Renal Effects of the Serine Protease Inhibitor Aprotinin in Healthy Conscious Mice

www.nature.com/aps ARTICLE OPEN Renal effects of the serine protease inhibitor aprotinin in healthy conscious mice Stefan Wörner1, Bernhard N. Bohnert1,2,3, Matthias Wörn1, Mengyun Xiao1, Andrea Janessa1, Andreas L. Birkenfeld1,2,3, Kerstin Amann4, Christoph Daniel4 and Ferruh Artunc 1,2,3 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 1234567890();,: function and counterregulatory stimulation of ENaC-mediated sodium transport. Keywords: aprotinin; serine proteases; renal dysfunction; epithelial sodium channel; sodium Acta Pharmacologica Sinica (2021) 0:1–10; https://doi.org/10.1038/s41401-021-00628-1 INTRODUCTION Serine proteases are involved in the intrarenal regulation of the Aprotinin is a broad-spectrum serine protease inhibitor that was epithelial sodium channel ENaC that is expressed in the first isolated from cow parotis gland and pancreas in the 1930s. It aldosterone-sensitive distal nephron and determines the final is an unselective inhibitor of trypsin-like serine proteases such as urinary sodium concentration [10, 11]. ENaC is a heterotrimer trypsin, plasmin, plasma and tissue kallikreins, coagulation factor composed of three subunits named α, β, and γ, each participating XII, and others [1]. Aprotinin was used as an antifibrinolytic agent in the formation of the channel pore [12]. A special feature of in cardiac surgery to reduce blood loss associated with the ENaC is its regulation by serine proteases, which remove inhibitory extracorporal circuit [2]. Due to its peptide character, aprotinin is tracts from the α- and γ-subunits to fully activate the channel freely filtered and almost completely excreted by the kidneys. [10, 13, 14]. During maturation intracellular furin cleaves α-ENaC Between 2006 and 2008, studies appeared that found an twice leading to the release of one inhibitory tract and γ-ENaC increased risk for renal dysfunction and even mortality in patients once. Final proteolysis and release of a second inhibitory tract undergoing cardiac surgery who were treated with aprotinin [3–6]. from the γ-subunit requires an additional serine protease. Several Renal dysfunction manifested as acute kidney injury ranging from renally expressed serine proteases such as membrane-anchored small increases in serum creatinine concentration to dialysis- prostasin [15, 16] or tissue kallikreins [17] have been shown to be dependent renal failure. After early termination of the BART trial involved in proteolytic ENaC activation. An important fact is that (Blood Conservation Using Antifibrinolytics in a Randomized Trial) they are all aprotinin-sensitive. Pathophysiologically, proteolytic due to increased mortality of aprotinin-treated patients [7], activation of ENaC by aberrantly filtered serine proteases or aprotinin was withdrawn from the market in 2007. Since then, proteasuria has been implicated in the sodium retention seen in the mechanisms leading to renal dysfunction remain ill-defined nephrotic syndrome [18]. Our group has shown that treatment of [8]. However, in the last years aprotinin is being reintroduced in nephrotic mice with the serine protease inhibitor aprotinin Europe and Canada after reevaluation of the safety data, prevented ENaC-mediated sodium retention and edema forma- highlighting several methodological limitations [9]. tion as did the ENaC blocker amiloride [19–21]. 1Division of Endocrinology, Diabetology and Nephrology, Department of Internal Medicine, University Hospital Tübingen, Tübingen, Germany; 2Institute of Diabetes Research and Metabolic Diseases (IDM) of the Helmholtz Center Munich at the University Tübingen, Tübingen, Germany; 3German Center for Diabetes Research (DZD) at the University Tübingen, Tübingen, Germany and 4Department of Nephropathology, Friedrich-Alexander University (FAU) Erlangen-Nürnberg (FAU), Erlangen, Germany Correspondence: Ferruh Artunc ([email protected]) These authors contributed equally: Stefan Wörner, Bernhard N. Bohnert Received: 10 December 2020 Accepted: 12 February 2021 © The Author(s) 2021 Aprotinin in healthy mice S Wörner et al. 2 Currently, the translation of the beneficial effects of aprotinin to measured from 5 µL urine samples incubated for 48 h at 37 °C patients with nephrotic syndrome is difficult due to the with- with 10 µL substrate (P-Check®: 1 mg/mL) and 85 µL Tris-Buffered drawal of aprotinin and its association with renal dysfunction. We Saline in a total volume of 100 µL. Increase in fluorescence counts hypothesized that the mechanism for the negative renal effects of was detected on a fluorescence reader (Tecan, Spark®10 M). aprotinin might be related to the inhibition of serine proteases Values were expressed as relative fluorescence units [23]. that are physiologically involved in ENaC regulation, thereby Creatinine, glucose, phosphorus, and urea concentrations were leading to kidney injury by sodium and volume loss. We therefore measured with colorimetric assays (Labor + Technik, Berlin, undertook this study to investigate the impact of aprotinin Germany) and urinary sodium concentrations with flame photo- treatment on sodium handling in healthy wild-type mice under metry (Eppendorf EFUX 5057, Hamburg, Germany). Plasma sodium control and low-salt conditions. concentrations were measured using an IL GEM® Premier 3000 blood gas analyzer (Instrumentation Laboratory, Munich, Ger- many). ELISA kits were used to measure plasma aldosterone (IBL, MATERIALS AND METHODS Germany), plasma and urinary cystatin C (R&D systems, USA), and Mouse studies urinary aprotinin (Cloud corp, PRC). Urinary albumin was Experiments were performed on 6-month-old wild-type 129S1/ measured using a fluorometric kit against mouse albumin as SvImJ mice of both sexes. Mice were kept up to five in standard (Active motif, Carlsbad, USA). Urinary amiloride concen- Eurostandard type II long cages (365 mm × 207 mm × 140 mm, tration was measured fluorometrically according to Baer et al. [24]. area 530 cm2) on a bedding from wood chips. Mouse houses made of plastic, wooden tunnels, and cellulose were also used as Western blot enrichment. The light–dark cycle was 12:12 h at an ambient Western blot analysis for γ-ENaC expression was performed from a temperature of 22–24 °C. Mice were fed a standard diet (ssniff, membrane protein preparation of kidney cortex collected from sodium content 0.24% corresponding to 104 µmol/g, Soest, placebo- or aprotinin-treated mice under a control or low-salt diet Germany) with tap water ad libitum. To study urinary sodium after 2 days. Half the kidney per mouse was sliced, and the cortex excretion, all mice were housed singly in metabolic cages was dissected using a scalpel. Homogenization was performed (diameter 20 cm, area 314 cm2, Tecniplast, Italy) for 2 days under using a Dounce homogenisator in 1 mL lysis buffer containing a control diet (C1000, Altromin, Lage, Germany, sodium content 250 mM sucrose, 10 mM triethanolamine HCl, 1.6 mM ethanola- 106 µmol/g). Thereafter, mice underwent implantation of custom- mine, and 0.5 mM EDTA at pH 7.4 (all Sigma) [21, 25]. During all made pellets with a matrix-driven sustained release of aprotinin or preparation steps, aprotinin (40 µg/mL) and a protease inhibitor placebo (Innovative Research of America, Florida, USA). These cocktail (final concentration 0.1 × stock; mini-complete, Roche) pellets were surgically implanted subcutaneously on the back of were present to avoid ENaC cleavage in vitro. Homogenates were the mice under isoflurane anesthesia. After this operation, the centrifuged at 1000 ×g for removal of the nuclei. Subsequently, wound conditions and the suture were checked several times. This the supernatant was centrifuged at 20,000 ×g for 30 min at 4 °C, route and mode of administration ensured that the kidneys were and the resulting pellet containing plasma membranes was constantly exposed to aprotinin, which otherwise would rapidly resuspended and diluted to a concentration of 5 mg/L. This be cleared by glomerular filtration. Subsequently, mice were yielded higher ENaC signals compared to centrifugation at studied in a

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