Inhibition of NF-kappaB-dependent Bcl-xL expression by clusterin promotes albumin-induced tubular cell apoptosis.

Apoptosis and inflammation, important contributors to the progression of chronic kidney disease, can be influenced by clusterin (a secreted glycoprotein that regulates apoptosis) and nuclear factor-kappaB (NF-kappaB, a transcription factor modifying the expression of inflammatory genes). We studied proteinuria-induced renal disease and its influence on clusterin-mediated apoptosis. Exposure of cultured mouse proximal tubule epithelial cells to bovine serum albumin (BSA) resulted in activation of NF-kappaB and activator protein-1 (AP-1) within hours followed by a decline in their activation, decreased activation of extracellular signal-regulated kinases (ERK1/2), decreased cell-associated antiapoptotic Bcl-xL protein but increased apoptosis. Clusterin progressively increased in the media over a 3 day period. Clusterin siRNA blocked protein production, increased NF-kappaB activation, and significantly increased cellular Bcl-xL protein, thereby reducing spontaneous and BSA-induced apoptosis. An siRNA to the NF-kappaB inhibitor IkappaBalpha had similar results. BSA-stimulated NF-kappaB activation reciprocally decreased AP-1 activity by preventing ERK1/2 phosphorylation. These in vitro studies suggest that clusterin inhibits NF-kappaB-mediated antiapoptotic effects by the apparent stabilization of IkappaBalpha switching from promoting inflammation to apoptosis during proteinuria.

Tranilast inhibits interleukin-1beta-induced monocyte chemoattractant protein-1 expression in rat mesangial cells.

Monocyte chemoattractant protein-1 (MCP-1), a member of the CC subfamily of chemokines, plays a crucial role in the progression of glomerulonephritis by recruitment of monocytes. Tranilast, a clinically used anti-allergic drug, has been demonstrated to have various anti-inflammatory and anti-proliferative effects, and recently has been reported to prevent restenosis after percutaneous transluminal coronary angioplasty. In this study, we investigated whether tranilast inhibits MCP-1 secretion in mesangial cells. Tranilast inhibited interleukin-1beta-induced MCP-1 secretion and mRNA expression in a concentration-dependent manner. Luciferase assay showed that tranilast suppressed interleukin-1beta-induced nuclear factor-kappaB (NF-kappaB)-dependent transcription. Interleukin-1beta-induced Jun N-terminal kinase (JNK) activation was also suppressed selectively by tranilast. These results indicate that tranilast inhibits interleukin-1beta-induced MCP-1 production, at least in part, by inhibiting NF-kappaB activity and that suppression of JNK activation might be involved in the inhibition of MCP-1 production. Tranilast may serve as a new therapeutic agent for glomerulonephritis through anti-chemokine property.

Overexpression of truncated I kappa B alpha potentiates TNF-alpha-induced apoptosis in mesangial cells.

BACKGROUND: Dysregulation of apoptosis is one of the likely underlying mechanisms of mesangial proliferative glomerulonephritis (GN), a disease in which proinflammatory cytokines exhibit a wide range of biological activities. Among them, tumor necrosis factor-alpha (TNF-alpha) induces two conflicting pathways, one leading to activation of the nuclear factor-kappa B (NF-kappa B), and the other leading to caspase-mediated apoptosis. We investigated whether or not specific inhibition of NF-kappa B affects TNF-alpha-induced apoptosis in rat mesangial cells (MCs). METHODS: To specifically inhibit NF-kappa B activation, we constructed a recombinant adenovirus vector expressing a truncated form of I kappa B alpha (AdexI kappa B delta N) that lacks the phosphorylation sites essential for the activation of NF-kappa B. Electrophoretic mobility shift assay was performed to evaluate NF-kappa B activity. Nuclear morphology was observed by staining with Hoechst-33258. DNA fragmentation was detected using an ELISA kit with an antihistone antibody. To investigate the regulation of apoptosis, we measured caspase-3 and caspase-8 activity by ELISA, and examined the Bcl-2 and Bax protein level by Western blot. RESULTS: TNF-alpha-induced NF-kappa B activation was blocked by overexpression of I kappa B delta N. Overexpression of I kappa B delta N potentiated TNF-alpha-induced apoptosis compared to mock transfection, and the potentiation was abolished by treatment with a caspase-3 inhibitor, Z-DEVD-FMK. Overexpression of I kappa B delta N augmented TNF-alpha-induced caspase-3 and caspase-8 activity, but did not affect Bcl-2 or Bax protein expression. CONCLUSION: Overexpression of I kappa B delta N potentiates TNF-alpha-induced apoptosis and augments caspase-8 and caspase-3 activity in rat MCs without changing Bcl-2 or Bax protein expression. These results suggest the potential usefulness of AdexI kappa B delta N to induce apoptosis in MCs under inflammatory conditions.

Neonatal diarrhoea in calves given milk-substitutes differing in fat source and fed by different procedures.

1. The incidence of diarrhoea, digestibilities of nutrients and the faecal bacterial flora were compared among three groups of Holstein male calves up to 3 weeks of age. Two groups of four calves were given a milk-substitute containing tallow by nipple-pail (group TN) and by open-bucket (group TB). The third group of four calves was nipple-fed a milk-substitute containing soya-bean oil (group SN). Each of the milk-substitutes contained approximately 300 g milk-protein and 100 g fat/kg dry matter (DM)., 2. Mean faecal DM contents (g/kg) were 217, 185 and 112 for groups TN, TB, and SN respectively and the corresponding pH values were 7.21, 7.00 and 6.50. The difference between groups TN and SN was statistically significant (P less than 0.05). 3. No difference was observed between groups TN and SN in the apparent digestibilities of DM, crude protein (CP, nitrogen X 6.25), diethyl ether extract (EE) and total reducing sugars. But in the group TB, the digestibility of EE was significantly lower (P less than 0.05), and that of CP tended to be, though not significantly, lower than in the other two groups. 4. Bacterial flora in faeces showed considerably wide quantitative variations among individual calves, but there was a tendency for increased viable counts of Lactobacilli in faeces of group SN. 5. The present suggested that an appreciable difference in the mechanism would exist between diarrhoea occurring when milk-substitute was offered by bucket and when highly-unsaturated vegetable oils were contained in it. Possible mechanisms were also discussed.