Aslam Khan1, Karen Byer2, Saeed R Khan3. 1. Department of Pharmacy, Shaheed Benazir Bhutto University, Sheringal, Dir Upper, Khyber Pakhtunkhwa, Pakistan. 2. Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL. 3. Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL. Electronic address: Khan@pathology.ufl.edu.
Abstract
OBJECTIVE: To investigate nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase activity in Madin-Darby canine kidney (MDCK) cells and the production of reactive oxygen species on exposure to oxalate (Ox) or calcium oxalate (CaOx) crystals. METHODS: Monolayers of confluent Madin-Darby canine kidney cells were exposed to 100, 300, 500 μmol, 1 mmol Ox or 33, 66, 132 μg/cm(2) CaOx crystals for 15 minutes, 30 minutes, 1 hour, 2 hours, or 3 hours. After specified periods of exposure to Ox and CaOx crystals, lactate dehydrogenase release, trypan blue exclusion, activation of NADPH oxidase, and superoxide production were determined using standard procedures. The production of Nox4, a membrane associated subunit of the NADPH oxidase enzyme, was determined by western blot analysis. RESULTS: Exposure to Ox and CaOx crystals leads to time- and concentration-dependent activation of NADPH oxidase. Western blot analysis showed an increase in the production of Nox4. The production of superoxide also changed in a time- and concentration-dependent manner, with maximum increases after 30-minute exposure to the highest concentrations of Ox and CaOx crystals. Longer exposures did not change the results or resulted in decreased activities. Exposure to higher concentrations also caused increased lactate dehydrogenase release and trypan blue exclusion indicating cell damage. CONCLUSION: Results indicate that cells of the distal tubular origin are equipped with NADPH oxidase that is activated by exposures to Ox and CaOx crystals. Higher concentrations of both lead to cell injury, most probably through the increased reactive oxygen species production by the exposed cells.
OBJECTIVE: To investigate nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase activity in Madin-Darby canine kidney (MDCK) cells and the production of reactive oxygen species on exposure to oxalate (Ox) or calcium oxalate (CaOx) crystals. METHODS: Monolayers of confluent Madin-Darby canine kidney cells were exposed to 100, 300, 500 μmol, 1 mmol Ox or 33, 66, 132 μg/cm(2) CaOx crystals for 15 minutes, 30 minutes, 1 hour, 2 hours, or 3 hours. After specified periods of exposure to Ox and CaOx crystals, lactate dehydrogenase release, trypan blue exclusion, activation of NADPH oxidase, and superoxide production were determined using standard procedures. The production of Nox4, a membrane associated subunit of the NADPH oxidase enzyme, was determined by western blot analysis. RESULTS: Exposure to Ox and CaOx crystals leads to time- and concentration-dependent activation of NADPH oxidase. Western blot analysis showed an increase in the production of Nox4. The production of superoxide also changed in a time- and concentration-dependent manner, with maximum increases after 30-minute exposure to the highest concentrations of Ox and CaOx crystals. Longer exposures did not change the results or resulted in decreased activities. Exposure to higher concentrations also caused increased lactate dehydrogenase release and trypan blue exclusion indicating cell damage. CONCLUSION: Results indicate that cells of the distal tubular origin are equipped with NADPH oxidase that is activated by exposures to Ox and CaOx crystals. Higher concentrations of both lead to cell injury, most probably through the increased reactive oxygen species production by the exposed cells.