Anethole Dithiolethione Increases Glutathione in Kidney by Inhibiting γ-Glutamyltranspeptidase: Biochemical Interpretation and Pharmacological Consequences

Aims. Anethole dithiolethione (ADT) is a marketed drug to treat xerostomia. Its mechanism of action is still unknown, but several preclinical studies indicate that it is able to increase intracellular glutathione (GSH) and protect against oxidative stress. Here, we investigated the molecular mechanisms behind these effects. Results. Oral treatment of rats confirmed the GSH enhancing properties of ADT; among the different organs examined in this study, only the kidney showed a significant GSH increase that was already observed at low-dose treatments. The increase in GSH correlated with a decrease in γ-glutamyltranspeptidase (γ-GT) activity of the different tissues. In vitro and ex vivo experiments with tubular renal cells and isolated perfused rat kidney showed that the cellular uptake of intact GSH was correlated with the extracellular concentrations of GSH. Conclusions. The prominent in vivopharmacological effect of ADT was a marked increase of GSH concentration in the kidney and a decrease of some systemic and renal biomarkers of oxidative stress. In particular, by inhibition of γ-GT activity, it decreased the production cysteinylglycine, a thiol that has prooxidant effects as the consequence of its autooxidation. The activity of ADT as GSH enhancer in both the circulation and the kidney was long-lasting. All these characteristics make ADT a promising drug to protect the kidney, and in particular proximal tubule cells, from xenobiotic-induced damage.

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 = 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 ≤ 0.04, except for sodium reabsorption under constant pressure [P = 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 ≤ 0.03 except for urine output with raxibacumab [P = 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 ± 1.06 ng/ml versus 1.51 ± 0.44 ng/ml [means ± standard errors of the means {SEM}; P = 0.0001). Edema toxin has renal effects that could contribute to extravascular fluid collection characterizing anthrax infection clinically.

Involvement of neutral sphingomyelinase in the angiotensin II signaling pathway

The possibility that angiotensin II (ANG II) exerts its effects through the activation of neutral sphingomyelinase (nSMase) has not been tested in kidneys. The results of the present study provide evidence for the activity and expression of nSMase in rat kidneys. In isolated perfused rat kidney, ANG II-induced renal vasoconstriction was inhibited by GW4869, an inhibitor of nSMase. We used nSMase for investigating the signal transduction downstream of ceramide. nSMase constricted the renal vasculature. An inhibitor of ceramidase (CDase), N-oleoylethanolamine (OEA), enhanced either ANG II- or nSMase-induced renal vasoconstriction. To demonstrate the interaction between the nSMase and cytosolic phospholipase A2 (cPLA2) signal transduction pathways, we evaluated the response to nSMase in the presence and absence of inhibitors of arachidonic acid (AA) metabolism: arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor of cPLA2; 5,8,11,14-eicosatetraynoic acid (ETYA), an inhibitor of all AA pathways; indomethacin, an inhibitor of cyclooxygenase (COX); furegrelate, a thromboxane A2 (TxA2)-synthase inhibitor; and SQ29548 , a TxA2-receptor antagonist. In these experiments, the nSMase-induced renal vasoconstriction decreased. ANG II or nSMase was associated with an increase in the release of thromboxane B2 (TxB2) in the renal perfusate of isolated perfused rat kidney. In addition, the coexpression of the ceramide with cPLA2, was found in the smooth muscle layer of intrarenal vessels. Our results suggest that ANG II stimulates ceramide formation via the activation of nSMase; thus ceramide may indirectly regulate vasoactive processes that modulate the activity of cPLA2 and the release of TxA2.