Endothelial cell damage in glomeruli and kidney arterioles appears to play a pivotal role in glomerular inflammatory diseases. Glomerular endothelial cells, a specialized microvascular cell type involved in the regulation of glomerular ultrafiltration, die by apoptosis in response to tumour necrosis factor‐α (TNF‐α), TNF‐α/basic fibroblast growth factor (bFGF), TNF‐α/cycloheximide, and bacterial lipopolysaccharide (LPS). Apoptotic cell death is characterized by extensive DNA cleavage, DNA ladder formation, and characteristic morphological alterations. In search for apoptosis‐preventing signals, we identified glucocorticoids as potent death preventing factors. Co‐treatment of cells with 10 nM dexamethasone and TNF‐α, TNF‐α/bFGF, TNF‐α/cycloheximide, or LPS blocked roughly 90% of apoptotic cell death in glomerular endothelial cells. Similarly to dexamethasone (TNF‐α‐ and LPS‐induced apoptosis are prevented with IC50 values of 0.8 and 0.9 nM, respectively), other synthetic and natural forms of glucocorticoids, such as fluocinolone, prednisolone, hydrocortisone, and corticosterone potently inhibited cell death with IC50 values of 0.2, 6, 50 and 1000 nM, for TNF‐α and 0.7, 8, 100 and 500 nM for LPS, respectively. Apart from glucocorticoids, mineralocorticoids such as aldosterone also blocked TNF‐α/LPS‐induced apoptosis (IC50 ∼500 nM for TNF‐α and ∼500 nM for LPS), whereas sex hormones, i.e. β‐estradiol and testosterone remained without effect. The protective effect of glucocorticoids (and mineralocorticoids) required glucocorticoid receptor binding as it could be antagonized by the glucocorticoid receptor antagonist RU‐486. Concerning TNF‐α and LPS signal transduction, we found that dexamethasone efficiently prevented TNF‐α‐ and LPS‐induced activation of caspase‐3‐like proteases. Therefore, we postulate inhibitory mechanisms upstream of terminal death pathways. British Journal of Pharmacology (1999) 127, 1633–1640; doi:

1 Tumour necrosis factor-a (TNF-a)-and lipopolysaccharide (LPS)-induced apoptosis of bovine glomerular endothelial cells is now recognized as an important part in the pathogenesis of glomerulonephritis characterized by early mitochondrial cytochrome c release, mitochondrial permeability transition, Bak protein upregulation, Bcl-X L protein downregulation and caspase-3 activation. 2 Co-treatment of cells with 10 nM dexamethasone and TNF-a or LPS blocked roughly 90% of apoptotic cell death in glomerular endothelial cells. The action of glucocorticoids could be documented in that they prevented all apoptotic markers such as DNA laddering, DNA fragmentation measured by the diphenylamine assay as well as morphological alterations. 3 To mechanistically elucidate the action of glucocorticoids we evaluated whether glucocorticoids elicit a time-dependent eect. For dexamethasone, to maximally inhibit DNA fragmentation a preincubation period was not required. Even if dexamethasone was supplemented 6 h following TNF-a or LPS we observed a maximal inhibitory eect. 4 Concerning its in¯uence on TNF-a and LPS signal transduction, we found that dexamethasone only partially prevented cytochrome-c-release as a ®rst sign of apoptotic cell death but eciently blocked mitochondrial permeability transition. Moreover, TNF-a-and LPS-induced Bak upregulation, Bcl-X L -downregulation, and the activation of caspase-3-like proteases, measured uorometrically using DEVD-AMC and PARP cleavage, were eciently blocked by dexamethasone. 5 We postulate that glucocorticoids exert their inhibitory action upstream of the terminal death pathways but downstream of primary receptor mediated signals by blocking pro-apoptotic signals pre-and/or post cytochrome c release and mitochondrial signalling.