Calcium phosphate-induced renal epithelial injury and stone formation: involvement of reactive oxygen species.
BACKGROUND
Crystal formation and retention are critical events for the formation of kidney stones. Oxalate and calcium oxalate (CaOx) crystals are injurious to renal epithelium, and membranes of injured cells promote crystal adherence and retention. Calcium phosphate (CaP) is the most common crystal in both urine and stones, most likely to form in the early segments of the nephron and can nucleate CaOx in a metastable solution. We hypothesized that CaP can also injure the renal epithelial cells.


METHODS
We exposed proximal tubular origin line derived from pig proximal tubules (LLC-PK1), and collecting duct origin Madin-Darby canine kidney (MDCK) cell lines to various concentrations of Brushite (Br) crystals and investigated staining with Trypan Blue and the release of lactate dehydrogenase (LDH) into the medium as an indicator of injury. In order to determine the involvement of reactive oxygen species, we also measured LDH release in the presence of superoxide dismutase (SOD) and production of hydrogen peroxide (H2O2) and 8-isoprostane (8-IP) in the presence of the catalase.


RESULTS
Exposure to Br crystals was associated with LDH release by both cell types, induced the production of H2O2 and 8-IP. Presence of SOD and catalase reduced LDH release as well as staining with trypan blue. Catalase was also associated with reduced production of H2O2 and 8-IP.


CONCLUSIONS
Brushite crystals are injurious to cells of both the proximal tubules as well as collecting ducts. Injury is mediated by reactive oxygen species. We propose that CaP crystals can independently interact with renal epithelium, promote sites for crystal attachment, and then either grow into mature CaP stones or create sites for CaOx crystal nucleation, retention, and stone development.
Aihara
Kinue
K
Department of Pathology, College of Medicine, University of Florida, Gainesville, Florida 32610, USA.
Byer
Karen J
KJ
Khan
Saeed R
SR
D000818:Animals; D002130:Calcium Phosphates / Q000494:pharmacology*; D002374:Catalase / Q000494:pharmacology; D002460:Cell Line; D004396:Coloring Agents; D015237:Dinoprost / Q000031:analogs & derivatives / Q000494:pharmacology; D004285:Dogs; D004357:Drug Synergism; D004848:Epithelium / Q000187:drug effects / Q000473:pathology; D006861:Hydrogen Peroxide / Q000378:metabolism; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology*; D007669:Kidney Calculi / Q000139:chemically induced*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D018374:LLC-PK1 Cells; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013194:Staining and Labeling; D013482:Superoxide Dismutase / Q000494:pharmacology; D013552:Swine; D014343:Trypan Blue
D002130:Calcium Phosphates; D004396:Coloring Agents; D017382:Reactive Oxygen Species; C075750:8-epi-prostaglandin F2alpha; D015237:Dinoprost; D006861:Hydrogen Peroxide; D007770:L-Lactate Dehydrogenase; D002374:Catalase; D013482:Superoxide Dismutase; D014343:Trypan Blue; C494366:calcium phosphate, dibasic, dihydrate