Polymyxin-B hemoperfusion inactivates circulating proapoptotic factors

Abstract

Objective: To test the hypothesis that extracorporeal therapy with polymyxin B (PMX-B) may prevent Gram-negative sepsis-induced acute renal failure (ARF) by reducing the activity of proapoptotic circulating factors.

Setting: Medical-Surgical Intensive Care Units.

Patients and interventions: Sixteen patients with Gram-negative sepsis were randomized to receive standard care (Surviving Sepsis Campaign guidelines) or standard care plus extracorporeal therapy with PMX-B.

Measurements and results: Cell viability, apoptosis, polarity, morphogenesis, and epithelial integrity were evaluated in cultured tubular cells and glomerular podocytes incubated with plasma from patients of both groups. Renal function was evaluated as SOFA and RIFLE scores, proteinuria, and tubular enzymes. A significant decrease of plasma-induced proapoptotic activity was observed after PMX-B treatment on cultured renal cells. SOFA and RIFLE scores, proteinuria, and urine tubular enzymes were all significantly reduced after PMX-B treatment. Loss of plasma-induced polarity and permeability of cell cultures was abrogated with the plasma of patients treated with PMX-B. These results were associated to a preserved expression of molecules crucial for tubular and glomerular functional integrity.

Conclusions: Extracorporeal therapy with PMX-B reduces the proapoptotic activity of the plasma of septic patients on cultured renal cells. These data confirm the role of apoptosis in the development of sepsis-related ARF.

Fig. 1

a Evaluation of tubular apoptosis (TUNEL) induced by incubation for 48 h with CONV or PMX plasmas in tubular cells. All PMX and CONV plasmas induced a significant increase of tubular apoptosis (*P < 0.05). Incubation of tubular cells with PMX T72 plasma resulted in a significant decrease of apoptosis compared to PMX T0 plasma (^† P < 0.05). LPS (30 ng/mL) was used as positive control. b Evaluation of tubular apoptosis (TUNEL) in tubular cells subjected to short interfering RNA (siRNA) for tumor necrosis factor-receptor 1 (TNF-R1) or for a noncoding control after incubation with CONV and PMX plasma. Compared to control siRNA, a significant decrease of tubular apoptosis was observed in siRNA TNF-R1 tubular cells incubated with CONV and PMX plasma (*P < 0.05). LPS (30 ng/mL) was used as positive control

Fig. 2

Top: Enzyme-linked immunoabsorbent (ELISA) evaluation of caspase-3, caspase-8, and caspase-9 activities on tubular cells cultured for 48 h with 5% CONV or PMX plasma. PMX T0 and CONV T0 and T72 plasmas induced a significant increase of all caspase activities (*P < 0.05 vs. healthy plasma). A significant decrease of all caspase activities was found with PMX T72 plasma compared to PMX T0 (^† P < 0.05 PMX T72 vs. PMX T0); however, caspase-3 and caspase-9 activities remained significantly higher than healthy plasma (*P < 0.05 PMX T72 vs. healthy plasma). Middle: Representative images of FACS and immunofluorescence (insets) analysis of Fas (CD95) expression on tubular cell surface after exposure to CONV or PMX plasma. PMX T0 and CONV T0 and T72 plasmas all induced a marked upregulation of Fas, which was significantly reduced in presence of PMX T72 plasma. ×400 magnification. Bottom: Representative western blot analysis of the mitochondrial proteins Bax and Bcl2 in tubular cells exposed to CONV or PMX plasma, and related densitometric analysis expressed as Bax/Bcl2 ratio. PMX T0, CONV T0, and CONV T72 plasmas induced a marked upregulation of the Bax/Bcl2 ratio that was reduced in the presence of PMX T72 plasma. (Lane 1 Vehicle; Lane 2 Healthy; Lane 3 PMX T0; Lane 4 PMX T72; Lane 5 CONV T0; Lane 6 CONV T72). Beta-actin was used as reference for protein loading

Fig. 3

Average fluorescence expression of megalin and ZO1 on tubular cells. PMX T0, CONV T0, and CONV T72 plasmas induced a marked reduction of megalin and ZO1 expression (*P < 0.05 vs. healthy plasma). After incubation with PMX T72 plasma, a significant increase of megalin and ZO1 staining was observed (^† P < 0.05 PMX T72 vs. PMX T0)

Fig. 4

Average fluorescence expression of nephrin and B7.1 on glomerular podocytes. PMX T0, CONV T0, and CONV T72 plasmas induced a marked reduction of nephrin and the upregulation of B7.1 (*P < 0.05 vs. healthy plasma). After incubation with PMX T72 plasma, an increased expression of nephrin and a reduced staining for B7.1 was observed (^† P < 0.05 PMX T72 vs. PMX T0)