AK154753 is AGGGAAATGTCCTAAACACAT. The virus was added to Bumpt cells, and cells were selected with puromycin (Beyotime, Shanghai, China) at 48 hours after transfection.
MiRNA mimics and inhibitors were purchased from Ribobio (Guangzhou, China). The miRNA mimics (concentration 10 nmol/L) or inhibitors (concentration 50 nmol/L) were transfected into cells, and cells were cultured at 5% CO2 and 37 ℃ for 24 to 48 hours. After 24 hours, the RNA was extracted from the cells for real-time reverse transcription PCR (real-time RT-PCR). The protein was extracted from the cells for Western blotting after 48 hours.
1.6 Western blotting
Western blotting was performed to detect apoptosis-related proteins including Bak, Bim, and cleaved-caspase3. The lysate was prepared and the protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a 0.22 μm polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with PBST containing 5% skim milk at room temperature for 1 hour. After incubating with the primary and secondary antibodies, the target protein signal in the PVDF membrane was detected. The commercially available antibodies and the dilutions used are as follows: anti-Bim (CST, USA; 1꞉1 000); anti-Bak (CST, USA; 1꞉1 000); anti-caspase3 (CST, USA; 1꞉1 000); anti-β-actin (Abcam, UK; 1꞉5 000); anti-GAPDH (Abcam, UK; 1꞉10 000); goat anti-rabbit IgG H&L (Abcam, UK, 1꞉20 000).
1.7 Real-time reverse transcription PCR
Trizol reagent (Invitrogen, USA) was used to extract total RNA from cells and renal tissues. The RNA was quantified using NanoDrop 2000 instrument (Thermo Fisher, USA). The total RNA was transferred into complementary DNA (cDNA) using cDNA reverse transcription Kit (Takara, Japan). U6, miR-345-3p and miR-708-5p primers were purchased from Ribobio (Guangzhou, China). The primers of GAPDH were 5'-TGGTGAAGGTCGGTGTGAAC-3' (forward) and 5'-GCTCCTGGAAGATGGTGATGG-3' (reverse), while 5'-GCCCAGAACTGGTAAGAAGC-3' (forward) and 5'-ACTTACAGTTATTTGCTGCCATC-3' (reverse) were of AK154753. The levels of AK154753, miR-345-3p and miR-708-5p were measured using SYBR® Green Premix Pro Taq HS qPCR Kit (Agbio, Hunan, China). The lncRNA expression level of each sample was normalized to the expression of GAPDH, the expression levels of miRNAs of each sample were normalized to the expression of U6, and the 3 biological replicates were compared with real-time RT-PCR)
1.8 Hematoxylin and eosin staining
Hematoxylin and eosin (HE) staining was performed on kidney tissue samples. The sections were put into xylene I for 10 minutes, xylene II for 10 minutes, absolute ethanol I for 5 minutes, and absolute ethanol II for 5 minutes, 95% alcohol for 5 minutes, 90% alcohol for 5 minutes, 80% alcohol for 5 minutes, 70% alcohol for 5 minutes, and finally the sections were rinsed with distilled water. The sections were stained with hematoxylin for 8 minutes, rinsed with tap water and finally stained in eosin staining solution for 3 minutes. The slices were put in 95% alcohol I for 5 minutes, 95% alcohol II for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, xylene I for 5 minutes, and finally xylene II for 5 minutes for dehydration. The slices were removed from the xylene and dried slightly. The slices were observed through a microscope, and performed image acquisition combined with analysis.
1.9 Detection of apoptosis
The morphological change of apoptotic cells was observed by TUNEL staining using In Situ Cell Death Detection Kit (Roche, Basel, Switzerland). Cells and kidney tissue samples were fixed in 4% paraformaldehyde at room temperature. After discarding 4% paraformaldehyde, wash slices with PBS 3 times for 5 minutes each time. Aspirate PBS, add PBS containing 0.3% Triton X-100, and incubate at room temperature for 5 minutes. After removing Triton X-100, wash with PBS 3 times for 5 minutes each time, and aspirate PBS. Preparation of TUNEL reaction solution (TdT꞉dUTP=1꞉10), drop the reaction solution on the glass slide and place it in a wet box, react at 37 °C for 1 hour, After washing, nuclei were stained with DAPI in the dark for 5 minutes, and anti-fluorescence quencher was added. The apoptotic cells were photographed under the fluorescence microscope.
Flow cytometry was also performed to detect cell apoptosis with Annexin V-FITC apoptosis detection kit (Keygentec, Nanjing, China). The cells were washed twice with cold PBS buffer, and then binding buffer was used to make a cell suspension. Annexin V and nucleic acid dye were added into cells, and the cell suspension was mixed gently and placed in the dark at room temperature 15 minutes. The cells were washed once with 1×binding buffer, and the supernatant was removed. Propidium iodide (PI) was added into cells and placed at room temperature in the dark for 15 minutes. The results were confirmed by the flow cytometer within 1 hour.
1.10 Microarray
Microarray-based transcriptome sequencing, including lncRNA sequencing, miRNA sequencing and mRNA sequencing, was carried out by Kangcheng Biological Co., Ltd. (Shanghai, China).
1.11 Luciferase reporter gene
We used miRWalk2.0 (Mannheim, Germany) software to predict the binding sites of AK154753/miR-345-3p/Bak and AK154753/miR-708-5p/Bim, respectively. The 3'-UTR sequences of AK154753, Bak, and Bim genes as well as the sequences of miR-345-3p and miR-708-5p, were inserted into the reporter gene vector. The luciferase reporter gene was detected by the Dual-Luciferase® Reporter Assay System (Promega, Madison, WI, USA).
1.12 Statistical analysis
SPSS 22.0 software was used for statistical analysis of the data. Data are summarized as means±standard error. All data points and animals were included in the statistical analyses. No formal test of normality or power analysis was performed, as the sample sizes were small and the data were assumed to follow an approximately normal distribution. Statistical analyses for animal and cell cultures experiments were performed by using ANOVA with the Bonferroni test for multiple comparisons or the independent-samples t test for two comparisons. P<0.05 was considered statistically significant.
2 Results
2.1 AK154753, Bak, and Bim were upregulated in kidney after IRI while miR-345-3p and miR-708-5p were downregulated
The results showed that levels of BUN and Scr in the IRI group were significantly higher than those in the sham group (all
P<0.05, Figure
1A and
1B). Moreover, IRI triggered massive cell death (
Figure 1C) and obvious tubular injury (
Figure 1D), as assessed by TUNEL staining and HE staining, respectively. Next, microarray-based transcriptome sequencing was performed using renal tissues from mice in the IRI group and the sham group. Compared with the sham group, miR-345-3p and miR-708-5p of the IRI group were down-regulated both in reperfusion for 24 and 48 hours (Supplementary
Figure 1A,
https://doi.org/10. 57760/sciencedb.xbyxb.00160). Through ceRNA analysis, miR-345-3p was predicted to bind to Bak, and miR-708-5p was predicted to bind to Bim, while AK154753 can simultaneously interact with both miR-345-3p and miR-708-5p (Supplementary
Figure 1B,
https://doi.org/10. 57760/sciencedb.xbyxb.00160). Furthermore, the levels of AK154753, Bak, and Bim were up-regulated in IRI group, while miR-345-3p and miR-708-5p in IRI group were down-regulated, which were verified by real-time RT-PCR (all
P<0.01, Figure
1E-
1I).
2.2 AK154753 was increased after OGD/R in renal tubular cell together with induced apoptosis and miR-345-3p/miR-708-5p were decreased
Real-time RT-PCR analysis showed that the mRNA of
AK154753 in the OGD/R group was significantly up-regulated (
P<0.01,
Figure 2A), while the levels of miR-345-3p (
P<0.01,
Figure 2B) and miR-708-5p (
P<0.01,
Figure 2C) were significantly down-regulated compared with the control group. The expression levels of Bak, Bim, and cleaved-caspase 3 were obviously up-regulated in the OGD/R group as shown by Western blotting results (all
P<0.01, Figure
2D-
2G). Flow cytometry revealed that cell apoptosis was significantly increased after OGD/R (
P<0.01, Figure
2H and
2I).
2.3 Silencing of AK154753 attenuates the apoptosis of BUMPT cells
After silencing the expression of AK154753 and establishing the OGD/R model in BUMPT cell, real-time RT-PCR results showed that the increased expression of AK154753 after OGD/R treatment was reduced after transfection with shRNA-AK154753 (
P<0.01,
Figure 3A). Western blotting revealed that AK154753 shRNA attenuated the level of cleaved-caspase 3 after OGD/R. Meanwhile, the expression levels of Bim and Bak were significantly reduced in the AK154753 shRNA group than those in the OGD/R+sh-NC group (all
P<0.01, Figure
3B-
3E). The apoptosis rate of BUMPT cells in the OGD/R group was increased, however, knock-down of AK154753 decreased the number of TUNEL-positive apoptotic cells induced by OGD/R (all
P<0.01, Figure
3F and
3G).
2.4 Targeting AK154753 mitigates IRI development in vivo
Real-time RT-PCR results demonstrated that AAV-sh-AK154753 administration significantly reduced AK154753 expression in the renal tissues of IRI mice (
P<0.05，
Figure 4A). Consistently, the levels of BUN and Scr in IRI mice were significantly decreased by AAV-sh-AK154753 (both
P<0.01，Figure
4B and
4C). HE staining showed that the IRI-induced kidney tubule damage was markedly reversed by AAV-sh-AK154753 (
P<0.01，Figure
4D and
4E). TUNEL analysis showed that AAV-sh-AK154753 reduced cell apoptosis in the renal tissues of IRI mice (
P<0.01，Figure
4F and
4G). Moreover, the levels of Bak, Bim, and cleaved-caspase3 were significantly up-regulated in IRI renal tissues, which were all down-regulated by AAV-mediated AK154753 silencing (all
P<0.01, Figure
4H-
4K).
2.5 AK154753 increases Bak and Bim through regulating miR-345-3p and miR-708-5p respectively
Mechanistically, AK154753 was mainly localized in the cytoplasm (
Figure 5A). The decreased levels of miR-345-3p and miR-708-5p in BUMPT cells after OGD/R were significantly rescued by AK154753 silencing (both
P<0.01, Figure
5B and
5C). In addition, AAV-sh-AK154753 administration significantly increased the levels of miR-345-3p and miR-708-5p in the renal tissues of IRI mice (both
P<0.01, Figure
5D and
5E). In addition, miR-345-3p and miR-708-5p can facilitate apoptosis through Bak and Bim, respectively. MiR-345-3p mimic inhibited the expression of Bak, while miR-345-3p inhibitor promoted the expression of Bak (both
P<0.01, Figure
5F and
5G). As the same, miR-708-5p mimic inhibited the expression of Bim, while miR-708-5p inhibitor promoted the expression of Bim in BUMPT cells (both
P<0.01, Figure
5H and
5I).
2.6 AK154753 increases cell apoptosis through targeting miR-345-3p/Bak and miR-708-5p/Bim
The luciferase reporter assay results showed that miR-345-3p had the binding sites with the 3'-UTR of both AK154753 and Bak (Supplementary Figure
2A and
2B,
https://doi.org/10.57760/sciencedb.xbyxb.00160). Similarly, miR-708-5p could bind to the 3'-UTR of both AK154753 and Bim (Supplementary Figure
2C and
2D,
https://doi.org/10.57760/sciencedb.xbyxb.00160). Importantly, the concomitant of miR-345-3p inhibitor can abrogate the anti-apoptosis effect of AK154753 silencing and miR-708-5p inhibitor can also attenuate the anti-apoptosis effect of AK154753 silencing (all
P<0.01; Supplementary Figure
2E and
2F,
https://doi.org/10. 57760/sciencedb.xbyxb.00160). The downregulation of Bak and Bim along with AK154753 silencing could also be blocked by the addition of miR-345-3p inhibitor and miR-708-5p inhibitor respectively (
P<0.01; Supplementary Figure
2G-
2J,
https://doi.org/10.57760/sciencedb.xbyxb. 00160).
3 Discussion
In recent years, an increasing number of studies
[13-14] have demonstrated that lncRNAs play critical regulatory roles in the development and progression of AKI. Previous study
[13] has shown that lncRNAs act as ceRNAs to interact with specific miRNAs, thereby modulating target gene expression and participating in various pathological processes such as inflammation, oxidative stress, apoptosis, autophagy, and ferroptosis. For instance, lncRNA TapSAKI was first reported to be highly expressed in the serum and renal tissues of AKI patients, positively correlated with disease severity, and proposed as a potential early prognostic biomarker
[14]. MALAT1 aggravates inflammation and tubular epithelial cell apoptosis by sponging miR-204 and activating the NF-κB signaling pathway
[7], whereas NEAT1 is upregulated in both septic and ischemic AKI models, promoting apoptosis through p53 or NF-κB dependent signaling via miR-27a-3p and miR-204
[15]. Moreover, TUG1, MEG3, SNHG14, and HOTAIR have been reported to modulate the PI3K-Akt, autophagy, or NF-κB pathways, exerting either pro-apoptotic or cytoprotective effects
[16-19]. Collectively, these studies indicate that lncRNAs function as key post-transcriptional regulators in AKI and possess considerable pathophysiological and potential clinical significance.
In this study, transcriptome profiling based on microarray analysis revealed that lncRNA AK154753 was markedly upregulated in the renal tissues of IRI mice. Further investigation demonstrated that AK154753 could simultaneously bind 2 miRNAs, miR-345-3p and miR-708-5p, which target the pro-apoptotic proteins Bak and Bim, respectively. Functional assays showed that silencing AK154753 significantly reduced OGD/R-induced apoptosis of renal tubular epithelial cells, decreased Bak and Bim protein levels, and alleviated renal dysfunction and histological damage in IRI mice, as evidenced by lower BUN and serum creatinine levels. Notably, unlike previously reported lncRNAs such as TapSAKI or NEAT1 that act through a single miRNA pathway, AK154753 exerts its pro-apoptotic effect by concurrently regulating two miRNA-target axes, suggesting that it may serve as an integrative hub in the apoptotic signaling network of AKI.
Bak and Bim, members of the Bcl-2 family, are pivotal regulators of the mitochondrial apoptotic pathway. Bim activates Bak and Bax to increase mitochondrial outer membrane permeability (MOMP), leading to cytochrome c release, activation of caspase-9 and caspase-3, and subsequent apoptotic execution
[20]. During renal ischemia-reperfusion, bursts of reactive oxygen species (ROS) and Ca²⁺ overload induce the upregulation of Bak and Bim, thereby promoting tubular epithelial cell apoptosis and contributing to renal dysfunction
[21]. Our data demonstrated that AK154753 silencing exerts a pronounced anti-apoptotic effect during OGD/R, while inhibition of miR-345-3p or miR-708-5p attenuated this protective effect. Notably, the changes in apoptosis rate, Bak, and Bim were prominent, whereas cleaved caspase-3 exhibited a relatively modest response. This discrepancy indicates that the AK154753/miR-345-3p/miR-708-5p axis primarily regulates the upstream mitochondrial events of apoptosis, while caspase-3 activation may be influenced by multiple factors, thus showing a weaker change.
Although no studies have directly reported the roles of miR-345-3p and miR-708-5p in AKI. However, numerous studies have demonstrated that both miRNAs participate in the regulation of apoptosis and stress responses in various diseases. For example, miR-503-3p enhances the radioresistance of oral squamous carcinoma cells by suppressing Bak expression
[22], while miR-148a downregulates Bim to promote glioblastoma cell survival
[23], indicating that Bak and Bim are negatively regulated by multiple miRNAs. In this study, IRI markedly decreased the expression of miR-345-3p and miR-708-5p, whereas AK154753 knockdown partially restored their levels, reduced Bak and Bim expression, and attenuated apoptosis. These findings suggest that AK154753 may function as a ceRNA integrating the miR-345-3p/Bak and miR-708-5p/Bim pro-apoptotic axes, thereby promoting tubular epithelial cell apoptosis during AKI.
Nonetheless, several limitations should be acknowledged. First, the present experiments have confirmed the interactions between AK154753 and miR-345-3p/miR-708-5p, but further studies are needed to verify their direct binding and dissect the detailed molecular mechanisms. Second, whether AK154753 participates in other biological processes, such as autophagy, ferroptosis, or inflammation, remains to be determined. Moreover, given the limited sequence conservation of lncRNAs across species, the translational relevance of AK154753 requires validation in human AKI samples. In future work, our group plans to perform single-cell transcriptomic analysis using AK154753-knockout mouse kidneys to systematically explore its function in renal IRI, identify potential regulatory pathways, and investigate its upstream transcriptional regulation and clinical correlation. These efforts may provide new insights into the molecular mechanisms and therapeutic strategies for AKI.
In conclusion, this study elucidates the pro-apoptotic role and molecular mechanism of AK154753 in AKI, thereby expanding the current understanding of lncRNA involvement in renal IRI. By functioning as a ceRNA that simultaneously regulates 2 pro-apoptotic pathways, AK154753 exemplifies how lncRNAs can serve as multilayered regulatory nodes that coordinate cell death signaling. Silencing AK154753 not only reduces apoptosis in vitro but also improves renal function and attenuates histological injury in vivo, suggesting its potential as a therapeutic target.
the National Natural Science Foundation of China(81770692)
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