Abstract
Oxidative stress is associated with many renal disorders, both acute and chronic, and has also been described to contribute to the disease progression. Therefore, oxidative stress is a potential therapeutic target. The human antioxidant α1-microglobulin (A1M) is a plasma and tissue protein with heme-binding, radical-scavenging and reductase activities. A1M can be internalized by cells, localized to the mitochondria and protect mitochondrial function. Due to its small size, A1M is filtered from the blood into the glomeruli, and taken up by the renal tubular epithelial cells. A1M has previously been described to reduce renal damage in animal models of preeclampsia, radiotherapy and rhabdomyolysis, and is proposed as a pharmacological agent for the treatment of kidney damage. In this paper, we examined the in vitro protective effects of recombinant human A1M (rA1M) in human proximal tubule epithelial cells. Moreover, rA1M was found to protect against heme-induced cell-death both in primary cells (RPTEC) and in a cell-line (HK-2). Expression of stress-related genes was upregulated in both cell cultures in response to heme exposure, as measured by qPCR and confirmed with in situ hybridization in HK-2 cells, whereas co-treatment with rA1M counteracted the upregulation. Mitochondrial respiration, analyzed with the Seahorse extracellular flux analyzer, was compromised following exposure to heme, but preserved by co-treatment with rA1M. Finally, heme addition to RPTE cells induced an upregulation of the endogenous cellular expression of A1M, via activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-pathway. Overall, data suggest that A1M/rA1M protects against stress-induced damage to tubule epithelial cells that, at least partly, can be attributed to maintaining mitochondrial function.
Highlights
Oxidative stress is a term often used to describe physiological conditions where there is an imbalance between oxidants and antioxidants that results in a biological stress response with disturbed redox signaling, and downstream molecular damage [1]
Cytochrome b-245 beta (CYBB) expression, 2.2. recombinant human A1M (rA1M) Counteracts Heme-Induced Upregulation of HSP70 and HMOX-1 Transcripts RNAscope, an in situ hybridization assay for the detection of RNA levels within intact cells, was used to evaluate expression of heat shock protein 70 (HSP70) and heme oxygenase 1 (HMOX-1) in HK-2 cells, following exposure to 10 μM heme, with or without the simultaneous addition of 10 μM Int
We have characterized the protective effects of rA1M in vitro, and show that it counteracts heme-induced cell death, mitochondrial deficiency and cellular stress in proximal tubule epithelial cells, both HK-2 cells and a primary human cell culture
Summary
Oxidative stress is a term often used to describe physiological conditions where there is an imbalance between oxidants and antioxidants that results in a biological stress response with disturbed redox signaling, and downstream molecular damage [1]. The production of oxidants, including reactive oxygen species (ROS) and free radicals, can be induced by external sources, such as radiation or smoke, as well as a wide variety of internal biological processes, such as inflammation, and may have detrimental effects on cells and tissues when not balanced by the antioxidation defense systems [2]. Both acute kidney injury (AKI) and chronic kidney disease (CKD) have been associated with decreased antioxidant defense and/or increased ROS that contribute to the disease progression [3,4]. Sci. 2020, 21, 5825 we have further characterized the renal protective mechanisms of A1M/rA1M by exposing human proximal tubule epithelial cells, both a cell line and primary cells, to heme-induced oxidative stress
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