Regulation of mesangial cell apoptosis and proliferation by intracellular Ca2+ signals

Background. In inflammatory glomerular diseases, proliferation, as well as apoptosis of mesangial cells (MCs), has been shown histomorphologically. Bo th processes may regulate the cellular content of the mesangium by closely influencing each other. In the present study, we examined whether the cytop lasmic free Ca2+ concentration [Ca2+](i) is involved as a key second messen ger in the regulation of proliferative and apoptotic events. Methods. Thapsigargin, an inhibitor of the endoplasmic Ca2+-Mg2+-ATPase, wa s used as a test substance to investigate the role of [Ca2+](i) in signalin g MC apoptosis and growth in vitro. Apoptosis was determined by nuclear chr omatin staining with Hoechst 33258, by a [H-3]-thymidine-based DNA fragment ation assay or by flow cytometry detecting binding of FITC-conjugated annex in V. Proliferation was measured by [H-3]- thymidine incorporation into aci d-precipitable material and corroborated by cell counting. Results. Thapsigargin significantly induced apoptosis and inhibited prolife ration dose dependently in nanomolar concentrations without evoking necroti c damage when administered not longer than 12 hours. Significant apoptosis was measurable after a six-hour treatment of MCs with thapsigargin. Determi nation of [Ca2+](i) by fura-2-dependent spectrofluorometry showed that thap sigargin was able to induce prolonged [Ca2+](i) rises that could be prevent ed by preincubation with the intracellular Ca2+ chelator 1,2-bis(2-aminophe noxy)-ethane-N,N,N', N'-tetra-acetic acid (BAPTA) acetomethyl ester (AM). B APTA had no influence on MC viability but reversed thapsigargin-induced apo ptosis to control levels. After thapsigargin treatment (100 nmol/L, 12 hour s), apoptotic MCs had a significantly higher [Ca2+](i) of 251 +/- 25 nmol/L (N = 41) as compared with MCs that were not or not yet apoptotic ([Ca2+](i ) of 116 +/- 20 nmol/L, N = 26, P < 0.05). Platelet-derived growth factor ( PDGF), a well-characterized growth factor for MCs, reversed the effects of thapsigargin on proliferation and apoptosis in a similar fashion as BAPTA. PDGF acutely stimulated increases of [Ca2+](i) but abolished thapsigargin-d ependent, but not angiotensin II- or ATP-induced Ca2+ rises when administer ed during a 12-hour preincubation. Conclusions. Our data suggest that a sustained increase of [Ca2+](i) may se rve as a signal to trigger MC apoptosis. Growth factors such as PDGF can ab olish apoptosis induced by elevations of [Ca2+](i) by altering intracellula r Ca2+ signaling.