Abstract
Hyperoxaluria is a risk factor for urolithiasis and can cause renal epithelial cell injury secondary to oxidative stress. Reactive oxygen species (ROS) produced during cell damage originate from different sources and play different roles. Here, we explored the potential sources of ROS production and investigated the role of ROS from various sources in oxalate-induced oxidative stress and cell injury in normal rat kidney-52 epithelial (NRK-52E) cells. Oxalate-induced injury was assessed by lactate dehydrogenase (LDH) release experiments. 2,7-dichlorodihydrofluorescein diacetate and mitoSOX Red were used to determine the intracellular and mitochondrial ROS (mtROS) production, respectively. The expression level of Nox4, Nox2, and p22 protein was detected by Western blotting to observe the effect of oxalate on nicotinamide adenine dinucleotide phosphate oxidase (NADPH) oxidase (Nox). Furthermore, a specific NADPH oxidase subtype inhibitor and targeted mitochondrial antioxidants were used to preliminarily identify the role of ROS from different sources in renal tubular epithelial cell injury induced by oxalate. We found that oxalate inhibited cell viability, induced LDH release, and prompted intracellular and mitochondrial ROS (mtROS) production. Oxalate also decreased the protein expression level of Nox4 and increased the protein expression level of p22. Mitochondria were also a source of ROS production. In addition, Nox2 inhibitor or mtROS scavenging prevented oxalate-induced cell injury, reversed by an inhibitor of Nox4/1. We concluded that ROS from different sources might play different roles in oxalate-induced renal tubular epithelial cell injury. We also identified new potential targets for preventing or alleviating oxalate-induced renal tubular epithelial cell injury.Graphic abstract
Highlights
Calcium oxalate calculi are the most common type of urinary calculi, accounting for approximately 70–80% of all cases
Cellular responses to the injury induced by Ox or calcium oxalate (CaOx) crystals can be inhibited by antioxidants as well as classical nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) inhibitors, such as diphenylene iodonium (DPI) [2, 5, 6]
After treatment with oxalate (700 μmol/L) for 3 h, the mitochondrial membrane potential of normal rat kidney-52 epithelial (NRK-52E) cells decreased distinctly compared to the control group (Fig. 2a)
Summary
Calcium oxalate calculi are the most common type of urinary calculi, accounting for approximately 70–80% of all cases. Cellular responses to the injury induced by Ox or CaOx crystals can be inhibited by antioxidants as well as classical NADPH oxidase (Nox) inhibitors, such as diphenylene iodonium (DPI) [2, 5, 6] Another classical NADPH oxidase inhibitor, apocynin, has been verified to protect against renal injury and decrease stone formation in animal models of hyperoxaluria [7,8,9]. NADPH oxidase is an important source of ROS in the kidney and is involved in several physiological and pathological responses [10, 11] It has been considered an important therapeutic target for oxalate-mediated renal tubular epithelial cell injury and stone formation [12, 13]. The Nox family comprises seven isoforms, and the expression of Nox, Nox, and Nox was found to be decreased gradually in the kidney [10, 16, 17]
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