Cell Death Patterns Due to Warm Ischemia or Reperfusion in Renal Tubular Epithelial Cells Originating from Human, Mouse, or the Native Hibernator Hamster

Abstract

Ischemia⁻reperfusion injury contributes to the pathogenesis of many diseases, with acute kidney injury included. Hibernating mammals survive prolonged bouts of deep torpor with a dramatic drop in blood pressure, heart, and breathing rates, interspersed with short periods of arousal and, consequently, ischemia⁻reperfusion injury. Clarifying the differences under warm anoxia or reoxygenation between human cells and cells from a native hibernator may reveal interventions for rendering human cells resistant to ischemia⁻reperfusion injury. Human and hamster renal proximal tubular epithelial cells (RPTECs) were cultured under warm anoxia or reoxygenation. Mouse RPTECs were used as a phylogenetic control for hamster cells. Cell death was assessed by both cell imaging and lactate dehydrogenase (LDH) release assay, apoptosis by cleaved caspase-3, autophagy by microtubule-associated protein 1-light chain 3 B II (LC3B-II) to LC3B-I ratio, necroptosis by phosphorylated mixed-lineage kinase domain-like pseudokinase, reactive oxygen species (ROS) fluorometrically, and lipid peroxidation, the end-point of ferroptosis, by malondialdehyde. Human cells died after short periods of warm anoxia or reoxygenation, whereas hamster cells were extremely resistant. In human cells, apoptosis contributed to cell death under both anoxia and reoxygenation. Although under reoxygenation, ROS increased in both human and hamster RPTECs, lipid peroxidation-induced cell death was detected only in human cells. Autophagy was observed only in human cells under both conditions. Necroptosis was not detected in any of the evaluated cells. Clarifying the ways that are responsible for hamster RPTECs escaping from apoptosis and lipid peroxidation-induced cell death may reveal interventions for preventing ischemia⁻reperfusion-induced acute kidney injury in humans.

Keywords: apoptosis; autophagy; ferroptosis; hibernation; ischemia–reperfusion; lipid peroxidation; necroptosis.

Figure 1

Sensitivity of human, mouse, and hamster renal proximal tubular epithelial cells (RPTECs) to death due to anoxia or reoxygenation. Human RPTECs were extremely sensitive to anoxia since they died after 4 h of anoxia. Mouse RPTECs were less sensitive than human RPTECs, since they died after 48 h of anoxia. On the contrary, hamster RPTECs were extremely resistant to anoxic conditions, since they remained alive even after 120 h of observation (A). During reoxygenation, human RPTECs deteriorated significantly, showing condensation and loss of adherence on a very large scale, after 8 h. Mouse RPTECs were even more sensitive to reoxygenation, since they deteriorated considerably after 4 h. On the contrary, hamster RPTECs were extremely resistant to reoxygenation, since they preserved their morphology after 48 h of observation (B). The photos are representative of one of the nine performed experiments. Hu: human; Mo: mouse; Hm: hamster.

Figure 2

Biochemical detection of cell death in human, mouse, and hamster RPTECs under anoxia or reoxygenation. Compared to the control conditions, both anoxia and reoxygenation increased cell death in human and mouse RPTECs. On the contrary, neither anoxia nor reoxygenation affected cell death significantly in hamster RPTECs. Hu: human; Mo: mouse; Hm: hamster. Asterisk indicates a p < 0.05 compared to the control cultured under normoxia cells, and error bars correspond to SD.

Figure 3

Apoptosis and expression of the pro-apoptotic p53 in human, mouse, and hamster RPTECs under anoxia or reoxygenation. Apoptosis was assessed by the level of cleaved caspase-3. Both cleaved caspase-3 and the pro-apoptotic p53 were measured by Western blotting and the results of three, representative of the nine performed experiments, are depicted in panel (A). Compared to the control group, in human RPTECs, both anoxia and reoxygenation increased the level of cleaved caspase-3. In mouse RPTECs, cleaved caspase-3 increased only under anoxia, whereas, in hamster RPTECs, apoptosis was not observed either under anoxia or reoxygenation (B). The alterations in cleaved caspase-3 fitted to the alterations of the pro-apoptotic protein tumor suppressor p53 (C). Hu: human; Mo: mouse; and Hm: hamster. Asterisk indicates a p < 0.05 compared to the control cultured under normoxia cells, and error bars correspond to SD.

Figure 4

Autophagy in human, mouse, and hamster RPTECs under anoxia or reoxygenation. Autophagy was evaluated by the LC3B-II to LC3B-I ratio, assessed by Western blotting. In panel (A) three representatives of the nine performed experiments are depicted. Autophagy was observed only in human RPTECs under both anoxia and reoxygenation. Neither in mouse nor in hamster RPTECs did anoxia or reoxygenation induce autophagy (B). Hu: human; Mo: mouse; and Hm: hamster. Asterisk indicates a p < 0.05 compared to the control cultured under normoxia cells, and error bars correspond to SD.

Figure 5

Necroptosis in human, mouse, and hamster RPTECs under anoxia or reoxygenation. Necroptosis was evaluated by the level of p-MLKL assessed by Western blotting. In panel (A), three representatives of the nine performed experiments are depicted. Necroptosis was not observed in any of the evaluated cells under anoxia or reoxygenation. Phosphorylated MLKL decreased in human and hamster RPTECs under both conditions, and remained unaltered in mouse RPTECs (B). Hu: human; Mo: mouse; and Hm: hamster. Asterisk indicates a p < 0.05 compared to the control cultured under normoxia cells, and error bars correspond to SD.

Figure 6

Reactive oxygen species production and lipid peroxidation in human, mouse, and hamster RPTECs under anoxia or reoxygenation. Anoxia did not alter ROS in any of the evaluated cells, whereas reoxygenation increased ROS in all of them (A). Lipid peroxidation, a known mechanism of cell death, was assessed by the level of cellular malondialdehyde (MDA). Anoxia did not affect MDA in any of the evaluated cells. Reoxygenation induced a dramatic increase in MDA level in human and mouse RPTECs, but it did not alter the MDA level in hamster RPTECs (B). Hu: human; Mo: mouse; and Hm: hamster. Asterisk indicates a p < 0.05 compared to the control cultured under normoxia cells, and error bars correspond to SD.