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Both bacterial LPS and TNF-alpha potently induced apoptotic cell death in glomerular endothelial cells. Direct endotoxin-induced apoptosis may therefore be relevant in the progression of acute renal failure, which is a frequent complication of gram-negative sepsis.

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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.

Bradykinin (BK), a known vasodilator in vivo, and arginine vasopressin (AVP), a vasoconstrictor in vivo, both stimulate a rise in cytosolic free Ca2+ ([Ca2+]i) in vascular smooth muscle cells (VSMC). The present study was undertaken to investigate this apparent paradox. The following three possibilities were examined, namely, 1) signaling events other than [Ca2+]i are different for BK and AVP; 2) BK, but not AVP, stimulates prostaglandins in VSMC, thus resulting in divergent effects on VSMC tone; and 3) AVP and BK exhibit qualitatively similar effects on VSMC signal transduction but divergent effects on VSMC tone are mediated by endothelial events. The results demonstrated that BK stimulated a rise in inositol trisphosphate (IP3), [Ca2+]i, 45Ca2+ efflux, and Ca2+ influx and a biphasic change in intracellular pH when N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered solution was used. The BK-induced VSMC contraction was also comparable to that observed with AVP. The cyclooxygenase inhibitor, meclofenamate, enhanced the effect of both BK and AVP on VSMC tone, as assessed by shape change, by a comparable degree. BK, but not AVP, stimulated endothelial cells to release a substance that blocked the contractile response of BK and AVP. Methylene blue, a blocker of cytosolic guanylate cyclase and therefore of the production of guanosine 3',5'-cyclic monophosphate (2nd messenger of endothelium-derived relaxing factor, EDRF), and nordihydroguaiaretic acid, an inhibitor of EDRF, both prevented this endothelium-dependent effect of BK. These results therefore indicate that BK is a constrictor of VSMC, an effect that can be overridden by the hormone's endothelial effect to stimulate the release of a vasodilator(s), which is most likely EDRF.

This study characterized the rapid desensitization induced by arginine vasopressin (AVP) in vascular smooth muscle cells (VSMC) in culture. The Ca2+ mobilization response and, in some experiments, the intracellular pH changes were used as a probe for the desensitization phenomenon. In VSMC, AVP desensitization was homologous, concentration dependent, and occurred in less than 30 s. The desensitization was complete with 10(-7) M AVP. Receptor occupancy was a critical factor in the maintenance of desensitization, since complete hormone washing by acid glycine buffer produced an earlier (less than 5 min) recovery of the cell response, whereas partial hormone washing with saline (pH 7.4) required 15 min to produce any significant recovery. Protein kinase C activation was a significant mechanism in AVP desensitization, because protein kinase C downregulation inhibited the desensitization phenomenon. Receptor internalization was, however, not important for the desensitization phenomenon, since it still occurred at 4 degrees C. Treatment with pertussis toxin did not affect the Ca2+ mobilization response but decreased the AVP-mediated intracellular alkalinization, therefore suggesting that a Gi or Go protein may be involved in some but not all the aspects of the AVP signal transduction and the desensitization phenomena.