Clusterin protects against oxidative stress in vitro through aggregative and nonaggregative properties

Perturbations of cell interactions, an carly event in acute renal injury, have important pathophysiologic consequences. We hypothesized that promotion of cell interactions protects cells from injury. To test this hypothesis, a single cell suspension of LLC-PK₁ cells (porcine proximal tubular cell line) treated with albumin (control) was compared to cells aggregated with fibrinogen or purified human clusterin (aggregation graded 0 to 4). Following aggregation, the cells were injured with 1.5 mM hydrogen peroxide (H₂O₂) for three hours. Cell aggregation induced by clusterin but not fibrinogen protected against oxidant injury by H₂O₂. Complete abrogation of cytotoxicity occurred at a clusterin concentration of 2.5 μg/ml, which resulted in an aggregation score of 1. In the absence of aggregation, clusterin at concentrations of 20 and 50 μg/ml, but not lower doses, partially protected against injury induced by H₂O₂. Cell aggregation induced by both clusterin and fibrinogen partially protected against endogenously generated oxidant stress induced by incubating LLC-PK₁ cells with aminotriazole and 1-chloro-2,4-dinitrobenzene (CDNB). In conclusion, clusterin protects against models of oxidant stress in vitro, whether generated by exogenously administered hydrogen peroxide, or from endogenously produced peroxide, and such protective effects can accrue from aggregative and nonaggregative properties of clusterin.