Myoglobin Induces Oxidative Stress and Decreases Endocytosis and Monolayer Permissiveness in Cultured Kidney Epithelial Cells without Affecting Viability

Abstract

Background: Muscle degradation caused by severe burn releases myoglobin (Mb), which accumulates in the kidney (termed myoglobinuria). Mb is a pro-oxidant. Aim: To demonstrate that Mb promotes oxidative stress and dysfunction in cultured Madin-Darby canine kidney type II (MDCK II) cells. Methods: The glutathione redox ratio was used to monitor oxidative stress. Regulation of antioxidant response genes was determined with RT-PCR. Propidium iodide and annexin V staining were markers of necrosis and apoptosis, respectively. Mitochondrial function was assessed by monitoring mitochondrial depolarisation. Endocytosis was determined with immune fluorescence microscopy, and monolayer permeability was monitored with labelled inulin. Results: Kidney epithelial cells exposed to (0–100 µM) Mb showed a dose-dependent decrease in the glutathione redox ratio indicative of enhanced oxidative stress. In parallel, the expression of antioxidant genes for superoxide dismutase (SOD)-1/2, inducible haemoxygenase (HO-1) and catalase (CAT) increased in MDCK II cells, coupled with increases in corresponding activity. Notably, apoptosis and necrosis remained unaffected. However, transferrin endocytosis and monolayer permeability decreased significantly, while clathrin distribution and mitochondrial function were unaffected. Conclusion: Low concentrations of Mb promote oxidative stress in kidney epithelial cells that manifest as subtle changes to function without decreasing viability. Whether this impairs kidney function in burns patients is not clear.

Oxidative stress elicited by modifying the ceramide acyl chain length reduces the rate of clathrin-mediated endocytosis

“…Finally, altering the SL acyl chain composition also affects intracellular trafficking of connexin 32 , again due to altered biophysical properties. Thus, SLs have direct effects on receptor internalization and trafficking but also indirect effects, such as those reported in the current study, whereby dysfunction of mitochondrial complex IV affects ROS levels and results in concomitant changes in pathways affected by ROS, such as endocytosis (Parry et al, 2008;Poumay and Ronveaux-Dupal, 1988). Whether similar changes in signaling pathways occur upon acute changes in SL levels (Morales et al, 2007) is currently unknown, but the current study highlights the need to pay attention to both direct effects of SLs on membrane biophysical properties and also to indirect effects caused by activation of downstream pathways, such as defective clathrin uncoating caused by oxidative stress.…”

Inhibition of cytochrome P450 2E1 and activation of transcription factor Nrf2 are renoprotective in myoglobinuric acute kidney injury

“…Myoglobin has been shown to induce oxidative stress in canine epithelial cells 35 and cytotoxicity in human proximal tubular-2 cells. 36 Exposure of LLC-PK1 cells to myoglobin resulted in CYP2E1 degradation (Figure 6a), increasing ROS generation and cytotoxicity as measured by lactate dehydrogenase (LDH) release in a dose-and time-dependent manner (Figures 6b and c).…”

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

“…Apply standard methods of reverse transcription of mRNA to complementary DNA (cDNA) and amplification of target genes [21][22][23] .…”