Hypoxia is a unique environmental stressor that widely influences the physiological functions of multiple systems, including respiratory, circulatory, urinary, and digestive systems, exerting varying degrees of impact at the individual, organ, and cellular levels. Severe hypoxia can cause tissue and organ damage and disease, involving morphological structure, metabolism, proliferation, autophagy, and apoptosis at the cellular level. The kidney, a crucial component of the urinary system essential for maintaining homeostasis, regulating water-electrolyte and acid-base balance, and eliminating metabolic waste, is sensitive to hypoxia. Currently, the effects of hypoxia on bovine kidney cells and their underlying molecular mechanisms remain unclear.

This study employed the Madin-Darby Bovine Kidney (MDBK) cell line as a cellular model to investigate the effects of varying hypoxic concentrations (11% O₂, 5% O₂, and 1% O₂) and treatment durations (24, 48, and 72 h) on the proliferative capacity, ultrastructure, mitochondrial function, and hypoxic stress response of bovine kidney cells, in order to provide experimental evidence for further exploration of the hypoxic adaptation mechanisms in bovine kidney and a foundation for understanding cellular survival strategies and the regulatory mechanisms of mitophagy under hypoxic conditions.

Different cells and organelles experience distinct oxygen partial pressures. Under normoxic conditions, atmospheric oxygen partial pressure was 159.22 mm Hg (20.95%), arterial oxygen partial pressure was 100 mmHg (13%), venous oxygen partial pressure (PaO₂) was 40 mmHg (5.2%), and mitochondrial oxygen partial pressure ranges from 4-20 mm Hg (0.52%-2.60%). Based on this, 11% O₂, 5% O₂, and 1% O₂ were selected as hypoxic treatment conditions. MDBK cells were seeded in DMEM complete medium supplemented with 10% fetal bovine serum and cultured at 37 ℃ with 5% CO₂ until reaching the logarithmic growth phase. Cells were then transferred to a tri-gas incubator and cultured under 11% O₂, 5% O₂, and 1% O₂ for 24, 48, and 72 h. The effects of hypoxia on bovine kidney cell proliferation were assessed by MTT assay. Ultrastructural changes in MDBK cells under hypoxic conditions were observed by transmission electron microscopy. Mitochondrial membrane potential was measured using the JC-1 fluorescent probe method. Reactive oxygen species (ROS) production was detected by the DCFH-DA fluorescent probe method. The expression of hypoxia-related and autophagy-related genes was analyzed by real-time quantitative PCR and Western blotting.

Cell proliferation was significantly inhibited with decreasing oxygen concentration and prolonged treatment time, with more pronounced inhibition at lower oxygen concentrations and longer durations. Hypoxia caused mitochondrial structural damage and functional impairment, manifested as mitochondrial swelling, disorganized or absent cristae, a significant reduction in mitochondrial number, condensation of some mitochondria, and the presence of mitochondria enveloped by membranous structures indicative of mitophagy. Concurrently, mitochondrial membrane potential decreased significantly, but intracellular ROS levels increased markedly. The expression of hypoxia-related genes EPAS1 and PPARα was significantly upregulated, while the expression levels of mitophagy-related genes PINK1, PRKN, BNIP3, and BNIP3L (NIX) were significantly elevated.

This study demonstrated that treatment with 5% and 1% oxygen concentrations significantly inhibited the proliferative activity of bovine kidney cells, causing mitochondrial structural damage and dysfunction, decreased mitochondrial membrane potential, and increased ROS production. In response to hypoxic stress, bovine kidney cells activate mitophagy to eliminate dysfunctional mitochondria, thereby alleviating oxidative stress and maintaining intracellular homeostasis.

Heat stress has seriously impaired the production and health of dairy cows, causing the subsequent limitation in sustainable development of dairy industry. DNA methylation is an important epigenetic regulatory mechanism involved in an animal’s heat stress response, but the potential functions and molecular mechanisms of which are not clear. The current study was conducted to detect the DNA methylation related to heat stress in dairy cows and to identify target genes related to DNA methylation, so as to provide a better insight into the epigenetics mechanism of heat stress in dairy cows.

In the study, 24 Chinese Holstein lactation cows (same lactation stage and same parity) in Sanyuan dairy farm were used for the blood samples collection in heat stress period (July in the summer of 2017) and non-heat stress period (April in spring 2017), respectively, followed by DNA extraction. To explore the DNA methylation differences in dairy cows from different heat stress period, 15 of 24 animals were randomly assigned to 3 groups (N=5 animals/group), 5 DNA samples in one group were mixed together to get a single pooled DNA sample, thus 6 pooled DNA samples including 3 from spring and 3 from summer were used for the DNA methylation detection by the whole-genome bisulfite sequencing (WGBS), then the differential methylation region (DMR; 1000 bp windows, 500 bp overlap, P<0.05) and key gene were identified. PROMO and Methprimer software were used to predict transcription factor binding sites and CpG islands, respectively. Then, the bovine mammary gland epithelial cells (Mac-T) were treated at 39 ℃ for 24 h, 48 h, and 72 h, and the cell viability were detected by MTT method. Finally, using the bisulfite sequencing PCR (BSP), the methylation levels of target gene promoter in 24 dairy cows in spring and summer and Mac-T cells treated in 39 ℃ were examined, respectively.

Based on the DNA methylation analysis of WGBS data, 49 861 differential methylation regions (DMRs) associated with heat stress were identified. One of DMRs was attributed to the promoter area of GNAS complex locus (GNAS), whose methylation level significantly increased in heat-stressed animals (P<0.001). Also, there was a 352 bp CpG island in the promoter of GNAS containing potential binding sites for Sp1, C/EBP and other important transcription factors. Further the methylation status of the GNAS gene promoter region in heat stressed dairy cows were verified by BSP, and the average methylation level in all cytosine of 31 CpG sites was higher in heat stress cows than that in control groups (P<0.05), which corresponding to the above WGBS results. Moreover, the 21 (-113 bp, Chr13:57532733) and 27 (-63 bp, Chr13:57532683) CpG sites showed significant differences between the spring and summer groups (P<0.05). In Mac-T cells, after 48 h and 72 h heat treatment, the cell viability decreased significantly (P<0.01), but the overall CG methylation level of 31 CpG sites in the GNAS gene promoter region increased significantly (P<0.05), and also the similar significant methylation changes appeared in the site 21 and 27 CpG in cell.

Heat stress increased the methylation levels of the promoter region of the GNAS in dairy cows as well as in cells, which indicated that GNAS was a potential target gene regulated DNA methylation in heat stress response of dairy cows.