Abstract
Mitochondrial dysfunction has been implicated in the early stages or progression of many renal diseases. Improving mitochondrial function and homeostasis has the potential to protect renal function. Serum- and glucocorticoid-induced kinase 1 (SGK1) is known to regulate various cellular processes, including cell survival. In this study, we intend to demonstrate the effect and molecular mechanisms of SGK1 in renal tubular cells upon oxidative stress injury and to determine whether regulation of mitochondrial function is implicated in this process. HK-2 cells were exposed to H2O2, and cell viability and apoptosis were dynamically detected by the CCK-8 assay and annexin-V/PI staining. The concentrations of cellular reactive oxygen species (ROS) and adenosine triphosphate (ATP) and the expression of the SGK1/GSK3β/PGC-1α signaling pathway were analyzed by flow cytometry or western blot. In addition, shRNA targeting SGK1 and SB216763 were added into the culture medium before H2O2 exposure to downregulate SGK1 and GSK3β, respectively. Cell viability and mitochondrial functions, including mitochondrial membrane potential (Δψm), Cytochrome C release, mtDNA copy number, and mitochondrial biogenesis, were examined. Protein levels and SGK1 activation were significantly stimulated by H2O2 exposure. HK-2 cells with SGK1 inhibition were much more sensitive to H2O2-induced oxidative stress injury than control group cells, as they exhibited increased apoptotic cell death and mitochondrial dysfunction involving the deterioration of cellular ATP production, ROS accumulation, mitochondrial membrane potential reduction, and release of Cytochrome C into the cytoplasm. Studies on SGK1 knockdown also indicated that SGK1 is required for the induction of proteins associated with mitochondrial biogenesis, including PGC-1α, NRF-1, and TFAM. Moreover, the deleterious effects of SGK1 suppression on cell apoptosis and mitochondrial function, including mitochondrial biogenesis, were related to the phosphorylation of GSK3β and partially reversed by SB216763 treatment. H2O2 leads to SGK1 overexpression in HK-2 cells, which protects human renal tubule cells from oxidative stress injury by improving mitochondrial function and inactivating GSK3β.
Highlights
Oxidative stress, characterized by excessive levels of reactive oxygen species (ROS) resulting from an imbalance between the oxidative and antioxidative systems, has been widely implicated in renal pathological conditions [1, 2]
We investigated the effect of different durations and doses of H2O2 exposure on the viability, apoptosis, and mitochondrial function of HK-2 cells
In agreement with the Cell Counting Kit-8 (CCK-8) assay results, in which the cell viability was approximately 75% when the HK-2 cells were treated with 250 μM H2O2 for 2 h, the annexin-V/PI apoptosis examination demonstrated that the proportion of apoptotic cells under the same conditions was approximately 25%
Summary
Oxidative stress, characterized by excessive levels of reactive oxygen species (ROS) resulting from an imbalance between the oxidative and antioxidative systems, has been widely implicated in renal pathological conditions [1, 2]. The kidney is one of the most energy-consuming organs in the human body [3]. Owing to their active reabsorption and secretion function, renal proximal tubular cells contain the second highest mitochondrial content after only cardiomyocytes [4]. Mitochondrial dysfunction leads to decreased ATP production, increased ROS levels and, loss of renal function [5, 6]. Growing evidence show that aging is a major contributor to the increasing incidence of kidney disease. Mitochondrial dysfunction is one of the most crucial hallmarks that contribute to the process of aging. Targeting mitochondrial function and homeostasis is a potential strategy to protect renal function and retard senescence [7]
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