Saleh H, Schlatter E, Lang D, Pauels H G, Heidenreich S
Department of Medicine, University of Münster, Münster, Germany.
Kidney Int. 2000 Nov;58(5):1876-84. doi: 10.1111/j.1523-1755.2000.00359.x.
In inflammatory glomerular diseases, proliferation, as well as apoptosis of mesangial cells (MCs), has been shown histomorphologically. Both processes may regulate the cellular content of the mesangium by closely influencing each other. In the present study, we examined whether the cytoplasmic free Ca(2+) concentration Ca(2+) is involved as a key second messenger in the regulation of proliferative and apoptotic events.
Thapsigargin, an inhibitor of the endoplasmic Ca(2+)-Mg(2+)-ATPase, was used as a test substance to investigate the role of Ca(2+) in signaling MC apoptosis and growth in vitro. Apoptosis was determined by nuclear chromatin staining with Hoechst 33258, by a [3H]-thymidine-based DNA fragmentation assay or by flow cytometry detecting binding of FITC-conjugated annexin V. Proliferation was measured by [3H]-thymidine incorporation into acid-precipitable material and corroborated by cell counting.
Thapsigargin significantly induced apoptosis and inhibited proliferation dose dependently in nanomolar concentrations without evoking necrotic damage when administered not longer than 12 hours. Significant apoptosis was measurable after a six-hour treatment of MCs with thapsigargin. Determination of Ca(2+) by fura-2-dependent spectrofluorometry showed that thapsigargin was able to induce prolonged Ca(2+) rises that could be prevented by preincubation with the intracellular Ca(2+) chelator 1, 2-bis(2-aminophenoxy)-ethane-N,N,N', N'-tetra-acetic acid (BAPTA) acetomethyl ester (AM). BAPTA had no influence on MC viability but reversed thapsigargin-induced apoptosis to control levels. After thapsigargin treatment (100 nmol/L, 12 hours), apoptotic MCs had a significantly higher Ca(2+) of 251 +/- 25 nmol/L (N = 41) as compared with MCs that were not or not yet apoptotic (Ca(2+) 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-dependent, but not angiotensin II- or ATP-induced Ca(2+) rises when administered during a 12-hour preincubation.
Our data suggest that a sustained increase of Ca(2+) may serve as a signal to trigger MC apoptosis. Growth factors such as PDGF can abolish apoptosis induced by elevations of Ca(2+) by altering intracellular Ca(2+) signaling.
在炎症性肾小球疾病中，组织形态学已显示系膜细胞（MCs）的增殖以及凋亡。这两个过程可能通过相互密切影响来调节系膜的细胞含量。在本研究中，我们检测了细胞质游离钙离子浓度[Ca(2+)]i是否作为关键的第二信使参与增殖和凋亡事件的调节。
毒胡萝卜素，一种内质网Ca(2+)-Mg(2+)-ATP酶抑制剂，用作测试物质来研究[Ca(2+)]i在体外调节MC凋亡和生长中的作用。通过用Hoechst 33258进行核染色质染色、基于[3H]-胸腺嘧啶核苷的DNA片段化分析或通过流式细胞术检测FITC偶联的膜联蛋白V的结合来确定凋亡。通过[3H]-胸腺嘧啶核苷掺入酸沉淀物质来测量增殖，并通过细胞计数进行证实。
当给药时间不超过12小时时，毒胡萝卜素在纳摩尔浓度下显著诱导凋亡并剂量依赖性抑制增殖，且不引起坏死性损伤。用毒胡萝卜素处理MCs 6小时后可检测到明显的凋亡。通过fura-2依赖性荧光分光光度法测定[Ca(2+)]i表明，毒胡萝卜素能够诱导[Ca(2+)]i长时间升高，而预先用细胞内Ca(2+)螯合剂1,2-双(2-氨基苯氧基)-乙烷-N,N,N',N'-四乙酸（BAPTA）乙酰甲酯（AM）孵育可预防这种升高。BAPTA对MC活力没有影响，但可将毒胡萝卜素诱导的凋亡逆转至对照水平。毒胡萝卜素处理（100 nmol/L，12小时）后，凋亡的MCs的[Ca(2+)]i显著高于未凋亡或尚未凋亡的MCs（[Ca(2+)]i为116±20 nmol/L，N = 26，P<0.05），为251±25 nmol/L（N = 41）。血小板衍生生长因子（PDGF），一种已充分表征的MC生长因子，以与BAPTA类似的方式逆转了毒胡萝卜素对增殖和凋亡的影响。PDGF急性刺激[Ca2+]i升高，但在12小时预孵育期间给药时可消除毒胡萝卜素依赖性但不能消除血管紧张素II或ATP诱导的Ca(2+)升高。
我们的数据表明，[Ca(2+)]i的持续升高可能作为触发MC凋亡的信号。诸如PDGF之类的生长因子可通过改变细胞内Ca(2+)信号传导来消除由[Ca(2+)]i升高诱导的凋亡。
