Oxalate at physiological urine concentrations induces oxidative injury in renal epithelial cells: effect of α-tocopherol and ascorbic acid.
OBJECTIVE
To test our hypothesis that physiological levels of urinary oxalate induce oxidative renal cell injury, as studies to date have shown that oxalate causes oxidative injury only at supra-physiological levels. To study the combined effect of α-tocopherol and ascorbic acid against oxalate-induced oxidative injury, as oxalate-induced oxidative cell injury is known to promote initial attachment of calcium oxalate crystals to injured renal tubules and subsequent development of kidney stones.


METHODS
Cultures of normal (antioxidant-undepleted) and antioxidant-depleted LLC-PK1 cells were exposed to oxalate at human physiological urine concentrations. After exposure, markers of oxidative stress and cell injury were measured in the cells and media, respectively. In addition, we also evaluated the combined effects of α-tocopherol and ascorbic acid on oxalate-induced oxidative cell injury.


RESULTS
Exposure of renal cells to oxalate at urinary physiological levels increased the oxidative cell injury as assessed by increased lactate dehydrogenase (LDH) leakage and increased lipid hydroperoxide in the renal cells; however, this effect was not seen until 24 h after oxalate exposure, at which point the injury was milder. On the other hand, when cellular reduced glutathione (GSH) and catalase were depleted in renal epithelial cells with pharmacological inhibitors, the physiological levels of urinary oxalate caused significant oxidative cell injury at 24 h, and remarkably, when additional endogenous antioxidants were depleted, the oxalate at the upper limit of normal 24 h urine caused a significant amount of cell injury in a shorter period of time, which was comparable to that seen in cells exposed to higher levels of oxalate. Exposure of LLC-PK1 cells to oxalate resulted in increased levels of H2 O2 and lipid hydroperoxide, correlating with increased release of cell injury markers, including LDH, alkaline phosphate, and γ-glutamyl transpeptidase from renal tubular epithelial cells. Oxalate exposure decreased the activity and protein expression of superoxide dismutase and glutathione peroxidase in a time-dependent manner. LLC-PK1 cells treated with oxalate and either α-tocopherol or ascorbic acid alone exhibited a significant decrease in oxidative cell injury and restored endogenous renal antioxidants towards normal levels, and interestingly, combined treatment with α-tocopherol and ascorbic was more efficient at preventing oxalate-induced toxicity than treatment with either agent alone.


CONCLUSIONS
To our knowledge this is the first study to show that oxalate alone at human physiological urine concentrations (in the absence of calcium oxalate crystal formation), induced oxidative renal injury in renal epithelial cells when endogenous antioxidants are depleted. Our data further suggests that a combination of α-tocopherol and ascorbic acid may be more effective than each individual agent in reducing oxalate-induced oxidative renal injury and subsequent calcium oxalate crystal deposition in recurrent stone formers.
Vattikuti Urology Institute, Henry Ford Health System, Detroit, MI, USA.
Vijayalakshmi
V
Thamilselvan
Mani
M
Menon
Sivagnanam
S
Thamilselvan
D000975:Antioxidants / Q000378:metabolism / Q000494:pharmacology*; D001205:Ascorbic Acid / Q000378:metabolism / Q000494:pharmacology*; D002374:Catalase / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008054:Lipid Peroxides / Q000378:metabolism; D010070:Oxalates / Q000009:adverse effects; D018384:Oxidative Stress / Q000187:drug effects*; D013997:Time Factors; D024502:alpha-Tocopherol / Q000378:metabolism / Q000494:pharmacology*
D000975:Antioxidants; D008054:Lipid Peroxides; D010070:Oxalates; D007770:L-Lactate Dehydrogenase; D002374:Catalase; D005978:Glutathione; D024502:alpha-Tocopherol; D001205:Ascorbic Acid
LLC-PK1 cells; antioxidants; calcium oxalate crystals; oxidative stress; renal epithelial cells; urolithiasis