5.1 Sample Collection
All our animal protocols, approved by the Institutional Animal Care and Use Committee of Gansu Agricultural University College of Veterinary Medicine((Ethic approval file NO.GSAU-Eth-VMC-2023-012). We collected kidney samples from healthy, disease-free adult yaks (age = 3 years) at the Qingyuan Slaughterhouse in Gannan Tibetan Autonomous Prefecture, Gansu Province, China; we also collected kidney samples from healthy, disease-free adult yellow cattle (age = 3 years) at a slaughterhouse in Lanzhou City, Gansu Province, China. All kidney samples from yaks and yellow cattle were collected immediately after slaughter.
All kidney tissues were rinsed with sterile physiological saline, transported in a preservation solution to our laboratory, and divided into three portions each. One portion was preserved in 4% paraformaldehyde and then used for immunohistochemistry and immunofluorescence experiments. Another portion was stored in liquid nitrogen and then used for Western blotting (WB) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses. Finally, the third portion was directly placed in sterile physiological saline and immediately sent to the laboratory for primary cell culture and animal cell experiments.
5.2 Tissue Immunohistochemical and Immunofluorescence Staining
Paraffin-embedded fixed yak kidney tissue samples were sliced into 4-μm-thick sections. They were then deparaffinized, dehydrated, and subjected to antigen retrieval. This was followed by blocking and sealing treatment using the SP kit (Bioss, Beijing, China), according to the manufacturer’s instructions. The sections were then incubated with anti-HIF1α (1:400; AF1009; Affinity), the primary antibody, at 4°C overnight; here, phosphate-buffered saline (PBS) was used in the negative control.
For immunohistochemical staining, the sections incubated with the primary antibody were washed with PBS, followed by exposure to the corresponding secondary antibody from the SP kit and incubation at 37°C for 10 min. Next, the sections were exposed to a horseradish peroxidase–labeled avidin working solution at 37°C for 10 min and then stained using DAB. This was followed by counterstaining with hematoxylin and then by dehydration in an increasing alcohol gradient.
For immunofluorescence staining, the sections incubated with the primary antibody were exposed to a fluorescent antirabbit immunoglobulin G Fab2 (1:1000; 8889S; Cell Signaling, Danvers, MA, USA), followed by incubation at 37°C for 1 h. For nuclear staining, the sections were stained with DAPI at room temperature for 3 min. After the sections were sealed, observation and image capture were performed under a microscope (DP73; Olympus, Tokyo, Japan).
5.3 Yak RTEC Isolation, Culture, and Counting
Kidney tissues were repeatedly rinsed with physiological saline (containing penicillin and streptomycin) to remove the capsule and obtain the cortex. Next, they were cut into blocks of approximately 1 mm3, transferred to a centrifuge tube, and centrifuged at 1,000 r·min−1 for 5 min. The obtained precipitate was placed in a culture dish, and 0.1% type I + II collagenase (Sigma-Aldrich, St. Louis, MO, USA) was added. The tissues were digested at 37°C for 2 h. Digestion was terminated using a complete culture medium. The liquid in the culture dish and the incompletely digested tissue were filtered through 70- and 40-μm sieves. The filtered liquid was centrifuged at 1,000 r·min−1 for 3 min three times. The cells in the precipitate were resuspended in the complete culture medium to prepare a cell suspension and then cultured at 37°C under 5% CO2 for 48 h, after which the culture medium was replaced.
Log-phase cells were harvested and used to prepare a cell suspension. Next, 1 mL of this suspension was diluted 104 times. For each test and blank control group, five replicate wells, with 100 μL of culture medium in each well, were set up in a 96-well plate and placed in a 37°C incubator. After cell attachment, 10 μL of the Cell Counting Kit-8 (CCK-8) reagent (Beyotime, Shanghai, China) was added every 24 h. After 3 h of incubation with the reagent, the optical density was measured on a microplate reader (Shanghai Bio-Chain Biological Technology, Shanghai, China). This process was repeated over 7 days to plot a growth curve.
5.4 Cell Immunofluorescence Staining
Cells were fixed with 4% paraformaldehyde for 30 min, permeabilized using 0.5% Triton X-100 (Beyotime) for 7 min, and blocked with immunofluorescence blocking solution (containing bovine serum albumin) at room temperature for 1 h. Subsequently, the cells were separately incubated with antibodies against cytokeratin (CK) 18 (1:400; bs2043R; Bioss), vimentin (1:400; ab8069; Abcam), E-cadherin (1:400; AF0131; Affinity), BECN1 (1:300; AF5128; Affinity), BAX (1:300; AF0120; Affinity), BCL2 (1:300; AF6139; Affinity), and LC3B (1:300; bs2912R; Bioss) at 4°C overnight. Next, the cells were washed and then incubated with goat antirabbit fluorescent secondary antibody (1:300) at 37°C for 1 h. Next, the cells were stained with a DAPI staining solution at room temperature for 5 min. The stained cells were covered and sealed with a coverslip and observed under an inverted microscope (DP71; Olympus).
5.5 qRT-PCR for mRNA Expression
Total RNA was extracted on ice using TRIzol reagent (Invitrogen, CA, USA) and then reverse-transcribed to cDNA by using a reverse transcription kit (Promega, Madison, USA). Next, qRT-PCR was performed with target and reference primer sequences designed on Primer Premier (version 6.0; Table 1). The volume of the reaction system was 20 μL (comprising 10 μL of SYBR Green Mix, 8 μL of ddH2O, 1 μL of cDNA, 0.5 μL each of the forward and reverse primers). For each gene, three replicates were set up. The mRNA relative expression level of the gene was calculated using the 2−ΔΔCt method.
5.6 WB
The cells were subjected to protein lysis on ice by using 1 mL of RIPA buffer and 10 μL phenylmethylsulfonyl fluoride. For protein denaturation, the extracted proteins were mixed with 4× protein buffer and placed in a 100°C constant temperature metal bath for 10 min. Next, 6 μL of the protein samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis; the separated proteins were transferred onto polyvinylidene fluoride membranes by using the wet transfer method. The membranes were then blocked with 5 mL·L−1 skim milk at room temperature for 2 h and then incubated with primary antibodies against HIF1α (1:800; AF1009; Affinity), BNIP3 (1:800; bs4239R; Bioss), CASP3 (1∶1,000; AF6311; Affinity), BECN1(1∶800; AF5128; Affinity), BAX (1:800; AF0120; Affinity), BCL2 (1:800; AF6139; Affinity), and ACTB (1:3,000; HC201-01; TransGen Biotech) at 4°C overnight. The membranes were incubated with the secondary antibody (1∶3,500) at room temperature for 50 min and then exposed to an enhanced chemiluminescence reagent (Beyotime) for the visualization of WB bands, which were analyzed using ImageJ.
5.7 Monodansylcadaverine Staining
In each well, RTECs were treated with 50 μmol·L−1 dimethyloxalylglycine (DMOG; MCE, NJ, USA) and 10 μmol·L−1 LW6 (MCE) for 24 h. Next, the cells were incubated with 1 mL of monodansylcadaverine (MDC) staining solution (Beyotime) at 37°C for 30 min in the dark. After the MDC staining solution was removed, cells were washed with 1 mL of the assay buffer three times. Next, 1 mL of assay buffer was added, and the cells were observed for green fluorescence under a fluorescence microscope at an excitation wavelength of 335 nm.
5.8 Flow Cytometry
RTECs were collected and fixed in 70% ethanol at 4°C overnight. Next, the cells were suspended in FACS buffer, and their concentration was adjusted to 3 × 105 cells·mL−1. This cell suspension was incubated with fluorescein isothiocyanate–labeled annexin V at room temperature for 10 min, followed by centrifugation and PBS washing. The cells were then resuspended in buffer, stained with propidium iodide for 5 min, and analyzed through flow cytometry.
5.9 Statistics Analyses
The data, presented as means ± standard errors of the means from three independent experiments, were analyzed using GraphPad Prism (version 8.3.0; GraphPad Software, San Diego, CA, USA). Differences between two groups were analyzed using a two-tailed Student’s t test. For multigroup comparisons with more than one variable, we used two-way analysis of variance followed by post hoc Tukey’s test. P values of <0.05 and <0.01 were considered to indicate statistical significance and extreme statistical significance, respectively.