Renal effects of the serine protease inhibitor aprotinin in healthy conscious mice

Abstract

Treatment with aprotinin, a broad-spectrum serine protease inhibitor with a molecular weight of 6512 Da, was associated with acute kidney injury, which was one of the reasons for withdrawal from the market in 2007. Inhibition of renal serine proteases regulating the epithelial sodium channel ENaC could be a possible mechanism. Herein, we studied the effect of aprotinin in wild-type 129S1/SvImJ mice on sodium handling, tubular function, and integrity under a control and low-salt diet. Mice were studied in metabolic cages, and aprotinin was delivered by subcutaneously implanted sustained release pellets (2 mg/day over 10 days). Mean urinary aprotinin concentration ranged between 642 ± 135 (day 2) and 127 ± 16 (day 8) µg/mL . Aprotinin caused impaired sodium preservation under a low-salt diet while stimulating excessive hyperaldosteronism and unexpectedly, proteolytic activation of ENaC. Aprotinin inhibited proximal tubular function leading to glucosuria and proteinuria. Plasma urea and cystatin C concentration increased significantly under aprotinin treatment. Kidney tissues from aprotinin-treated mice showed accumulation of intracellular aprotinin and expression of the kidney injury molecule 1 (KIM-1). In electron microscopy, electron-dense deposits were observed. There was no evidence for kidney injury in mice treated with a lower aprotinin dose (0.5 mg/day). In conclusion, high doses of aprotinin exert nephrotoxic effects by accumulation in the tubular system of healthy mice, leading to inhibition of proximal tubular function and counterregulatory stimulation of ENaC-mediated sodium transport.

Transmembrane serine protease 2 (TMPRSS2) proteolytically activates the epithelial sodium channel (ENaC) by cleaving the channel’s γ-subunit

“…Indeed, aprotinin has been reported to inhibit ENaC-mediated sodium transport to different degrees in toad urinary bladder (91), A6 Xenopus kidney epithelial cells (43), mpkCCDc14 mouse cortical collecting duct cells (50) and primary nasal epithelial cells (57). Intriguingly, aprotinin treatment resulting in high urinary aprotinin concentrations failed to prevent basal proteolytic ENaC cleavage in kidney of healthy mice (92). This suggests predominant intracellular proteolytic ENaC activation in the distal nephron under physiological conditions consistent with previously reported findings that ENaC is present mainly in its cleaved form in the apical membrane of mouse and rat distal nephron (93).…”

ENaC activation by proteases

“…This was explained by a counterregulatory stimulation of ENaC-mediated sodium transport. 90 Further studies are needed to unveil the in vivo effect of aprotinin on sodium transport independent of the proteolytic ENaC activation.…”

“…In this context, it is interesting to note that aprotinin exerted nephrotoxic effects in healthy mice by inhibiting proximal tubular function and unexpectedly led to increased proteolytic ENaC activation. This was explained by a counterregulatory stimulation of ENaC‐mediated sodium transport 90 . Further studies are needed to unveil the in vivo effect of aprotinin on sodium transport independent of the proteolytic ENaC activation.…”

Rodent models to study sodium retention in experimental nephrotic syndrome

“…Aprotinin's effect in nephrotic mice was later found to be paralleled by the normalization of the expression of fully cleaved γ‐ENaC (Figure 2G, 108 ). In healthy mice, aprotinin was found to accumulate in the kidney, thereby inducing injury to the proximal tubule 109 . In theory, this could contribute to the natriuretic effects of aprotinin seen in nephrotic mice.…”

“…In healthy mice, aprotinin was found to accumulate in the kidney, thereby inducing injury to the proximal tubule. 109 In theory, this could contribute to the natriuretic effects of aprotinin seen in nephrotic mice. However, proteolytic ENaC activation was only prevented in aprotinin-treated nephrotic but not in healthy mice, which might be explained by the dominant effect of soluble proteases in NS.…”