Transcription Factor Sox9 Exacerbates Kidney Injury through Inhibition of MicroRNA-96-5p and Activation of the Trib3/IL-6 Axis

Abstract

Introduction

Our study investigated the possible mechanisms of the role of the transcription factor Sox9 in the development and progression of kidney injury through regulation of the miR-96-5p/Trib3/IL-6 axis.

Methods

Bioinformatics analysis was performed to identify differentially expressed genes in kidney injury and normal tissues. An in vivo animal model of kidney injury and an in vitro cellular model of kidney injury were constructed using LPS induction in 8-week-old female C57BL/6 mice and human normal renal tubular epithelial cells HK-2 for studying the possible roles of Sox9, miR-96-5p, Trib3, and IL-6 in kidney injury.

Results

Sox9 was highly expressed in both mouse and cellular models of kidney injury. Sox9 was significantly enriched in the promoter region of miR-96-5p and repressed miR-96-5p expression. Trib3 was highly expressed in both mouse and cellular models of kidney injury and promoted inflammatory responses and kidney injury. In addition, Trib3 promoted IL-6 expression, which was highly expressed in kidney injury, and promoted the inflammatory response and extent of injury in kidney tissue. In vivo and in vitro experiments confirmed that the knockdown of Sox9 improved the inflammatory response and fibrosis of mouse kidney tissues and HK-2 cells, while the ameliorative effect of silencing Sox9 was inhibited by overexpression of IL-6.

Conclusion

Collectively, Sox9 up-regulates miR-96-5p-mediated Trib3 and activates the IL-6 signaling pathway to exacerbate the inflammatory response, ultimately promoting the development and progression of kidney injury.

Introduction

Clinically, performance renal injury is mainly characterized by a rapid increase in serum creatinine (SCr) and a decrease in urine output. In addition, proximal tubular epithelial cell death is a common cause of renal injury. The main causes of renal injury include inadequate renal perfusion [1], type 1 cardiorenal syndrome [2], sepsis [3], major surgery [4], intra-abdominal hypertension [5], acute progressive glomerulonephritis [6], acute interstitial nephritis [7], and post-renal renal injury [8]. Renal injury is diagnosed mainly by testing SCr or urine output. Still, renal injury usually occurs as part of other syndromes (e.g., heart failure, liver failure, and sepsis), which themselves lead to significant morbidity and mortality and therefore tend to overlook the importance of renal injury as a marker and determinant of disease severity [9].

Sox9 is a transcription factor involved in various biological processes, such as cell proliferation, differentiation, migration, and apoptosis. Diseases associated with Sox9 include the search for abnormal curved limb development [10] and X/Y sex reversal [11]. It plays a key role in chondrocyte differentiation and skeletal development [12]. Zhang et al. [13] found a significant increase in Sox9 expression in kidney injury tissues. In addition, using a rat kidney injury model, the investigators found that Sox9 was closely associated with fibrosis [14].

Trib3 is a pseudokinase that regulates cellular signaling pathways affecting cell survival and death. Trib3 has been reported to be associated with type 2 diabetes [15], and its related pathways include insulin receptor signaling cascade and CD28 co-stimulation. Currently, Trib3 is less commonly reported to be associated with kidney injury. However, two datasets, GSE30718 and GSE139061, have been screened for differentially expressed genes, including TRIB3, which is upregulated in kidney injury [16] by the overlap of iron death associated with kidney injury. In addition, studies have revealed a potential oncogenic role for TRIB3 in the pathogenesis of renal cell carcinoma, with HIF-1α regulating TRIB3 expression by targeting the MAPK signaling pathway to enhance cell viability and invasiveness [17].

miR-96-5p is a microRNA involved in many biological processes, such as cell proliferation, migration, invasion, apoptosis, and autophagy [18]. miR-96-5p has been reported to target FN1 to attenuate TGF-β1-induced renal fibrosis [19]. In addition, miR-96-5p inhibited UUO-induced renal fibrosis and ECM protein accumulation in the kidney by suppressing TGF-β1-induced expression of type I collagen, type III collagen, and fibronectin in BUMPT cells [20]. In conclusion, miR-96-5p plays an important role in kidney disease and has many biological functions.

The main objective of this study was to reveal the mechanism of Sox9-mediated miR-96-5p/Trib3/IL-6 signaling axis in the development and progression of renal injury. First, we will determine the expression and distribution of Sox9 in kidney injury tissues and explore its role in the onset and progression of kidney injury. We will then also validate the changes in miR-96-5p expression in kidney injury and explore its role in the process of kidney injury and its association with Sox9. Then, we will delve into the expression and function of Trib3 in renal injury and verify whether it is a direct target gene of miR-96-5p. Finally, the expression and role of IL-6 in kidney injury will be explored, and its regulatory role in the Sox9-mediated miR-96-5p/Trib3/IL-6 signaling axis will be elaborated, providing a new theoretical basis and target for the early diagnosis, treatment, and prevention of kidney injury.

Materials and Methods

Bioinformatics Analysis

The GEO database was used to search the GSE192532 (Sham group, n = 3; Model group, n = 3) and GSE71647 (Sham group, n = 2; Model group, n = 2) GSE11783 gene expression microarrays, which contained 10 kidney injury samples and 6 normal samples, with |logFC|>2 and p value <0.05 as the differential mRNA screening criteria, and the R language “limma” package was used to screen for significantly different genes in normal control mice and mouse models with kidney injury. TargetScan (https://www.targetscan.org/vert_71/) and miRDB database (http://www.mirdb.org/mirdb/index.html) were then used to predict the downstream target genes of miR-96-5p. Finally, the database prediction results were compared with the GSE192532 microarray of significantly highly expressed intersected genes.

Construction of a Mouse Model of Kidney Injury with Lentivirus Transfection

Twenty-four 8-week-old male C57BL/6 mice (Beijing Viton Lever Laboratory Animal Technology Co., Ltd., Beijing, China) were housed in SPF-grade animal laboratories in separate cages at 60–65% humidity and 22–25°C and provided with free food and water under a 12-h light and dark cycle for 1 week after acclimatization and observation of their health status before the experiments. All experiments and procedures were carried out following the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals, in compliance with animal ethics standards, and every effort was made to minimize animal suffering.

The mice were anesthetized by intraperitoneal injection of 2% pentobarbital sodium (50 mg/kg), fixed, and injected intraperitoneally with 10 mg/kg LPS. The sh-NC, sh-Sox9, sh-Sox9+, and sh-IL-6 lentiviruses were transfected according to the instructions for lentiviral infection. oe-IL-6 lentiviruses were transfected (Shanghai Jima synthesized sequences), and all lentiviral vectors carried luciferase.

The model group mice were randomly divided into 3 groups (6 mice in each group): model+sh-NC group (model mice were injected intraperitoneally with 10 μL of negative control lentivirus sh-NC), model+sh-Sox9 group (model mice were injected intraperitoneally with 10 μL of sh-Sox9 lentivirus), model+sh-Sox9+IL-6 group (model mice were injected intraperitoneally with 10 μL of sh-Sox9 lentivirus and 400 ng/100 μL of IL-6) were injected intraperitoneally into the model mice.

Groups of mice were given the negative control lentivirus sh-NC and sh-Sox9 lentivirus via intraparenchymal injection at a lentivirus titer of 5 × 10⁷ TU/only and treated with LPS 1 week after injection [21]; for IL-6 treatment, mice were given 400 ng IL-6 via intraperitoneal injection (5 h after LPS injection, mice were anesthetized with sodium pentobarbital (1%, 50 mg/kg, Sigma, USA). Blood was collected from the heart in 0.8 mL, and serum was collected. In addition, 300 μL of serum was used to determine inflammatory factor levels. The mice were then executed, and the tissues were taken from each group; part of the tissues was used for H&E staining, while the other part was stored in liquid nitrogen for subsequent RT-qPCR and Western blot assays.

Mouse Renal Function Test

Whole blood specimens were taken 24 h after the injection of LPS to establish renal injury, left at 37°C for 1 h, and centrifuged at 4,000 r/min for 8 min. Serum was taken for renal function testing, and SCr and urea nitrogen (BUN) were measured using a fully automated biochemical instrument (Siemens ADVIA 1800 Chemistry System, Germany).

H&E Dyeing

Sections of mouse kidney tissue were paraffined and placed sequentially in xylene I for 10 min, xylene II for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% alcohol for 5 min, 90% alcohol for 5 min, 80% alcohol for 5 min, 70% alcohol for 5 min, distilled water wash, and at the end of dewaxing and dehydration, the sections were immersed in Harris hematoxylin (G1120, Solarbio, China) stain for 10–30 min, tap water wash, 1% hydrochloric acid alcohol fractionation for a few seconds, tap water rinse, 0.6% ammonia return to blue, running water rinse. Next, the sections were stained in an eosin staining solution for 1–3 min. The sections were placed sequentially in 95% alcohol I for 5 min, 95% alcohol II for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, xylene I for 5 min, and xylene II for 5 min to dehydrate and clarify. A microscopic examination was performed, and the renal tubular lesions in each field of view were scored.

The degree of tubular vacuolar degeneration in renal cortical tubular cells was analyzed and scored according to the degree of tubular vacuolar degeneration [22, 23]. A score of 4 was assigned for more than 75% tubular vacuolar degeneration, 3 for more than 51–75%, 2 for 26–50%, 1 for 10–25%, and 0 for less than 10%.

Toluidine Blue Staining

The paraffin sections were dehydrated and washed. Sections were removed from the water and plunged into 0.1% toluidine blue (G3668, Solarbio, China) solution for 10 min. Sections were rewashed and divided in glacial acetic acid until nuclei and granules were visible. Sections were washed, dried at room temperature, cleared with xylene, and sealed with neutral resin. Whole sections were observed under a high magnification field (×400). Four areas of the sectioned specimen were selected: upper, lower, left, and right, with five randomly selected high magnification fields in each area to observe the number of mast cells, which could be observed under the microscope with the tissue-stained light blue, nuclei stained dark blue, and mast cells purplish red; data was recorded. The number of MC-positive cells per 1 mm² specimen area was calculated [24].

ELISA

To detect TNF-α (ab208348-mouse, ab181421-human, Abcam, UK), IL-1β (ab197742-mouse, ab214025-human, Abcam, UK) and IL-6 (ab222503-mouse, ab178013-human, Abcam, UK) were expressed using the ELISA kit (Abcam, UK), according to the kit instructions, 0.1 mL of urine or cell supernatant was taken in the reaction wells of the 96-well plate in the kit, incubated at 37°C for 1 h, then washed (while doing the blank wells), the washing solution was discarded and slightly cooled and dried. Next, add 0.1 mL of freshly diluted enzyme antibody (titrated dilution) to each well, incubate for 0.5–1 h at 37°C, wash, discard the wash solution, and allow to cool, then add 0.1 mL of TMB substrate solution to each well and incubate for 10–30 min at 37°C. Finally, the reaction was terminated by adding 0.05 mL of 2 m sulfuric acid to each well. The OD of each well was measured at 450 nm on an enzyme marker, and the concentrations of TNF-α, IL-1β, and IL-6 were calculated [25]. Each experiment was repeated three times.

RT-qPCR

To extract mRNA or miRNA from tissues or cells, tissue RNA was extracted using the Trizol method, referring to the instructions of Trizol (16096020, AM1561, Thermo Fisher Scientific, NY, USA). 5 µg of tissue RNA was extracted according to the instructions of RT-qPCR kit (RR047A, Takara, Japan). The mRNA was reverse transcribed into cDNA, and the synthesized cDNA was subjected to RT-qPCR using Fast SYBR Green PCR kit (Applied Biosystems) and ABI PRISM 7300 RT-PCR system (Applied Biosystems) – qPCR assays. cDNA for miRNA was synthesized according to the instructions for using the TaqMan microRNA assay kit (Applied Biosystems, Foster City, USA). RT-qPCR reactions were performed using FastStart Universal SYBR Green Master Mix (Roche, Indianapolis, USA), and three replicates were set up for each well. U6 was the internal reference for miR-96-5p, and GAPDH was the internal reference for the other genes. 2^−ΔΔCt indicates the fold relationship between the expression of the target gene in the experimental group and the control group with the following formula: ΔΔCT = (average Ct value of the target gene in the experimental group – average Ct value of the housekeeping gene in the experimental group) – (average Ct value of the target gene in the control group – average Ct value of the housekeeping gene in the control group). The number of amplification cycles required for the real-time fluorescence intensity to reach a set threshold, where amplification increases in the logarithmic phase [25]. The experiment was repeated three times. The primer design is shown in online supplementary Table S1 (for all online suppl. material, see https://doi.org/10.1159/000533544).

Immunohistochemical Staining

Mouse kidney tissue was paraffin-embedded in 4 μm serial sections and routinely dewaxed. The sections were rinsed with PBS and then closed by adding normal goat serum in a drop. Normal tissues were used as negative controls and stained with a HistostainTMSP-9000 immunohistochemical staining kit (Zymed). Primary antibodies were Sox9 (ab185966, 1:300, Rabbit, Abcam, UK), Collagen I (ab34710, 1:200, Rabbit, Abcam, UK), Collagen III (ab6310, 1:200, Mouse, Abcam, UK), snail (ab180714, 1:200, Rabbit, Abcam, UK), and α-SMA (#19245, 1:500, Rabbit, CST, USA), which were treated overnight at 4°C. After rewarming and rinsing with PBS, the secondary antibodies Rabbit (ab6802, 1:1,000, Donkey, Abcam, UK) and Mouse (ab6820, 1:1,000, Donkey, Abcam, UK) were added dropwise and reacted at room temperature for 30 min. The staining time was adjusted under the microscope, hematoxylin was restained for 1 min, and the slices were sealed with gum. Positive criteria: 5 representative high magnification fields were selected and counted, and those with brown or yellow cytoplasm were considered positive [26]. The immunostained samples’ integrated optical density was evaluated using image processing software (Image-Pro Plus version 6; Media Cybernetics, Silver Spring, MD, USA).

In vitro Culture and Lentiviral Transfection of HK-2 Cells

Human renal tubular epithelial cells HK-2 (SCSP-511, NCACC, China) cells and HEK-293T (GNHu 43, NCACC, China) cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), HK-2 cells were treated with Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (1:1, Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum and 1% penicillin/streptomycin. HEK-293T cells were cultured in DMEM medium (12800017, Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) in an incubator (Thermo Fisher Scientific, USA) at 37°C with 5% CO₂. Cells were digested with trypsin when HEK-293T cells were in the log phase, and cells were inoculated in 6-well plates at 0.2 × 10⁵ per well. After 24 h of conventional culture, when cell fusion reached about 20–30%, cells were infected according to the instructions for lentivirus infection, and cells were grouped into the sh-NC group, sh-Sox9-1 group, sh-Sox9-2 group, sh-Trib3-1 group, sh-Trib3-2 group, oe-NC group, oe-Sox9 group, NC mimic group, miR-96-5p mimic group, NC inhibitor group, and miR-96-5p inhibitor group, and the cells were collected after 72 h of infection for subsequent experiments.

HK-2 cells were digested with trypsin in the log phase, and the cells were inoculated in 6-well plates at 1 × 10⁵ per well. After 24 h of conventional culture, when cell fusion reached about 75%, the cells were infected with lentivirus and divided into the Control group: 24 h intervention with PBS of equal volume with LPS; LPS group: 24 h intervention with 100 ng/mL LPS (Escherichia coli serotype O55:B5, Sigma-Aldrich, St. Louis, MO) for 24 h to construct an in vitro cell model of kidney injury.

LPS+sh-NC group: HK-2 cells infected with sh-NC lentivirus for 48 h followed by adding 100 ng/mL LPS intervention for 24 h. LPS+sh-Sox9 group: HK-2 cells infected with sh-Sox9 lentivirus for 72 h followed by 100 ng/mL LPS intervention for 24 h. LPS+sh-Sox9+NC inhibitor group: HK-2 cells infected with sh-Sox9 lentivirus and NC inhibitor plasmid for 2 h followed by 100 ng/mL LPS intervention for 24 h; LPS+sh-Sox9+miR-96-5p inhibitor group: HK-2 cells infected with sh-Sox9 lentivirus and miR-96-5p inhibitor plasmid for 72 h followed by 100 ng/mL LPS intervention for 24 h; LPS+NC mimic group: HK-2 cells infected with sh-Sox9 lentivirus and miR-96-5p inhibitor plasmid for 72 h followed by 100 ng/mL LPS intervention for 24 h; LPS+NC mimic group: HK-2 cells infected with NC mimic plasmid for 72 h followed by 100 ng/mL LPS intervention for 24 h; LPS+miR-96-5p mimic group: HK-2 cells infected with oe-NC lentivirus for 72 h followed by 100 ng/mL LPS intervention for 24 h; LPS+miR-5p mimic group: HK-2 cells infected with oe-NC lentivirus for 72 h followed by 100 ng/mL LPS intervention for 24 h. Virus for 72 h followed by 100 ng/mL LPS intervention for 24 h; LPS+miR-96-5p mimic+oe-NC group: HK-2 cells infected with oe-NC lentivirus and miR-96-5p mimic plasmid for 72 h followed by 100 ng/mL LPS intervention for 24 h; LPS+miR-96-5p mimic+oe-NC group: HK-2 cells infected with oe-NC lentivirus and miR-96-5p mimic plasmid for 72 h followed by 100 ng/mL LPS intervention for 24 h; LPS+miR-96-5p mimic+oe-Trib3 group: HK-2 cells infected with oe-Trib3 lentivirus and miR-96-5p mimic plasmid for 72 h followed by 100 ng/mL LPS intervention for 24 h; LPS+sh-NC group: HK-2 cells infected with sh-NC lentivirus for 72 h followed by 100 ng/mL LPS intervention for 24 h; LPS+sh-Trib3 group. HK-2 cells infected with sh-Trib3 lentivirus for 72 h followed by 100 ng/mL LPS intervention for 24 h; LPS+sh-Trib3+rh-IL-6 group: HK-2 cells infected with sh-Trib3 lentivirus followed by rh-IL-6 (human recombinant interleukin 6, 50 ng/mL, BioLegend, 570804) intervention 48 h and 100 ng/mL LPS intervention for 24 h; LPS+sh-Sox9+rh-IL-6 group: HK-2 cells infected with sh-Sox9 lentivirus followed by rh-IL-6 intervention for 48 h and 100 ng/mL LPS intervention for 24 h.

Cells were collected at the end of the intervention for subsequent experiments. The Sox9 overexpression plasmid was obtained by cloning, and the core plasmid fragment was transfected into 293T cells using pcDNA3.1 empty vector (pcDNA, Invitrogen, Carlsbad, CA, USA). The supernatant was collected after 48 h of cell culture. The virus was mixed twice, and the titer was determined. Lentiviruses were purchased from Shanghai Gikai Gene with the sequences sh-NC (5′-UUC​UCC​GAA​CGU​GUC​ACG​U-3′), sh-Sox9-1 5′-UCA​ACG​GCU​CGA​GCA​AGA​AUU-3′, sh-Sox9-2 forward, 5′-GCA​UCC​UUC​AAU​UUC​UGU​AUA-3′, sh-Trib3-1 (5′- CGA​GCU​CGA​AGU​GGG​CCC​C-3′) and sh-Trib3-2 (5′- GCC​GUG​CUC​UUC​CGC​CAG​AUG-3′).

ChIP Assay

To detect Sox9 binding to the miR-96-5p promoter region, chromatin immunoprecipitation assays were performed using the EZ-Magna ChIP TMA kit (Millipore, Billerica, MA, USA). Different subgroups of HEK-293T cells at the logarithmic growth stage were cross-linked with 1% formaldehyde for 10 min, and the crosslinking was terminated with 125 mm glycine for 5 min at room temperature. The cells were washed twice with pre-cooled PBS and centrifuged at 2,000 rpm for 5 min to collect the cells. The cells were resuspended in cell lysate [150 mm NaCl, 50 mm Tris (pH 7.5), 5 mm EDTA, 0.005% NP40, 0.01% Triton X-100] to achieve a final concentration of 2 × 10⁶ cells per 200 mL. The protease inhibitor mixture was added, centrifuged at 5,000 rpm for 5 min, resuspended in nuclear separation buffer, lysed in an ice-water bath for 10 min, and sonicated until 200–1,000 bp chromatin fragments were obtained. Centrifuge at 14,000 g for 10 min at 4°C and aspirate the supernatant. Add 100 μL of supernatant (DNA fragment) to 900 μL of ChIP Dilution Buffer and 20 μL of 50 × PIC. 60 μL of ProteinA Agarose/Salmon Sperm DNA was added to each group and mixed upside down at 4°C for 1 h. The supernatant was left to stand for 10 min at 4°C and centrifuged at 700 rpm for 1 min. In the experimental group, the supernatant was added to 1 μL of Sox9 (ab137676, Rabbit, Abcam, UK) and 1 μL of rabbit anti-IgG (ab172730, Abcam, UK) in the negative control group. 60 μL of ProteinA Agarose/Salmon Sperm DNA was added to each tube. Sperm DNA was inverted at 4°C for 2 h. After 10 min of standing time, the tubes were centrifuged at 700 rpm for 1 min. The supernatant was removed, and the residue was washed with l mL of low salt buffer, high salt buffer, LiCl solution, and TE (2 times). Each tube was eluted twice with 250 mL of ChIP Wash Buffer. 20 mL of 5 m NaCl was used to uncrosslink the DNA, and after uncrosslinking, the DNA was recovered, and the promoter of miR-96-5p in the complex (primer sequence F: 5′- TGG​CCG​ATT​TTG​GCA​CTA​GC-3′; R: 5′- CCA​TAT​TGG​CAC​TGC​ACA​CC-3′) was analyzed by fluorescent quantitative PCR. CCA​TAT​TGG​CAC​TGC​ACA​TGA-3′) was quantified [27].

Dual-Luciferase Reporter Gene Assay

To verify the transcriptional regulation of miR-96-5p by Sox9, the miR-96-5p 3′UTR gene fragment was synthesized artificially using the luciferase reporter method and introduced into the reporter plasmid pMIR-reporter (Beijing Hua Yueyang Biotechnology Co., Ltd., Beijing, China) using endonuclease sites SpeI and Hind III. The seed sequence was designed on the miR-96-5p wild type. Complementary sequence mutation sites were designed on the miR-96-5p wild type. The target fragment was inserted into the pMIR-reporter reporter plasmid by restriction endonuclease digestion using T4 DNA ligase. The correctly sequenced luciferase reporter plasmids WT and MUT were cotransfected with Sox9 into HEK-293T cells, and the cells were collected after 48 h. The proteins were extracted and assayed for luciferase activity using a luciferase assay kit (K801-200, Biovision), and a Lomax 20/20 luminometer fluorescence detector (Promega) was used to detect luciferase activity. To verify the targeting relationship between miR-96-5p and Trib3, the Trib3 3′UTR gene fragment was synthesized artificially, the complementary sequence mutation site of the seed sequence was designed on the wild type of Trib3 and WT, and MUT was cotransfected with miR-96-5p into HEK-293T cells [28] respectively. Subsequent experiments were performed as above, and each set of experiments was repeated three times.

Western Blot

Groups of cells in culture were collected by trypsin digestion and lysed with an enhanced RIPA lysis solution containing protease inhibitors, followed by protein concentration determination using a BCA protein quantification kit. 10% SDS-PAGE separated the proteins, and the separated proteins were electrotransferred to PVDF membranes, and 5% BSA was closed at room temperature for 1 h. Diluted primary antibody Sox9 (ab185966, 1:1,000, Rabbit, Abcam, UK), Trib3 (ab75846, 1:1,000, Rabbit, Abcam. UK), KIM-1 (#14971, 1:1,000, Rabbit, Cell Signaling Technology, USA), NGAL (A2092, 1:1,000, Rabbit, ABclonal, China), with GAPDH antibody (ab8245, 1:5,000, Mouse, Abcam, UK) as an internal reference, incubated overnight at 4°C, the membrane was washed 3 times with TBST, HRP-labeled secondary antibody (goat anti-rabbit, item No. ab205718, 1:10,000; goat anti-mouse, 1:10,000; item No. ab205719, Abcam, UK) was added, incubated for 1 h at room temperature, and ECL working solution (WBULS0100. USAEMD Millipore), incubated for 1 h at room temperature, the membrane was washed 6 times with TBST. ECL chemiluminescence was developed (Shanghai Baumann Biotechnology Co., Ltd., Shanghai, China). Image J [29] analyzed the bands’ gray scale values. The experiment was repeated 3 times.

Statistical Analysis

Statistical analyses of the data in this study were performed using SPSS 21.0 (IBM, USA) statistical software. Measures were expressed as mean ± standard deviation. Normality and χ² tests were first performed for normal distribution and χ². Unpaired t tests were used between groups. One-way ANOVA or ANOVA with repeated measures was used for comparison between multiple groups. Tukey’s post hoc test was performed. p < 0.05 indicates a statistically significant difference.

Results

Sox9 Expression Is Upregulated in Kidney Tissues of Kidney-Injured Mice

GSE192532 and GSE71647, the gene expression microarrays associated with kidney injury, were retrieved from the GEO database, and 305 significantly up-regulated and 311 screened to 419 and 845 differentially expressed genes, respectively, in the kidney injury samples (Fig. 1a, b). Meanwhile, we retrieved 842 transcription factors through the JASPAR database and further intersected the differentially expressed genes with transcription factors to obtain three intersected genes, namely Sox9, Fos, and Fosl1 (Fig. 1c). Several studies have reported an association between Sox9 and kidney injury [30, 31]. Furthermore, GEO microarray analysis showed that Sox9 expression was significantly upregulated in the mouse model of kidney injury compared to normal controls (Fig. 1d, e). Therefore, we selected Sox9 as the target gene for subsequent experiments.

Fig. 1.

Bioinformatics analysis and in vivo animal studies to screen for the target gene Sox9. a Volcano plots of differentially expressed mRNAs screened by GSE192532 microarray (Sham group, n = 3; Model group, n = 3). b Volcano plots of differentially expressed mRNAs screened by GSE71647 microarray (Sham group, n = 2; Model group, n = 2). c Venn diagram of GSE192532 and GSE71647 microarray analysis. c Venn diagram of the intersection of GSE192532 and GSE71647 microarray results with the JASPAR database search results. d GSE192532 microarray data-based analysis of differential expression of Sox9 in normal control and kidney-injured mouse model groups (Sham group, n = 3; Model group, n = 3). e GSE71647 microarray data-based analysis of differential expression of Sox9 in normal control and kidney-injured mouse model groups (Sox9). e Differential expression of Sox9 in normal control and kidney-injured mice based on GSE71647 microarray data (Sham group, n = 2; Model group, n = 2). f Detection of SCr and urea in mice. g H&E staining for kidney histopathology in both groups (×200, 50 μm). h Toluidine blue staining for kidney inflammation in both groups (×400, 25 μm). i ELISA for expression of TNF-α, IL-1β, and IL-6 in the urine of two groups of mice. j RT-qPCR to detect the expression of Sox9 in the kidney tissues of two groups of mice. k Immunohistochemistry to detect the expression of Sox9 in the kidney tissues of two groups of mice (×400, 25 μm); n = 6, *indicates p < 0.05 compared to the sham group.

Next, we used LPS-induced male C57BL/6 mice to construct a mouse model of kidney injury and performed kidney function tests on the Sham and Model groups of mice. As shown in Figure 1f, the SCr, and BUN in the Model group were significantly higher than in the Sham group. In addition, the renal histopathology of the Sham and Model mice was examined by H&E staining. The results showed that the renal tissues of the Model mice exhibited severe renal pathological damage, including inflammatory cell infiltration and severe tubular vacuolar degeneration, compared to the Sham group (Fig. 1g). In addition, toluidine blue staining showed a significant increase in the number of degranulated mast cells and a significant increase in the inflammatory index in the kidney tissue of the Model mice compared to the Sham group (Fig. 1h).

We further used ELISA to detect the expression of TNF-α, IL-1β, and IL-6 in the urine of mice in the Sham and Model groups. The results showed that the expression of TNF-α, IL-1β, and IL-6 in the urine of mice in the Model group was significantly increased compared to the Sham group (Fig. 1i). In addition, RT-qPCR and immunohistochemical assays showed that Sox9 was significantly more highly expressed in the Model group compared to the Sham group (Fig. 1j, k). The above results indicate that our LPS-induced kidney injury model in mice was successfully constructed, and the kidney tissues of kidney-injured mice showed significant inflammatory responses.

Sox9 Could Promote Kidney Injury in the LPS-Induced HK-2 Cell Model by Downregulating the Expression of miR-96-5p

It has been reported that Sox9 inhibits the transcription of miR-96-5p and thereby downregulates its expression [32]. Also, miR-96-5p was significantly under-expressed in a mouse kidney injury model [33]. Therefore, we hypothesize that Sox9 plays a role in kidney injury by inhibiting the expression of miR-96-5p.

To further investigate the regulatory relationship between Sox9 and miR-96-5p in kidney injury, we examined the expression of miR-96-5p in the kidney tissues of Sham and Model groups of mice using RT-qPCR. As shown in Figure 2a, miR-96-5p was significantly low expressed in the kidney tissues of the Model group mice. The enrichment of Sox9 in the promoter region of miR-96-5p was detected in HEK-293T cells using ChIP assay (Fig. 2b). The reporter plasmid with miR-96-5p promoter region sequence and overexpression of Sox9 or null plasmid were cotransfected into cells HEK-293T. The transcriptional regulatory activity of Sox9 on miR-96-5p was assayed using a dual-luciferase reporter gene assay. As shown in Figure 2c, Sox9 inhibited the promoter activity of miR-96-5p.

Fig. 2.

Sox9 regulates miR-96-5p to affect LPS-induced kidney injury. a RT-qPCR assay of miR-96-5p expression in kidney tissues of Sham and Model groups of mice. b ChIP assay of Sox9 enrichment in the promoter region of miR-96-5p in HEK-293T cells. c Dual-luciferase reporter gene assay of Sox9 in HEK-293T cells on miR-96-5p transcriptional regulatory activity in HEK-293T cells. d Sox9 and miR-96-5p expression by RT-qPCR after silencing or overexpression of Sox9 in HEK-293T cells. e ELISA to detect the expression of IL-6, TNF-α, and IL-1β in the supernatant of HK-2 cells in Control and LPS groups. f Western blot to detect KIM-1 and NGAL, an index related to kidney injury in HK-2 cells of Control and LPS groups. g RT-qPCR and Western blot to detect the expression of Sox9 and miR-96-5p in HK-2 cells of Control and LPS groups. h RT-qPCR and Western blot for the expression of Sox9 and miR-96-5p in HK-2 cells of each group; *indicates p < 0.05; ^#indicates p < 0.05.

Next, after silencing or overexpressing Sox9 in HEK-293T cells, the expression of Sox9 and miR-96-5p was detected by RT-qPCR. As shown in Figure 2d, the silencing effect of sh-Sox9-2 was the best and could be used for subsequent experiments; after knocking down the expression of Sox9, the expression of miR-96-5p also increased; when the expression of Sox9 was upregulated, the expression of miR-96-5p decreased significantly. The above results suggest that Sox9 could bind to the promoter region of miR-96-5p to inhibit its transcriptional expression.

To further investigate whether Sox9 influences the progression of kidney injury by inhibiting the expression of miR-96-5p, we constructed an in vitro cell model of kidney injury using LPS-induced human normal epithelial cells HK-2. We divided the cells into Control and LPS groups. The expression of IL-6, TNF-α, and IL-1β in the supernatant of the LPS group was significantly higher than that of the Control group (Fig. 2e). In addition, the expression levels of KIM-1 and NGAL were significantly higher in the LPS group than in the Control group (Fig. 2f). The results indicated that we successfully constructed an in vitro cell model of kidney injury using LPS induction.

Further, RT-qPCR and Western blot were used to detect the expression of Sox9 or miR-96-5p in the cells of the Control and LPS groups. As shown in Figure 2g, the expression of Sox9 in the LPS group was higher than that in the Control group, while the expression of miR-96-5p was significantly lower than that in the Control group. Next, we intervened in the expression of Sox9 and miR-96-5p in an in vitro cell model of kidney injury, dividing the cells into LPS+sh-NC group, LPS+sh-Sox9 group, LPS+sh-Sox9+NC inhibitor group, and LPS+sh-Sox9+miR-96-5p inhibitor group. RT-qPCR and Western blot assays showed that, compared with the LPS+sh-NC group, the expression of Sox9 was decreased in the cells of the LPS+sh-Sox9 group. The expression of miR-96-5p was increased in the cells of the LPS+sh-Sox9+NC inhibitor group, compared with the LPS+sh-Sox9+miR-96-5p The expression of Sox9 was unchanged, and the expression of miR-96-5p was significantly downregulated in the inhibitor group (Fig. 2h).

Downregulation of Sox9 Can Inhibit LPS-Induced Pro-Inflammatory Response in an HK-2 in vitro Cell Model by Upregulating the Expression of miR-96-5p

The ELISA results showed that the expression of IL-6, TNF-α, and IL-1β was decreased in the cells of the LPS+sh-Sox9 group compared to the LPS+sh-NC group. The expression of IL-6, TNF-α, and IL-1β was increased in the cells of the LPS+sh-Sox9+NC inhibitor group compared to the LPS+sh-Sox9+miR-96-5p inhibitor group (Fig. 3a). IL-6, TNF-α, and IL-1β expression was increased in the LPS+sh-Sox9+NC inhibitor group compared to the LPS+sh-Sox9+miR-96-5p inhibitor group (Fig. 3b). The expression of KIM-1 and NGAL, an indicator related to kidney injury, was detected in each group of cells using the kit. The results showed that KIM-1 and NGAL were reduced in cells of the LPS+sh-Sox9 group compared to the LPS+sh-NC group and in cells of the LPS+sh-Sox9+NC inhibitor group compared to the LPS+sh-Sox9+miR-96-5p inhibitor group cells showed elevated expression of KIM-1 and NGAL.

Fig. 3.

The effect of Sox9 knockdown on miR-96-5p expression and LPS-induced inflammation in an in vitro HK-2 cell model. a ELISA for the expression of IL-6, TNF-α, and IL-1β in the supernatant of HK-2 cells of each group. b Western blot for the expression of KIM-1 and NGAL, an index related to kidney injury in HK-2 cells of each group; *indicates p < 0.05; ^#indicates p < 0.05.

miR-96-5p Attenuates LPS-Induced Renal Injury through Targeted Inhibition of Trib3

Next, we used TargetScan and the miRDB database to predict the downstream target genes of miR-96-5p. Finally, we intersected the database predictions with the genes significantly highly expressed in the GSE192532 chip to obtain one intersected gene: Trib3 (Fig. 4a).

Fig. 4.

miR-96-5p regulates Trib3 expression to affect LPS-induced kidney injury. a Venn diagram of the intersection of TargetScan and miRDB database prediction results and GSE192532 microarray analysis results. b RT-qPCR detection of Trib3 expression in kidney tissues of mice in Sham and Model groups. c Immunohistochemistry detection of Trib3 expression in kidney tissues of mice in Sham and Model groups. d RT-qPCR for Trib3 expression in HK-2 cells from Control and LPS groups. e Western blot for Trib3 expression in HK-2 cells from Control and LPS groups. f Dual-luciferase reporter gene assay to verify the relationship between miR-96 5p target binding to Trib3. g RT-qPCR to detect the expression of miR-96-5p and Trib3 in HEK-293T cells after silencing or overexpression of miR-96-5p. h Western blot to detect the expression of Trib3 in HEK-293T cells after silencing or overexpression of miR-96-5p. i RT-qPCR to detect the expression of miR-96-5p and Trib3 in each group of HK-2 cells. j Western blot to detect the expression of Trib3 in each group of HK-2 cells. k ELISA to detect the expression of IL-6, TNF-α, and IL-1β in the supernatant of each group of HK-2 cells. l Western blot to detect the expression of KIM-1 and NGAL, the indexes related to kidney injury in each group of HK-2 cells; *compared with the control group (Sham group; Control group; NC mimic group; LPS+NC mimic group), p < 0.05; ^#compared with the NC inhibitor group or LPS+miR-96-5p mimic+oe-NC group, p < 0.05.

The expression of Trib3 was further validated in an in vivo animal model of kidney injury and in vitro cellular model. Figure 4b–e shows that Trib3 was significantly overexpressed in LPS-induced kidney injury mouse cells. A dual-luciferase reporter gene assay was used to verify the targeted binding of miR-96-5p to Trib3 in HEK-293T cells. The results showed that the miR-96-5p mimic had no significant effect on the intensity of luciferase activity in the Trib3 mutant group compared to the NC mimic group. Still, the intensity of luciferase activity in the Trib3 wild-type group was significantly downregulated (Fig. 4f). In addition, after silencing or overexpressing miR-96-5p in HEK-293T cells, RT-qPCR, and Western blot were used to detect the expression of miR-96-5p and Trib3. As shown in Figures 4g, h, the expression of Trib3 was significantly upregulated after inhibition of miR-96-5p expression and significantly downregulated after overexpression of miR-96-5p. The above results suggest that miR-96-5p could target and inhibit the expression of Trib3.

To further verify whether miR-96-5p affects the progression of kidney injury by targeting and regulating the expression of Trib3, we intervened in the expression of miR-96-5p and Trib3 in an in vitro cell model of LPS-induced kidney injury. We divided the cells into the LPS+NC mimic group, LPS+miR-96-5p mimic group, LPS+miR-96-5p mimic+oe-NC group, and LPS+miR-96-5p mimic+oe-Trib3 group. RT-qPCR and Western blot assays showed that, compared with the LPS+NC mimic group, the expression of miR-96-5p was increased in the LPS+miR-96-5p mimic group. The expression of Trib3 In the LPS+miR-96-5p mimic+oe-NC group, miR-96-5p expression was not significantly changed, and Trib3 expression was significantly increased in the LPS+miR-96-5p mimic+oe-Trib3 group compared to the LPS+miR-96-5p mimic+oe-NC group (Fig. 4i, j).

In addition, ELISA was performed to detect the expression of IL-6, TNF-α, and IL-1β in the cell supernatant of each group. The results showed that the expression of IL-6, TNF-α, and IL-1β was decreased in the LPS+miR-96-5p mimic group compared to the LPS+NC mimic group and in the LPS+miR-96-5p mimic+oe-NC group compared to the LPS+miR-96-5p mimic+oe-NC group. The expression of IL-6, TNF-α, and IL-1β was significantly higher in the LPS+miR-96-5p mimic+oe-Trib3 group (Fig. 4k). Western blot detected the expression of KIM-1 and NGAL, an indicator related to kidney injury, in each group, and the results showed that compared to the LPS+NC mimic group, the expression of IL-6, TNF-α, IL-1β in the LPS+miR-96-5p group was increased. The expression of KIM-1 and NGAL was decreased in the LPS+miR-5p mimic group compared to the LPS+miR-5p mimic+oe-NC group, and was significantly increased in the LPS+miR-96-5p mimic+oe-Trib3 group compared to the LPS+miR-5p mimic+oe-NC group (Fig. 4l).

Trib3 Exacerbates LPS-Induced Inflammatory Responses in Renal Tubular Cells by Upregulating IL-6 Expression

It has been shown that Trib3 promotes IL-6 production [34] and that activation of the IL-6 signaling pathway promotes inflammation [35]. Therefore, we hypothesized that Trib3 could promote inflammation in kidney injury by promoting the expression of IL-6.

To test the above conjecture, we examined the expression of IL-6 in mouse models of kidney injury and cellular models. As shown in Fig. 5a–d, the results showed that IL-6 was significantly more highly expressed in the in vivo animal model of kidney injury and the in vitro cellular model, in the same way as Trib3 was expressed in the in vivo animal model of kidney injury and the in vitro cellular model.

Fig. 5.

Trib3 regulation of IL-6 expression affects LPS-induced inflammatory responses in renal tubular epithelial cells. a RT-qPCR to detect IL-6 expression in kidney tissues of mice in Sham and Model groups. b Immunohistochemistry to detect IL-6 expression in kidney tissues of mice in Sham and Model groups (×400, 25 μm). c RT-qPCR to detect IL-6 expression in HK-2 cells of Control and LPS groups. d ELISA to detect the expression of IL-6 in the supernatant of HK-2 cells in Control and LPS groups. e RT-qPCR and Western blot to verify the silencing effect of Trib3 in HEK-293T cells. f RT-qPCR to detect the expression of Trib3 and IL-6 in each group of HK-2 cells. g Western blot to detect the expression of Trib3 in each group of HK-2 cells. h ELISA to detect the expression of IL-6, TNF-α, and IL-1β in the supernatant of each group of HK-2 cells. i Western blot to detect the expression of KIM-1 and NGAL, kidney injury-related factors in each group of HK-2 cells; *indicates p < 0.05; *compared with the control group (Sham group; Control group; sh-NC group; LPS+sh-NC group), p < 0.05; ^#compared to the LPS+sh-Trib3 group, p < 0.05.

To further verify this regulatory relationship, we first infected HEK-293T cells with two silencing sequences of Trib3 lentivirus. Then, RT-qPCR and Western blot verified the silencing effect of Trib3. The results showed that sh-Trib3-2 had the best silencing effect (Fig. 5e). Therefore, we used the sh-Trib3 lentivirus with the best silencing effect and human recombinant IL-6 in HK-2 cells treated with LPS intervention and divided the cells into the LPS+sh-NC group, the LPS+sh-Trib3 group, and the LPS+sh-Trib3+rh-IL-6 group. RT-qPCR and Western blot assay showed that inhibition of Trib3 expression was followed by downregulation of IL-6 expression, and when IL-6 increased, Trib3 expression was unchanged (Fig. 5f, g).

ELISA measured the expression of IL-6, TNF-α, and IL-1β in the supernatant of each group of cells, and the results showed that the expression of IL-6, TNF-α, and IL-1β decreased after silencing Trib3, while overexpression of IL-6 reversed the down-regulation of IL-6, TNF-α, and IL-1β caused by Trib3 silencing (Fig. 5h). In addition, Western blot assayed the expression of KIM-1 and NGAL, associated with kidney injury, in each group of cells. The results showed that the silencing of Trib3 decreased the expression of KIM-1 and NGAL, while overexpression of IL-6 reversed the silencing effect of Trib3 (Fig. 5i).

Knockdown of Sox9 Mediates the miR-96-5p/Trib3/IL-6 Signaling Axis to Attenuate LPS-Induced Inflammatory Responses in Renal Tubular Epithelial Cells

To further demonstrate that Sox9 mediates the miR-96-5p/Trib3/IL-6 signaling axis affecting LPS-induced renal injury, we treated HK-2 cells after LPS intervention and divided the cells into the LPS+sh-NC group, the LPS+sh-Sox9 group, and the LPS+sh-Sox9+rh-IL-6 group. RT-qPCR and Western blot showed that knockdown of Sox9 resulted in down-regulation of Sox9 expression, upregulation of miR-96-5p expression, and decreased expression of Trib3 and IL-6, while upregulation of IL-6 expression resulted in unchanged expression of Sox9, miR-96-5p, and Trib3 and increased expression of IL-6 (Fig. 6a, b). In addition, ELISA results showed that the expression of TNF-α, IL-1β, and IL-6 in the cell supernatant was significantly reduced after overexpression of Sox9. In contrast, overexpression of IL-6 promoted the expression of TNF-α, IL-1β, and IL-6 (Fig. 6c). Western blot assay of the renal injury-related factors KIM-1 and NGAL in each group of cells showed that knockdown of Sox9 decreased the expression of KIM-1 and NGAL. In contrast, overexpression of IL-6 reversed the Sox9-induced upregulation of KIM-1 and NGAL (Fig. 6d).

Fig. 6.

Knockdown of Sox9 regulates the miR-96-5p/Trib3/IL-6 signaling axis to influence the inflammatory response to LPS-induced kidney injury. a RT-qPCR for the expression of Sox9, miR-96-5p, Trib3, and IL-6 in each group of HK-2 cells. b Western blot for the expression of Sox9 and Trib3 in each group of HK-2 cells. c ELISA for the expression of TNF-α, IL-1β, and IL-6 in the supernatant of each group of HK-2 cells. d Western blot for the expression of KIM-1 and NGAL in each group of HK-2 cells. *indicates p < 0.05; *compared with the LPS+sh-NC group, p < 0.05; ^#compared with the LPS+sh-Sox9 group, p < 0.05.

Knockdown of Sox9 Regulates the miR-96-5p/Trib3/IL-6 Signaling Axis to Attenuate LPS-Induced Renal Injury

To further reveal the mechanism of Sox9 regulation of miR-96-5p/Trib3/IL-6 signaling axis involved in the occurrence and development of kidney injury, we injected sh-Sox9 lentivirus and IL-6 intraperitoneally into kidney-injured mice. We divided the mice into the Sham group, model+sh-NC group, model+sh-Sox9 group, and model+sh-Sox9+IL-6 group. The results of RT-qPCR and Western blot showed that the expression of Sox9 was downregulated, the expression of miR-96-5p was increased, and the expression of Trib3 and IL-6 was decreased in the sh-Sox9 group compared with the sh-NC group; compared with the sh-Sox9 group, the expression of Sox9, miR-96-5p, and Trib3 was decreased in the sh-Sox9+IL-6 group. 5p and Trib3 expression was unchanged and IL-6 expression was increased in the sh-Sox9+IL-6 group (Fig. 7a, b).

Fig. 7.

Sox9 regulates the miR-96-5p/Trib3/IL-6 signaling axis to promote kidney injury. a RT-qPCR to detect the expression of Sox9, miR-96-5p, Trib3, and IL-6 in the kidney tissue of each group of mice. b Western blot to detect the expression of Sox9 and Trib3 in the kidney tissue of each group of mice. c kits to detect the levels of serum creatinine (SCr) and urea nitrogen (BUN) in mice. d H&E staining to detect kidney tissue pathology in mice (×200, 50 μm). e Toluidine blue staining to detect inflammation in mouse kidney tissue (×400, 25 μm). f ELISA to detect the expression of TNF-α, IL-1β, and IL-6 in mouse urine. g Immunohistochemistry to detect the expression of KIM-1 and NGAL, indicators related to kidney injury, in mouse kidney tissue (×400, 25 μm); *indicates p < 0.05; *compared with the Sham group, p < 0.05; ^#compared with the model+sh-NC group, p < 0.05; ^&compared to a model+sh-Sox9 group, p < 0.05.

Next, SCr and BUN were measured in each group of mice, and the results showed that: SCr and BUN were significantly higher in the sh-Sox9 group compared to the sh-NC group; SCr and BUN were significantly lower in the oe-Sox9+IL-6 group compared to the oe-Sox9 group (Fig. 7c). Furthermore, H&E staining showed that the extent of kidney tissue lesion was reduced after overexpression of Sox9, while the extent of kidney tissue lesion was increased again after upregulation of IL-6 expression (Fig. 7d). In addition, toluidine blue staining showed that the degranulated mast cells in the kidney tissues of the sh-Sox9 group were significantly reduced. As a result, the inflammation index was significantly lower than in the sh-NC group. In comparison, the degranulated mast cells in the kidney tissues of the sh-Sox9+IL-6 group were significantly increased, and the inflammation index was significantly higher compared to the sh-Sox9 group (Fig. 7e).

ELISA was performed to detect the expression of TNF-α, IL-1β, and IL-6 in the urine of mice. The results showed that the expression of TNF-α, IL-1β, and IL-6 in the urine of mice in the sh-Sox9 group was significantly lower compared to the sh-NC group. The expression of TNF-α, IL-1β, and IL-6 in the urine of mice in the sh-Sox9+IL-6 group was significantly higher compared to the sh-Sox9 group (Fig. 7f). In addition, the expression of TNF-α, IL-1β, and IL-6 in the urine of the sh-Sox9+IL-6 group was significantly higher (Fig. 7f). In addition, immunohistochemistry was performed to detect the positive expression of KIM-1 and NGAL in the kidney tissues of mice. The results showed that the expression of KIM-1 and NGAL in the kidney tissues of mice in the sh-Sox9 group was significantly lower than in the sh-NC group. In comparison, the expression of KIM-1 and NGAL in the kidney tissues of mice in the sh-Sox9+IL-6 group was significantly higher than in the sh-Sox9 group (Fig. 7g).

Discussion

We found through bioinformatic analysis and in vivo zoological experiments that Sox9 expression was up-regulated in kidney tissues of kidney-injured mice. The results suggest that Sox9 molecules may play an important role in the kidney injury and repair. Previous studies have shown that Sox9 is induced by renal fibroblast TGF-β and is an important downstream mediator of the TGF-β signaling pathway for renal fibrosis [36]. Another single-cell sequencing study found high expression of Sox9 in kidney injury tissue [13]. A recent study also showed an increase in the number of Sox9-positive cells after kidney injury and the vicinity of inflammatory cell infiltration [37]. Studies of kidney injury (renal injury) and renal fibrosis in rats support the finding in mouse models that persistent expression of Sox9 is associated with fibrosis after kidney injury [14].

We confirmed by in vitro cellular assays that the knockdown of Sox9 attenuated LPS-induced kidney injury by upregulating miR-96-5p expression. The present results suggest that miR-96-5p is closely associated with kidney injury and that miR-96-5p is a downstream gene of Sox9. miR-96-5p is closely associated with kidney injury and is downregulated in a mouse model of hypoxia-induced kidney injury [33]; furthermore, an increase in miR-96-5p expression was observed in patients with recovered kidney function after kidney injury. In addition, an increase in miR-96-5p expression level was observed in patients who recovered from renal injury. Sox9 has been reported to bind to the miR-96-5p promoter region, thereby inhibiting miR-96-5p expression and ultimately exacerbating sepsis-induced myocardial injury [32].

Further bioinformatic analysis and in vivo and in vitro experiments confirmed that miR-96-5p could attenuate LPS-induced kidney injury by targeting and inhibiting Trib3. The results suggest that Trib3 is a key downstream target gene of miR-96-5p and that Trib3 is involved in LPS-induced renal injury. miR-96-5p (formerly known as miR-96) regulates vascular smooth muscle cell contraction, colon cancer development, and other processes by affecting Trib3 expression [38, 39]. Trib3 is an apoptosis-associated gene that increases expression in chronic kidney disease [40]. In addition, renal fibrosis is a common form of kidney injury, and it was found that knocking down Trib3 significantly improved renal fibrosis by reducing ERK phosphorylation levels [41]. In addition, we found that Trib3 exacerbated LPS-induced inflammatory responses in renal tubular cells by up-regulating IL-6 expression.

This result suggests that IL-6 is involved in LPS-induced inflammatory responses in renal tubular cells as a downstream effector molecule of Trib3. IL-6 and Trib3 are upregulated in various inflammatory diseases such as chronic kidney disease and polytrauma [40, 42], and their relationship has been previously reported, with one study showing that Trib3 gene polymorphism is strongly associated with circulating IL-6 levels [43]. Another study showed that the knockdown of Trib3 reduced serum levels of inflammatory factors such as IL-6 and improved diabetic nephropathy [44]. In addition, it has also been reported that IL-6 promotes Trib3 expression through the Arid5a/AU021063 axis, suggesting that Trib3/IL-6 is a positive feedback loop and that activation of this loop could exacerbate the renal injury and trap it in a vicious circle [45].

Finally, in vitro cellular assays and in vivo animal experiments confirmed that the knockdown of Sox9 mediated the miR-96-5p/Trib3/IL-6 signaling axis to attenuate LPS-induced inflammatory responses in renal tubular epithelial cells and kidney injury in mice. This result suggests that Sox9 regulates renal injury by modulating the miR-96-5p/Trib3/IL-6 signaling axis. A recent study also showed that the number of Sox9-positive cells increased after kidney injury and in the vicinity of inflammatory cell infiltration [37]. Sox9 expression is elevated in kidney-injured cells, and Sox9 inhibits miR-96-5p expression by binding to the miR-96-5p promoter region, promoting NLRP3 expression, and exacerbating the sepsis-induced myocardial injury and myocardial cell death [32]. In contrast, TRIB3 siRNA knockdown ameliorated renal fibrosis with reduced ERK phosphorylation [41]. Silencing of TRB3 by siRNA reduced serum levels of cytokines, including TNF-α, IL-1β, and IL-6 epitopes. Furthermore, it alleviated diabetic nephropathy symptoms, indicating that TRB3 silencing significantly ameliorated nephropathy in diabetic rats [44]. In this study, we established that miR-96-5p/Trib3/IL-6 signaling axis molecules are jointly involved in the renal inflammatory injury, thus establishing that low expression level of Sox9 mediates miR-96-5p/Trib3/IL-6 signaling axis to alleviate LPS-induced inflammatory response in renal tubular epithelial cells and mouse kidney injury model, adding a new therapeutic direction for renal inflammatory injury.

Conclusion

Summing up the bioinformatics, in vitro cellular assays, and in vivo animal experiments, we could draw the following preliminary conclusions: Sox9 activates the IL-6 signaling pathway and exacerbates the inflammatory response by inhibiting the transcriptional upregulation of the downstream target gene Trib3 expression through miR-96-5p, ultimately promoting the development and progression of kidney injury (Fig. 8). This study thoroughly examined how the transcription factor Sox9 regulates the signaling pathway of miR-96-5p/Trib3/IL-6 in the molecular mechanism of kidney injury development. The study provides a potential strategy for preventing and treating kidney injury. Kidney injury commonly occurs in various clinical settings, such as severe infections, surgery, trauma, and numerous chronic diseases, such as hypertension and diabetes. Effective drugs for treating kidney injury are currently scarce, exacerbating the poor prognosis of this condition and further burdening the medical system. Furthermore, this study establishes a new molecular mechanism that deepens our comprehension of the development and occurrence of kidney injury. These outcomes guide research other kidney diseases, including renal fibrosis, nephritis, and renal cancer. Subsequent studies can use the results of this study as a foundation to explore the potential mechanisms involved in other kidney diseases.

Fig. 8.

Diagram of the mechanism of Sox9-mediated miR-96-5p/Trib3/IL-6 signaling axis in the development and progression of kidney injury.

Of course, the present study has the following limitations. First, the kidney injury models used in this study were LPS-induced C57BL/6 mice and human normal tubular epithelial cells HK-2; there are many causes of kidney injury, such as renal ischemia-reperfusion injury and calcium oxalate stones. This study did not involve other types of kidney injury models, so the role of the Sox9-mediated miR-96-5p/Trib3/IL-6 signaling axis in other types of kidney injury The role of the Sox9-mediated miR-96-5p/Trib3/IL-6 signaling axis in other types of kidney injury needs further investigation. Secondly, the present study investigated the role of Sox9-mediated miR-96-5p/Trib3/IL-6 signaling axis in kidney injury, mainly based on animal and cellular experiments. Although these findings are significant, the role of this signaling axis in human kidney injury needs further validation due to the need for more direct evidence from clinical patients with kidney injury. Finally, although this study found that the knockdown of Sox9 improved the inflammatory response and extent of injury in mouse kidney tissue and HK-2 cells, the safety and efficacy of this strategy in the clinical application need to be further evaluated.

Statement of Ethics

All the experiments were approved by Fuyang People’s Hospital, Anhui Medical University (approval number: 2023008/date: May 05, 2022) and in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Adequate measures were taken to minimize suffering of the included animals.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This study was supported by the Jiangxi Provincial Natural Science Foundation, Key Project (20212ACB206023).

Author Contributions

Xiao Wang, Guang Chen, and Yongqiang Du wrote the paper and conceived and designed the experiments; Jiajia Yang and Wei Wang analyzed the data; and Xiao Wang and Guang Chen collected and provided the sample for this study. All authors have read and approved the final submitted manuscript.

Funding Statement

This study was supported by the Jiangxi Provincial Natural Science Foundation, Key Project (20212ACB206023).

Data Availability Statement

Data are not publicly available due to ethical reasons. Further inquiries can be directed to the corresponding author.