Apoptosis caused by excessive mitochondrial albumin uptake in renal cells is initiated by increased mitochondrial calcium concentration

Abstract

Recent advances in the development of organelle-targeted low affinity Ca2+ sensitive dyes have lead to new understanding of the role of Ca2+ in subcellular compartments. Here we report that the onset of the mitochondrial apoptotic pathway is preceded by an increase in mitochondrial Ca2+. Excessive Ca2+ uptake in renal cells causes apoptosis and is a major contributor to the progression of chronic kidney disease. Primary rat proximal tubule cells (RPTC) were incubated with 10mg/ml lipid-free albumin and monitored with regard to mitochondrial Ca2+ concentration ([Ca2+]mt), mitochondrial membrane potential (Δψm) and mitochondrial accumulation of the apoptotic factor Bax. To monitor changes in [Ca2+]mt we expressed RCaMP1h, a mitochondria targeted genetic Ca2+ indicators in RPTC. [Ca2+]mt started to increase significantly after 5 min of albumin incubation and continued to increase during the observation period. At 30 min it was 20 % higher than during control conditions. The increase in [Ca2+]mt was followed by a significant Δψm depolarization after 45 min, and mitochondrial accumulation of the apoptotic factor Bax, which became significant after 30 min. Low concentration of ouabain (10 nM) protected from depolarization and Bax accumulation by activating a Na+, K+-ATPase/IP3R signalling pathway. Thapsigargin, an inhibitor of the SERCA pump causes apoptosis in all cells but its mechanism of action has been elusive. Here we found that exposure of RPTC to cyclopiazonic acid, an analogue of thapsigargin, caused an increase in mitochondrial Ca2+ that paralleled the increase in cytosolic Ca2+. Conclusion Selective sustained increase of mitochondrial Ca2+ concentration may be a major factor responsible for the onset of the intrinsic apoptotic pathway.