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.