Endogenous cardiotonic steroids in chronic renal failure

Abstract

Background: Previous reports demonstrated that digitalis-like cardiotonic steroids (CTS) contribute to the pathogenesis of end-stage renal disease. The goal of the present study was to define the nature of CTS in patients with chronic kidney disease (CKD) and in partially nephrectomized (PNx) rats.

Methods: In patients with CKD and in healthy controls, we determined plasma levels of marinobufagenin (MBG) and endogenous ouabain (EO) and erythrocyte Na/K-ATPase activity in the absence and in the presence of 3E9 anti-MBG monoclonal antibody (mAb) and Digibind. Levels of MBG and EO were also determined in sham-operated Sprague-Dawley rats and in rats following 4 weeks of PNx.

Results: In 25 patients with CKD plasma, MBG but not EO was increased (0.86 ± 0.07 versus 0.28 ± 0.02 nmol/L, P < 0.01) and erythrocyte Na/K-ATPase was inhibited (1.24 ± 0.10 versus 2.80 ± 0.09 μmol Pi/mL/h, P < 0.01) as compared to that in 19 healthy subjects. Ex vivo, 3E9 mAb restored Na/K-ATPase in erythrocytes from patients with CKD but did not affect Na/K-ATPase from control subjects. Following chromatographic fractionation of uremic versus normal plasma, a competitive immunoassay based on anti-MBG mAb detected a 3-fold increase in the level of endogenous material having retention time similar to that seen with MBG. A similar pattern of CTS changes was observed in uremic rats. As compared to sham-operated animals, PNx rats exhibited 3-fold elevated levels of MBG but not that of EO.

Conclusions: In chronic renal failure, elevated levels of a bufadienolide CTS, MBG, contribute to Na/K-ATPase inhibition and may represent a potential target for therapy.

Fig. 1.

Displacement of binding of anti-MBG 4G4 (A) and 3E9 (B) mAbs, anti-ouabain S antiserum (C), anti-ouabain M antiserum (D) and Digibind (E) to conjugated antigens by cardiotonic steroids in a competitive fluoroimmunoassays.

Fig. 2.

Plasma levels of MBG measured using 4G4 anti-MBG mAb (A) and of EO measured using anti-ouabain antiserums M (B) and S (C) in healthy controls (Ctrl) and in patients with CKD. (D) Determination of levels of CTS in pooled samples of plasma from Ctrl and CKD using competitive fluoroimmunoassay based on Digibind. Means ± SEM. Two-tailed t-test. Determination of MBG immunoreactivity (4G4 mAb) (E) and of ouabain immunoreactivity determined using anti-ouabain antiserums M and S (F) in pooled plasma samples from healthy controls spiked with MBG (0.125, 0.25, 0.5, 1.0 and 2 nmol/L).

Fig. 3.

Pattern of elution of endogenous MBG and ouabain immunoreactivity following fractionation of extracts of normal human plasma (A, C, E and G) and of human uremic plasma (B, D, F and H) on reverse-phase HPLC column in the linear gradient of acetonitrile (dotted line). The retention times of MBG and ouabain (OU) standards are indicated by arrows.

Fig. 4.

(A) Erythrocyte Na/K-ATPase in healthy control subjects and in patients with CKD in the absence and in the presence of Digibind and 3E9 anti-MBG mAb. Means ± SEM. One-way analysis of variance followed by Newman–Keuls test. (B) Correlation between plasma levels of MBG and activity of Na/K-ATPase in erythrocytes in patients with CKD. Two-tailed t-test.

Fig. 5.

CTS in rats with renal failure. Plasma levels of MBG measured using 4G4 anti-MBG mAb (A) and of EO measured using antiserums M (B) and S (C) in sham-operated (Sham) and PNx rats. (D) Determination of levels of CTS in pooled samples of plasma from Sham and PNx using competitive fluoroimmunoassay based on Digibind. Means ± SEM. Two-tailed t-test.