Protamine Sulfate and Oxidant Stress Cause Similar Changes in Hsp27 Intracellular Distribution in Cultured Podocytes

Abstract 1983 Poster Session II, Sunday, 5/2 (poster 189)

Nephrotic syndrome (NS) is characterized by massive proteinuria and dramatic structural changes in podocyte foot processes of the glomerular filtration barrier. These changes have been associated in vivo with oxidant injury to podocytes, and increased glomerular expression and phosphorylation of the small heat shock protein, hsp27, a reported regulator of actin polymerization. Protamine sulfate has been used as a model of NS in vivo because it has been shown to reproduce the structural and pathophysiological changes that occur during NS. To determine if well-described agents for inducing NS, such as PS, might act via induction of podocyte oxidant injury, we compared the changes in the intracellular distribution of hsp27 in cultured "differentiated" podocytes following treatment with PS to that following treatment with two known oxidants, menadione or hydrogen peroxide. Treated podocytes were extracted into a cytoskeleton-stabilizing buffer and cellular components were separated by centrifugation into a low-speed pellet (LSP, 10,000 × g), a high-speed pellet (HSP, 100,000 × g), and a high-speed supernatant (HSS) containing, respectively, large cytoskeletal components and protein aggregates, smaller filaments, and soluble components. Comparison of the hsp27 content of these fractions following treatment for 1 h with 1 mg/mL PS, 20 µM menadione, or 1 mM hydrogen peroxide, revealed a dramatic increase in the amount of hsp27 in the LSP and HSP after all treatments (340% PS, 250% menadione, 300% H₂O₂ in LSP; 200% PS, 180% menadione, 190% H₂O₂ in HSP) while there was a striking decrease in the amount of hsp27 in the HSS (60% PS, 52% menadione, 58% H₂O₂. Because of the relative distribution of protein between the pellet fractions and supernatant, these changes in hsp27 within each fraction translate to a major redistribution of the soluble vs. insoluble ratio of total cellular hsp27 following all treatments, from 70% of hsp27 being soluble (i.e. in the HSS) prior to treatment to approximately 35% remaining soluble after treatment with PS or either oxidant. These dramatic alterations in the intracellular distribution of hsp27 in response to PS, and their similarity to that following treatment with two different oxidants, suggest that: 1) PS induces a dramatic intracellular redistribution of hsp27 in podocytes, and 2) This change may occur either via induction of podocyte oxidant injury or by a common or parallel pathway to that seen following direct oxidant injury. Whether the reduction in hsp27 solubility following these treatments reflects aggregation of hsp27 or interaction of hsp27 with the disrupted actin filaments known to develop during podocyte foot process effacement is currently being analyzed.