Effect of calbindin-D28K on cyclosporine toxicity in cultured renal proximal tubular cells.
Cyclosporine A (CsA) is known to have direct toxicity to renal tubular cells. Its toxicity may be mediated by intracellular calcium because CsA increases intracellular calcium concentration and enhances the activities of calcium-dependent calpains and caspases. Calbindin-D28k, a cytosolic calcium binding protein, has been used as an intracellular Ca2+ buffer to reduce calcium-mediated cytotoxicity in non-renal cells such as neuronal cells. We investigated the effects of gene transfer of calbindin-D28k cDNA on CsA cytotoxicity and intracellular calcium concentration ([Ca2+]i) in cultured murine proximal tubular (MCT) cells. A plasmid containing calbindin-D28k cDNA under the control of CMV promoter was transfected to MCT cells with liposomes. Cytotoxicity was assessed by LDH release and cell viability assay, and [Ca2+]i was measured ratiometrically with fura-2. Compared with MCT cells, cells transfected with calbindin-D28k cDNA showed a reduction in LDH release by 27, 30, 32, 33, and 19% (all P < 0.05), respectively, after 24 h exposure to 1, 2.5, 5, 10, and 25 microM CsA. Cell viability after CsA treatment was also significantly higher in CB cells. A mock transfection using plasmid without calbindin-D28k cDNA insert did not affect the LDH release or cell viability after CsA treatment. CsA treatment did not affect the protein and mRNA abundance of transfected calbindin-D28k cDNA. The expression of calbindin-D28k did not affect the baseline [Ca2+]i, but significantly suppressed CsA-induced elevation in [Ca2+]i. The expression of calbindin-D28k in renal tubular cells provides cytoprotective effects against CsA toxicity, probably through its buffering effects on [Ca2+]i.
Wu
Ming-Ju
MJ
Division of Nephrology, Taichung Veterans General Hospital, Chung Shan Medical University, Taiwan.
Lai
Li-Wen
LW
Lien
Yeong-Hau H
YH
D000818:Animals; D064092:Calbindin 1; D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D016572:Cyclosporine / Q000633:toxicity*; D019610:Cytoprotection; D016257:Fura-2; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000032:analysis / Q000378:metabolism; D051379:Mice; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D064030:S100 Calcium Binding Protein G / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D018675:Toxicity Tests
C576499:Calb1 protein, mouse; D064092:Calbindin 1; D064026:Calbindins; D012333:RNA, Messenger; D064030:S100 Calcium Binding Protein G; D016572:Cyclosporine; D007770:L-Lactate Dehydrogenase; D002154:Calpain; D020169:Caspases; D002118:Calcium; D016257:Fura-2