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.

Glomerular complement regulation is overwhelmed in passive Heymann nephritis.

BACKGROUND: An injection of anti-Fx1A antibodies in rats leads to passive Heymann nephritis (PHN), a model of membranous nephropathy. Fx1A is a crude extract of renal cortex that contains megalin as a principal component. However, when rats are given anti-megalin antibodies, abnormal proteinuria does not occur. Because of the established complement dependence of PHN, we hypothesized that antibodies neutralizing complement regulatory proteins in the rat glomerulus also were required to induce PHN. Two likely targets are Crry and CD59, proteins abundant on the rat podocyte and contained within Fx1A that inhibit the C3 convertase and C5b-9 assembly, respectively. METHODS: Rats were injected with anti-megalin monoclonal antibodies, followed by anti-Crry and/or anti-CD59 F(ab')(2) antibodies five days later. In a second group of experiments, rats were injected with anti-Fx1A or anti-Fx1A immunodepleted of reactivity against Crry and/or CD59. RESULTS: In the setting of podocyte-associated anti-megalin monoclonal antibodies, simultaneous neutralization of Crry and CD59 function led to the development of significant proteinuria (11.0 +/- 2.1 mg/day, P < 0.001 vs. all other groups). In contrast, animals that had neither or only one of these complement regulators inhibited had normal urinary protein excretion (< or =6 mg/day). In animals given anti-Fx1A depleted of anti-Crry and/or anti-CD59, all groups developed typical PHN, characterized by heavy proteinuria and extensive glomerular deposition of C3 and C5b-9. CONCLUSION: Crry and CD59 play an important role in restraining complement-mediated injury following subepithelial immune complex deposition; however, in PHN, their regulatory capacity is overwhelmed.

A protein with characteristics of factor H is present on rodent platelets and functions as the immune adherence receptor.

Complement-coated particles interact with specific immune adherence receptors (IAR). In primates, this function is served by complement receptor 1 (CR1) on erythrocytes. In contrast, rodent platelets bear IAR distinct from CR1, the identity of which was studied here. A 150-kDa C3b-binding protein was isolated from rat platelets, which had immunochemical and biochemical identity to plasma factor H. Immunofluorescence microscopy and flow cytometry demonstrated that factor H was present on the surface of rat and mouse platelets, which could be removed by treatment with neuraminidase. Sheep erythrocytes bearing C3b underwent immune adherence with rat and mouse platelets, which was blocked with anti-factor H F(ab')(2) antibodies, but not with antibodies binding to the complement regulator, Crry, on the platelet surface. By reverse transcription-polymerase chain reaction using rat platelet RNA and primers designed from mouse factor H, a 472-base pair product was generated that was identical in sequence to that produced from rat liver RNA. The translated protein product was 85% similar to mouse liver factor H. The 3'-nucleotide sequence from platelets predicted a soluble factor H protein. By Northern analysis, liver and platelets had identically sized factor H mRNA. Thus, rat and mouse platelets have a membrane protein with characteristics of factor H that is linked via sialic acid residues and functions as the IAR. Whether platelet factor H is acquired by passive adsorption from sera and/or is produced by platelets remains to be determined.

Complement is activated in kidney by endotoxin but does not cause the ensuing acute renal failure.

BACKGROUND: Acute renal failure (ARF) in sepsis occurs when the release of multiple inflammatory mediators is induced by bacterial endotoxins. C3 mRNA is markedly up-regulated in mouse kidney after exposure to lipopolysaccharide (LPS). We hypothesized that LPS could induce tubular synthesis and secretion of C3, leading to activation of the complement cascade and direct renal tubular injury. METHODS: ARF was induced in mice by intravenous injection of LPS and was confirmed by an acute rise in blood urea nitrogen (BUN) and histologically by acute tubular necrosis. Three separate strategies were used to investigate the role of the complement system in this model of ARF: (1) Crry-Ig, a recombinant protein containing the potent murine complement C3 activation inhibitor Crry was injected at the same time as LPS (N = 8). (2) LPS was injected into transgenic mice overexpressing Crry in glomeruli and tubules (N = 8), and (3) LPS was injected into C3-deficient mice (N = 5). RESULTS: Compared with unmanipulated mice, C3 staining by immunofluorescence (IF) microscopy in mice injected with LPS was greater in renal cortical tubular cells (IF score of 2. 1 +/- 0.1 vs. 1.4 +/- 0.2 in controls, P = 0.013), most prominently at the basolateral surface. LPS injection led to a 16- to 42-fold increase in urinary C3 excretion. Despite reduction or complete elimination of renal C3 with maneuvers suppressing complement activation, BUN values were not statistically different across all groups. In no experiment did BUN values correlate with the extent of C3 staining. CONCLUSION: Although LPS up-regulates renal C3 synthesis, resulting in basolateral tubular C3 deposition, this is not responsible for LPS-induced ARF in mice.