Bacillus anthracis Edema Toxin Increases Fractional Free Water and Sodium Reabsorption in an Isolated Perfused Rat Kidney Model

Abstract

Bacillus anthracis edema toxin (ET) consists of protective antigen (PA), necessary for host cell toxin uptake, and edema factor (EF), the toxic moiety which increases host cell cyclic AMP (cAMP). Since vasopressin stimulates renal water and sodium reabsorption via increased tubular cell cAMP levels, we hypothesized the ET would also do so. To test this hypothesis, we employed an isolated perfused rat kidney model. Kidneys were isolated and perfused with modified Krebs-Henseleit buffer. Perfusate and urine samples were obtained at baseline and every 10 min over 150 min following the addition of challenges with or without treatments to the perfusate. In kidneys perfused under constant flow or constant pressure, compared to PA challenge (n ϭ 14 or 15 kidneys, respectively), ET (13 or 15 kidneys, respectively) progressively increased urine cAMP levels, water and sodium reabsorption, and urine osmolality and decreased urine output (P Յ 0.04, except for sodium reabsorption under constant pressure [P ϭ 0.17]). In ET-challenged kidneys, compared to placebo treatment, adefovir, an EF inhibitor, decreased urine cAMP levels, water and sodium reabsorption, and urine osmolality and increased urine output, while raxibacumab, a PA-directed monoclonal antibody (MAb), decreased urine cAMP levels, free water reabsorption, and urine osmolality and increased urine output (P Յ 0.03 except for urine output with raxibacumab [P ϭ 0.17]). Upon immunohistochemistry, aquaporin 2 was concentrated along the apical membrane of tubular cells with ET but not PA, and urine aquaporin 2 levels were higher with ET (5.52 Ϯ 1.06 ng/ml versus 1.51 Ϯ 0.44 ng/ml [means Ϯ standard errors of the means {SEM}; P ϭ 0.0001). Edema toxin has renal effects that could contribute to extravascular fluid collection characterizing anthrax infection clinically.KEYWORDS anthrax, edema toxin, kidney, water reabsorption B acillus anthracis infection is characterized clinically by the marked retention and extravasation of fluid, which can compromise cardiopulmonary function and impair host defense and tissue repair (1-6). Although the loss of vascular integrity and lymphatic obstruction contribute to this fluid accumulation, the significant hyponatremia in patients in the 2001 U.S. inhalational and 2009 UK soft tissue outbreaks suggests that alterations in renal water and sodium handling may also contribute (3, 5, 7). Notably, of the two toxins produced by B. anthracis, edema toxin (ET) but not lethal toxin (LT) infusion reduced serum sodium levels in canines (8). While ET is known to produce localized edema when injected subcutaneously, understanding whether it alters renal water and sodium handling has important therapeutic implications for the fluid management of patients with severe B. anthracis infection.Edema toxin consists of protective antigen (PA), the component necessary for host cell uptake of edema factor (EF), the toxic moiety (9, 10). Edema factor has calmodulin-