Cell cultivation and treatment

Human renal tubular epithelial cells (HK-2) were purchased from Shanghai Institute of Cell Research, Chinese Academy of Sciences. HK-2 cells were cultivated in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin at 37°C with 5% CO₂. Cell model of sepsis was induced by lipopolysaccharide (LPS). HK-2 cells were treated with 1μg/mL LPS (Sigma Aldrich, St Louis, MO, USA) for 24h and then seeded into a culture plate. The cells were incubated in the plate for 16h and then subjected to transfection.

Apart from the control group, LPS-induced HK-2 cells were accordingly divided into the following groups: LPS group, LPS+inhibitor group (transfected with 30nM miR-34b-5p inhibitor), LPS+inhibitor NC group (transfected with the negative control of miR-34b-5p inhibitor), LPS+mimic group (transfected with 30nM miR-34b-5p mimic), LPS+mimic NC group (transfected with the negative control of miR-34b-5p mimic), LPS+pcDNA3.1 group (transfected with empty pcDNA3.1), LPS+pcDNA3.1-AQP2 group (transfected with 2μg of pcDNA3.1-AQP2), LPS+si-AQP2 group (transfected with 2μg of si-AQP2), LPS+si-NC group (transfected with the negative control of si-AQP2), LPS+inhibitor NC+si-NC group (co-transfected with the negative controls of miR-34b-5p inhibitor and si-AQP2), LPS+inhibitor+si-NC group (co-transfected with miR-34b-5p inhibitor and the negative control of si-AQP2), and LPS+inhibitor+si-AQP2 group (co-transfected with miR-34b-5p inhibitor and si-AQP2). All plasmids were obtained from GenePharma (Shanghai, China) and transfected into the cells via LipoFiter^TM (Hanbio, Shanghai, China). The transfection experiment was performed in triplicate and the following experiments were conducted 24h after the transfection.

Dual-luciferase reporter assay

The binding site between miR-34b-5p and AQP2 was predicted by the online software Jefferson (https://cm.jefferson.edu/rna22/Precomputed/OptionController?species=HomoSapiens&type=mRNA&version=MB21E78v2). Wild and mutated sequences of the binding site on AQP2 (mut-AQP2 and wt-AQP2) were designed and synthesized according to the prediction. The sequences were inserted into vectors (pGL3-Basic, Promega, MADISON, WI, USA) and then co-transfected with miR-34b-5p mimic or the corresponding negative control into HEK293T cells. Luciferase intensity in the cells was tested using a dual-luciferase reporter kit (Promega) for determining the binding between miR-34b-5p and AQP2. The HEK293T cells were divided into mimic+mut-AQP2 group, mimic+wt-AQP2 group, mimic NC+mut-AQP2 group, and mimic NC+wt-AQP2 group. The assay was performed in triplicate.

qRT-PCR

TRIzol reagents (Invitrogen, Carlsbad, CA, USA) were used to extract total RNA from blood samples and HK-2 cells. The extraction process of the total RNA from blood samples was as follows. An RNase-free centrifuge tube was added with 0.5mL of blood and 1.5mL of TRIzol reagent, and maintained at a still position at room temperature after vibration. The mixture was centrifuged at 4°C, 12,000g/min for 15min. Supernatant obtained after the centrifugation was transferred to another RNase-free centrifuge tube using a pipette. This tube was added with chloroform (TRIzol:chloroform = 5:1), and placed at a still position for 3min after 15s of vortex. After that, the mixture was centrifuged at 4°C, 12,000g/min for 15min. Supernatant obtained (450μL) and pre-chilled isopropanol (TRIzol:isopropanol = 2:1) were added into a clean 1.5-mL RNase-free centrifuge tube. The well-mixed liquid was maintained stationary at room temperature for 10min, followed by centrifugation at 4°C, 12,000g/min for 15min. Supernatant was carefully removed, and the remaining sediments were mixed with 1mL of 75% ethanol prepared with DEPC (diethyl pyrocarbonate)-treated water, followed by centrifugation at 4°C, 8000g/min for 5min. Supernatant obtained after the centrifugation was discarded, and the tube was put upside down on a clean filter paper for a while. After ethanol volatilization for 2–3min, the tube was added with 15μL of DEPC-treated water. The mixture was treated with a water bath at 55–60°C for 10min. HK-2 cells collected for RNA extraction were centrifuged at 800 r/min for 3–5min. The culture medium was discarded, and the cells were mixed with 1mL of TRIzol reagent. The cell suspension was transferred to a dry EP tube that had been soaked in DEPC-treated water. The tube was placed on ice for 10min. The remaining process was the same as the extraction of total RNA from blood samples.

Concentration and purity of the RNA samples were measured after the extraction. Qualified RNA samples were adjusted to an appropriate concentration and then reverse-transcribed by reverse transcription kits (TaKaRa, Tokyo, Japan) using random primers. LightCycler 480 (Roche, Indianapolis, IN, USA) was used to detect gene expressions, and the experiments were carried out based on the instruction of SYBR Green Mix (Roche Diagnostics, Indianapolis, IN) as described below: 5min at 95°C followed by 40 cycles of 10s at 95°C, 10s at 60°C, and 20s at 72°C. Quantitative PCR of each RNA sample was performed in triplicate. U6 and GAPDH acted as the internal references of miRNA and mRNA, respectively. The 2^−ΔΔCt method was used for data analysis. Sequences of the primers are exhibited in Table 1.

Western blot

HK-2 cells were digested by trypsin and centrifuged at 1,000rpm for 5min. Supernatant was discarded and the cells were washed twice with pre-cooled phosphate buffered saline (PBS). The PBS was then discarded, and the petri dish was added with an appropriate volume of RIPA buffer (Beyotime, Shanghai, China) containing protease inhibitors and gently shaken to let the solution completely cover the bottom of the petri dish. After that, the dish was placed on ice for a few minutes. The cells were scraped from the bottom of the dish using a cell scraper, and the cell lysates were transferred into an EP tube using a pipette. The cell lysis and transfer of cell lysates were operated on ice. The EP tube was placed on ice for 15min. The lysates were centrifuged at 32,000rpm for 15min at 4°C. Supernatant was carefully pipetted into a new EP tube and centrifuged to acquire protein samples. GAPDH acted as the internal reference. The concentration of each protein sample was measured using a BCA kit (Beyotime) to ensure the same loading amount of each sample. The BCA working reagent was made by mixing Reagent A and Reagent B at a ratio of 50:1. The newly prepared BCA working reagent was stable at room temperature for 24h. Concentration of the standards was set to 0, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5mg/ml. The 96-well plate was added with an appropriate volume of protein samples, and each well was added with 200μL of BCA working reagent. The protein samples were incubated at 37°C for 30min. The absorbance value was measured at 562nm by a microplate reader. Concentration of the protein samples was calculated according to the standard curve. After the measurement of concentration, proteins of corresponding volumes were mixed with loading buffer (Beyotime) and denatured in boiling water for 3min. The proteins were separated by 10% SDS-PAGE which was run at 80V until bromophenol blue came into the gel and then continued at 120V for 1–2h. The transfer of proteins was conducted in an ice bath at 300mA for 60min. After that, the membrane was rinsed for 1–2min and then soaked in blocking buffer at room temperature for 60min or at 4°C overnight. The proteins were incubated with primary antibodies of GAPDH (rabbit anti-human, 1:1000, Cell Signaling, Boston, USA), Bax (rabbit anti-human, 1:2000, Abcam, Cambridge, UK), Bcl-2 (rabbit anti-human, 1:800, Abcam), cleaved caspase-3 (rabbit anti-human, 1:1000, Abcam) and AQP2 (rat anti-human, 1:500, Santa Cruz, TX, USA) on a shaking bed for 1h. The membrane was washed for 3×10min and then transferred to horseradish peroxidase-marked secondary antibody solution (goat anti-rabbit IgG, 1:10,000 or goat anti-rat IgG, 1:500) (Thermo Fisher Scientific, MA, USA) to incubate the proteins at room temperature for 1h. After that, the membrane was subjected to another round of washing, and added with color-developing solution. Protein expression was detected by a chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA).

TUNEL staining

HK-2 cells were fixed in 4% paraformaldehyde for 30min and in 70% cold ethanol for 15min. The cells were incubated in PBS containing 0.3% Triton X-100 at room temperature for 5min and then stained in TUNEL solution (Beyotime, Shanghai, China) in dark at 37°C for 60min. After three PBS washes, the cells were mounted with antifluorescence quenching sealing solution and observed under a fluorescence microscope. Cell nuclei were dyed with DAPI. Apoptosis rate = (TUNEL-positive cell number/total cell number) × 100%.

ELISA

The levels of TNF-α, IL-1β, IL-6, SCr, and blood urea nitrogen (BUN) in the serum of septic AKI patients and the levels of TNF-α, IL-1β, and IL-6 in the supernatant of HK-2 cells were measured using ELISA kits (Abcam, Cambridge, UK).

miR-34b-5p promotes LPS-induced renal cell apoptosis and inflammatory response

miR-34b-5p was up-regulated in the LPS group compared to the control group (Figure 2(A), p<0.01). After transfection of miR-34b-5p inhibitor or miR-34b-5p mimic, miR-34b-5p was successfully inhibited or overexpressed in LPS-induced HK-2 cells (Figure 2(B), p<0.05). TUNEL staining showed that LPS increased the apoptosis in HK-2 cells (Figure 2(C), p<0.01, vs the control group). The apoptosis rate was decreased in the LPS+inhibitor group while increased in the LPS+mimic group (Figure 2(C), p<0.01, vs the LPS+inhibitor NC group and LPS+mimic NC group respectively). The expression levels of apoptosis-related proteins (Bax, Bcl-2, and cleaved caspase-3) in HK-2 cells were detected by Western blotting. Bax and cleaved caspase-3 were increased while Bcl-2 was decreased in the LPS group (Figure 2(D), p<0.01, vs the control group). The expression levels of Bax and cleaved caspase-3 were down-regulated in the LPS+inhibitor group while up-regulated in the LPS+mimic group (Figure 2(D), p<0.01, vs the LPS+inhibitor NC group and LPS+mimic NC group, respectively). In contrast, Bcl-2 was increased in the LPS+inhibitor group while decreased in the LPS+mimic group (Figure 2(D), p<0.05, vs the LPS+inhibitor NC group and LPS+mimic NC group, respectively). TNF-α, IL-1β, and IL-6 were increased in the LPS group in comparison with the Control group (Figure 2(E–G), p<0.01). TNF-α, IL-1β, and IL-6 were down-regulated in the LPS+inhibitor group while up-regulated in the LPS+mimic group (Figure 2(E–G), p<0.05, vs the LPS+inhibitor NC group and LPS+mimic NC group, respectively). The above experiment results reveal that miR-34b-5p promotes LPS-induced apoptosis and inflammatory response in HK-2 cells.

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Figure 2.

miR-34b-5p promotes LPS-induced apoptosis and inflammatory response in HK-2 cells. Notes: (A) qRT-PCR detected the expression of miR-34b-5p in LPS-induced HK-2 cells; **P<0.01, compared to the control group. After transfection of miR-34b-5p inhibitor or miR-34b-5p mimic in LPS-induced HK-2 cells, (B) qRT-PCR detected the expression of miR-34b-5p in HK-2 cells; (C) TUNEL staining measured the apoptosis rate of HK-2 cells; (D) Western blotting detected the expressions of Bax, Bcl-2 and cleaved caspase-3 in HK-2 cells; ELISA measured the levels of TNF-α (E), IL-1β (F), and IL-6 (G) in the supernatant of HK-2 cells; scale bar = 50μm; *P<0.05, **P<0.01, ***P<0.001, compared to the control group, LPS+inhibitor NC group or LPS+mimic NC group. LPS: lipopolysaccharide.

AQP2 suppresses LPS-induced renal cell apoptosis and inflammatory response

AQP2 was detected to be down-regulated in LPS-induced HK-2 cells in comparison with the cells in the control group (Figure 3(A,B), p<0.01). AQP2 was up-regulated in LPS-induced HK-2 cells after the transfection of pcDNA3.1-AQP2 (Figure 3(C,D), p<0.001). The LPS+pcDNA3.1-AQP2 group obtained less apoptotic cells than the LPS+pcDNA3.1 group (Figure 3(E), p<0.01). Furthermore, Bax and cleaved caspase-3 were reduced, and Bcl-2 was increased in the LPS+pcDNA3.1-AQP2 group compared to the LPS+pcDNA3.1 group (Figure 3(F), p<0.05). The expression levels of TNF-α, IL-1β, and IL-6 also declined in the LPS+pcDNA3.1-AQP2 group compared to the LPS+pcDNA3.1 group (Figure 3(G–I), p<0.05). From the above, AQP2 can inhibit LPS-induced apoptosis and inflammatory response in HK-2 cells.

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Figure 3.

Overexpressing AQP2 suppresses LPS-induced apoptosis and inflammatory response in HK-2 cells. Notes: qRT-PCR (A) and Western blotting (B) detected the expression of AQP2 in LPS-induced HK-2 cells; **P<0.01, compared to the control group. After transfection of pCDNA3.1-AQP2 in LPS-induced HK-2 cells, qRT-PCR (C) and Western blotting (D) detected the expression of AQP2 in the cells; (E) TUNEL staining measured the apoptosis rate of HK-2 cells; (F) Western blotting detected the expressions of Bax, Bcl-2 and cleaved caspase-3 in HK-2 cells; ELISA measured the levels of TNF-α (G), IL-1β (H). and IL-6 (I) in the cell supernatant; scale bar = 50μm; *P<0.05, **P<0.01, ***P<0.001, compared to the LPS+pcDNA3.1 group. LPS: lipopolysaccharide.

miR-34b-5p targets AQP2

Plasmids inserted with wild and mutated three prime untranslated region (3′-UTR) of AQP2 were constructed for dual-luciferase reporter assay of the binding between miR-34b-5p and AQP2 (Figure 4(A)). Luciferase activities of the mimic+mut-AQP2 group, mimic NC+mut-AQP2 group, and mimic NC+wt-AQP2 group had no significant differences. The mimic+wt-AQP2 group obtained a significantly reduced luciferase activity (Figure 4(B), p<0.01, vs the mimic NC+wt-AQP2 group). qRT-PCR and Western blotting were applied to detect the expression of AQP2 in LPS-induced HK-2 cells after transfection with miR-34b-5p inhibitor or miR-34b-5p mimic. AQP2 was up-regulated in the LPS+inhibitor group while down-regulated in the LPS+mimic group (Figure 4(C,D), p<0.05), indicating that miR-34b-5p negatively regulated AQP2 in HK-2 cells.

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Figure 4.

miR-34b-5p targets AQP2. Notes: (A) 3′UTR of AQP2 where miR-34b-5p binds to; (B) dual-luciferase reporter assay for verifying the targeting relationship between miR-34b-5p and AQP2; **P<0.01, compared to the mimic NC+wt-AQP2 group. qRT-PCR (C) and Western blotting (D) detected the expression of AQP2 in LPS-induced HK-2 cells after transfection of miR-34b-5p inhibitor or miR-34b-5p mimic; *P<0.05, **P<0.01, compared to the LPS+inhibitor NC group or LPS+mimic NC group.

Thanks for all the participants and contributors.