Cytoprotective effects of calbindin-D(28k) against antimycin-A induced hypoxic injury in proximal tubular cells.
Intracellular calcium plays an important role on the pathogenesis of hypoxia-induced cellular injury. Calbindin-D(28k), a cytosolic vitamin D-dependent calcium binding protein, can serve as a buffer to limit a surge in intracellular Ca2+ concentration ([Ca2+]i) induced by various stimulations. To evaluate the possible cytoprotective effect of calbindin-D(28k) against hypoxic injury in proximal tubular cells, a plasmid containing calbindin-D(28k) cDNA under the control of CMV immediate-early gene promoter was transfected into the murine proximal tubular epithelial (MCT) cells. The expression of calbindin-D(28k) in the transfected cells was verified with Northern blot analysis, Western blot analysis, and immunofluorescent staining. The non-transfected and transfected MCT cells were subjected to chemical hypoxia induced by antimycin A (10 microM) and glucose deprivation for 30-120 min. The transfection of calbindin-D(28k) reduced lactate dehydrogenase (LDH) release by 41%, 41%, 24%, and 24%, respectively, at 30, 60, 90 and 120 min after hypoxia when compared to the non-transfected cells (all p < 0.05). Cell viability after hypoxic injury was also significantly higher in transfected cells than non-transfected cells. Transfection with the plasmid without calbindin-D(28k) cDNA did not affect LDH release or cell viability after chemical hypoxic injury. [Ca+2]i was measured ratiometrically with fura-2 after exposure to chemical hypoxia. The rate of initial rise in [Ca2+]i and final [Ca+2]i at 30-120 min were significantly lowered in transfected cells. In conclusion, this study demonstrated that transfection of calbindin-D(28k) gene into MCT cells provide protective effects against chemical hypoxic injury probably through its buffering effects on [Ca+2]i.
Wu
Ming-Ju
MJ
Department of Medicine, Nephrology Section, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA.
Lai
Li-Wen
LW
Lien
Yeong-Hau H
YH
D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D015687:Cell Hypoxia; D002460:Cell Line; D019610:Cytoprotection; D005947:Glucose / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D011401:Promoter Regions, Genetic; D064030:S100 Calcium Binding Protein G / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
D064026:Calbindins; D064030:S100 Calcium Binding Protein G; D000968:Antimycin A; D005947:Glucose; D002118:Calcium