The pepcans previously identified in biological tissues (pepcan-12, -14, -15, -17, -20 and -23)²⁷ were tested in classical radioactivity-based hCB2 receptor binding experiments using [³H]CP55,940 and [³H]WIN55,212-2 as high-affinity orthosteric CB2 receptor radioligands on stably hCB2 receptor transfected CHO cell membranes. Rather unexpectedly, we did not detect radioligand displacement but increased binding in both cases. As shown in Fig. 1A, at the screening concentrations of 100 nM and 1 μM, pepcan-12 was the most potent enhancer of [³H]CP55,940 and [³H]WIN55,212-2 receptor binding, respectively, inducing an overall significant 20–40% binding increase over vehicle control. Pepcan-14, pepcan-15 and pepcan-17 showed weaker yet still significant effects, while the longest peptides pepcan-20 and pepcan-23 did not exhibit any receptor interaction. In subsequent experiments, pepcan-12 displayed concentration-dependent CB2 receptor binding interactions, as reflected by the concomitant sigmoidal increase of [³H]CP55,940 and [³H]WIN55,212–2 binding (Fig. 1B). This potent and limited increase (approximately 140%) in CB2 receptor agonist binding suggested a positive allosteric modulation of the orthosteric ligands by pepcan-12 as opposed to the mutual full competition measured between CP55,940 and WIN55,212-2 (Fig. 1B). Therefore, the concentration binding curves obtained with pepcan-12 were analyzed with the ternary complex model that typically describes allosteric modulation³⁰. The results provided a cooperativity factor (α) of 1.294 with a K_b of 58 nM for [³H]CP55,940 and α = 2.513 with a K_b of 53 nM for [³H]WIN55,212-2. Statistical analyses confirmed that α values were significantly higher than the unit (1), thus confirming the positive cooperation between pepcan-12 and both orthosteric ligands. In order to better characterize the allosteric mechanism postulated, we measured the dissociation kinetics of [³H]CP55,940 from CB2 receptors in the presence of pepcan-12. The experiments were performed using an isotopic dilution approach in which 1 μM of CP55,940 was added to the CB2 receptor-bound [³H]CP55,940 in the presence of 300 nM pepcan-12 or vehicle. As shown in Fig. 1C, CP55,940 induced a significantly slower dissociation of the bound [³H]CP55,940 when incubated in the presence of pepcan-12 as compared to vehicle. Corresponding data analyses provided higher values of both fast k ₁ and slow k ₂ dissociation rate constants (Table 1).Figure 1

Pepcan binding to hCB2 receptors increases binding of orthosteric ligands and positive cooperativity. (A) Pepcan-mediated enhancement of [³H]CP55940 and [³H]WIN55212-2 binding, respectively, to stably transfected hCB2 receptor CHO-K1 cell membranes at 100 nM and 1 µM. (B) Concentration-dependent potentiation of radioligand binding by pepcan-12 (PC-12) versus mutual competition by the orthosteric ligands showing pK_B values and the cooperativity factor α. (C) Isotopic dilution in which 1 μM of CP55,940 was added to the CB1 receptor-bound [³H]CP55,940 in the presence of 300 nM pepcan-12 or vehicle. Data show mean values ± SEM of at least six independent experiment. *p < 0.05 vs. vehicle (two-tailed student t-test).

This reduction of [³H]CP55,940 dissociation from CB2 receptors was in line with the increased binding of the radioligands by pepcan-12 in the equilibrium binding experiments (Table 1). Both results indicated that pepcan-12 binds to the CB2 receptor at an allosteric binding site. With the intention to further characterize pepcan binding to CB2 receptors, we exploited a C-terminal fluorescence-labeled pepcan-12 conjugate (pepcan-F4), which was previously used to measure pepcan CB1 receptor binding affinities²⁷. The apparent K _d value (122 ± 41 nM) for pepcan-F4 for hCB2 receptors was calculated from a normalized (specific) saturation curve (Fig. 2A). As shown in Fig. 2B–F, pepcans differentially displaced pepcan-F4, matching the distinct biding interactions measured with the radioligands. Consequently, pepcan-12, −14, 15, and −17 showed nanomolar CB2 receptor binding affinities comparable to the previous experiments, pepcan-12 exhibiting the lowest K _i value of 44 ± 12 nM. The longest peptide tested (pepcan-20) showed no significant displacement of Pepcan-F4 up to 10 μM (Fig. 2F) in agreement with the lack of CB2 receptor radioligand binding modulation by the N-terminally extended peptide species.Figure 2

Displacement of fluorescent pepcan-F4 by endogenous pepcans. (A) Specific binding of pepcan-F4 to hCB2 expressing CHO-K1 cells measured by fluorescence, showing nonspecific binding in CHO-K1 WT cells. (B–F) Differential dose-dependent displacement of pepcan-F4 by pepcan-12, -14, -15, -17 and -20. Data show mean values of at least three independent experiments each performed in triplicates.

Given the allosteric CB2 receptor binding interaction observed with pepcan-12 (Figs 1 and 2), we studied its effects on key signaling events triggered by the CB2 receptor agonists CP55,940 and 2-AG. CP55,940 and the major endocannabinoid 2-AG were selected because both ligands have been shown to act as full agonists at CB2 receptors but display differential signaling bias^{31, 32}.

The effects of pepcan-12 on the modulation of CB2 receptor-mediated signaling was studied by measuring [³⁵S]GTPγS receptor binding, cAMP production and β-arrestin2 recruitment. In absence of the orthosteric ligands, pepcan-12 did not trigger or inhibit any of the CB2 receptor-mediated signaling pathways measured (Suppl. Fig. S1A). However, when pepcan-12 was incubated in presence of CP55,940 or 2-AG, it induced a significant potentiation of cAMP inhibition and [³⁵S]GTPγS binding (Fig. 3A,B). Pepcan-12 was tested at 10 nM, 30 nM and 100 nM. In line with the estimated K _d value, the concentration of 30 nM induced significant but submaximal effects, while 100 nM elicited maximal effects in both assays.Figure 3

Pepcan-12 potentiates the signaling of CP55,940 and 2-AG. (A) Concentration-dependent increase of CB2 receptor agonist [³⁵S]GTPγS binding by pepcan-12 in CHO-K1-hCB2 cell membranes. (B) Concentration-dependent potentiation of cAMP inhibition of CB2 receptor agonists by pepcan-12 in CHO-K1-hCB2 cells Data show mean values ± SEM of at least six independent experiments. *p < 0.05 vs. vehicle (two-tailed student t-test).

At 100 nM, pepcan-12 increased CP55,940 and 2-AG affinity for [³⁵S]GTPγS binding by approximately 3-fold and led to a higher maximal binding (Fig. 3A, Table 2). In the cAMP assay, the effect of 100 nM pepcan-12 increased the CP55,940-mediated effect by significantly reducing the IC₅₀ value from 3.1 nM to 1.0 nM. Intriguingly, pepcan-12 exerted an even stronger potentiation of 2-AG-mediated cAMP inhibition, reducing the EC₅₀ value approximately by 8-fold, from 92.1 nM to 12.4 nM (Fig. 3B, Table 2). The other members of the pepcan family did not significantly affect the agonist-induced cAMP inhibition, in line with their lower binding affinities (Suppl. Fig. S2). Unlike for cAMP and [³⁵S]GTPγS assays, pepcan-12 (100 and 300 nM) did not influence CP55, 940-induced β-arrestin2 recruitment at the time-point measured (Suppl. Fig. S3). Collectively, these data suggest that pepcan-12 might act as a signaling-specific positive allosteric modulator for CB2 receptor-mediated G protein recruitment and cAMP inhibition (G_αi), possibly without affecting β-arrestin2 recruitment and receptor internalization.Table 2

Summary of pepcan-12 modulation of CP55,940- and 2-AG-induced G protein activation and cAMP formation.

For the quantification of pepcans, we initially applied a cELISA method with the IgG2A mAb 1A12 against the C-terminal part (FKLLSH) of pepcans²⁷. Using this method we measured an increase of pepcans after 2 h from lipopolysaccharides (LPS) injection (endotoxemia model) in kidney and liver, while no significant changes were detected in brain, spleen and lung (Suppl. Fig. S4). However, using cELISA it is not possible to discriminate among the individual members of this family of peptides. Therefore, a specific LC-MS/MS method was developed in order to measure the individual peptides, i.e. pepcan-12 to pepcan-23. Although pepcan-14, -15, -17, -20 and -23 have previously been detected using a very sensitive immunoaffinity MALDI TOF-TOF LC-MS/MS method²⁷, LC-MS/MS measurements using optimized protein precipitation and solid phase extraction indicated that only pepcan-23 and pepcan-12 occur at quantifiable and physiologically relevant levels in tissues. The high amounts of pepcan-12 in liver, adrenals, spleen and kidney are noteworthy as they exceed the levels in brain (Fig. 4A). As shown in Fig. 4B, the levels of pepcan-23 were about three times higher than pepcan-12 in the brain. Importantly, the nonapeptide hemopressin was not identified in any of the tissues as acidic conditions during workup were avoided (see discussion). Given the reported protective role of CB2 receptors in the acute immune response and in sepsis^{33, 34}, we measured the peptides upon LPS challenge in Swiss albino female mice (eight weeks old). The LC-MS/MS quantification displayed the same trend of pepcan-12 levels as observed with the cELISA quantification (Suppl. Fig. S4), showing a significant increase induced by LPS treatment in kidney, liver and adrenals (Fig. 4A), but no effect in spleen and brain. Overall, pepcan-23 levels were highest in liver and spleen (Fig. 4B). In contrast to pepcan-12, pepcan-23 levels were not affected to the same degree by LPS treatment, despite a minor increase in the liver (Fig. 4B). These data exhibit a relatively uniform pepcan tissue distribution in liver, adrenals, spleen and kidney in the range of 20–130 pmol/g tissue. Interestingly, Pepcan-23 was below the limit of detection in adrenals, which however contained high amounts of pepcan-12. Noteworthy, the spleen expressing high levels of CB2 receptor contained comparably high amounts of pepcan-12 and pepcan-23 as the liver.Figure 4

LC-MS/MS quantification of pepcan-12 and pepcan-23 in peripheral tissues upon LPS challenge (6 h) in Swiss albino female mice. Quantification of (A) PC-12 and (B) PC-23 in tissues of wild type Swiss albino female mice in normal, saline (empty circles) and endotoxemic, 5 mg/kg LPS E.coli 055:B5 serotype (closed circles) conditions after 2 h. Data show means and SD. Groups were compared using two-tailed t-Student’s; *p < 0.05; **p < 0.001; ***p < 0.0001; ns = not significant. < LOD, below limit of detection.

In order to investigate whether a change in pepcan levels occurs in established models of tissue injury, pepcans were quantified in mice subjected to sham operation and ischemia/reperfusion (I/R) injury in both liver and kidney (Fig. 5), where CB2 receptors are known to exert protective roles.11,35 As shown in Fig. 5A, renal levels of pepcan-12 increased upon 6 h and 12 h of I/R injury, returning to basal level after 24 h. This concurred with the pathophysiological data (increase of BUN and neutrophil granulocyte infiltration) (Fig. 5B and C). The same was observed upon hepatic I/R damage (Fig. 5D) where the kinetics of pepcan production partially correlated with the pathophysiological parameters (increase and decrease of ALT and neutrophil granulocyte infiltration) (Fig. 5E and F). Because data from cELISA (Suppl. Fig. S4) showed a comparable kinetics of pepcan-12 production (Fig. 5), pepcan-12 seems to be the most relevant pepcan generated upon tissue damage in these tissues. However, in both I/R models the levels of pepcan-23 significantly increased at early times of reperfusion, returning to basal level (liver) or below limit of detection (kidney) as in the sham-operated mice (Fig. 5A and D).Figure 5

LC-MS/MS measurements of pepcan-12 and pepcan-23 in kidney and liver upon ischemia-reperfusion (I/R) damage and associated pathophysiology. (A) Time-dependent increase of PC-12 and PC-23 in kidney upon I/R damage in C57BL6 male mice. Bars show mean values ± SD., ***p < 0.001 vs. sham. < LOD, below limit of detection. (B) Time-course of renal I/R injury related blood urea nitrogen (BUN) levels, data show mean values ± SEM, n = 5, one-way ANOVA, *p < 0.05, **p < 0.01 vs. sham operated. (C) Representative micrographs of kidney samples after renal I/R injury at different time points after reperfusion (6 h, 12 h, 24 h). Arrowheads show Ly6G positive neutrophil granulocytes infiltrating the kidney following ischemic insult. Scale bar: 200 µm. (D) Time-dependent increase of PC-12 and PC-23 in liver upon I/R damage. Bars show mean ± SD. *p < 0.05, **p < 0.01 vs. sham. < LOD, below limit of detection. (E) Time-course of hepatic I/R injury related alanine aminotransferase (ALT) levels (units/liter), data show mean values ± SEM, n = 5–6, one-way ANOVA, **p < 0.01 vs. sham operated. (F) Representative micrographs of liver samples after hepatic I/R injury at different time points after reperfusion (2 h, 6 h, 24 h). Arrowheads show Ly6G positive neutrophil granulocytes infiltrating the liver following ischemic insult. Scale bar is 200 µm.

Role of adrenals as endocrine gland in the secretion of pepcan-12 under healthy and pathophysiological conditions The exclusive presence of pepcans in cell bodies and axons of a discrete population of noradrenergic neurons in the CNS and in the adrenal medulla was recently shown.28 To address the question whether the adrenals are the site of production of peripheral pepcans, e.g. acting as endocrine gland for pepcan-12, we quantified pepcan-12 in bilaterally adrenalectomized (ADX) mice three weeks post operation. As shown in Fig. 6A and B, in female Swiss albino and female C57BL6 adrenalectomized mice (ADX) the pepcan-12 levels dropped to approximately 10% of the levels measured in control littermates (WT) in liver, kidney and spleen (in C57BL6 only 50%) or disappeared completely in the lung. Remarkably, also levels of pepcan-12 in the brain were significantly reduced upon adrenalectomy, from 2.7 pmol/g to 0.8 pmol/g (Swiss Albino) and 1.8 to below limit of detection (C57BL6) versus WT controls. To further study the modulation of pepcan-12 production under pathological conditions, we compared pepcan levels measured in WT and ADX C57BL6 mice both subjected to renal I/R. Unexpectedly, upon I/R the levels of pepcan-12 in peripheral tissues did not change significantly between WT and ADX mice (Fig. 6C and D). Pepcan-12 concentration in liver was approximately 2–5 times higher upon I/R than in basal condition in both WT and ADX mice. However, compared to ADX mice under normal conditions, the levels of pepcan-12 in the liver of ADX mice after I/R injury were more than 50 times higher, indicating additional production in the liver. Noteworthy, the highest amount of pepcan-12 in renal I/R was measured in liver of C57BL6 R mice after 6 h renal I/R injury (approximately 500 pmol/g tissue) and not in the kidney (Fig. 6C).Figure 6

LC-MS/MS quantification of pepcan-12 in different mouse tissues in wild type and adrenalectomized mice. Levels of PC-12 quantified in wild type (WT) and adrenalectomized (ADX) female Swiss albino mice (A) and female C57/BL6 mice (B). Tissue levels of pepcan-12 quantified in an animal model of renal ischemia reperfusion (renal I/R) after (C) 6 h and (D) 24 h of reperfusion in ADX C57/BL6 male mice. Data shows means and SD. Groups were compared using two-tailed t-Student’s; *p < 0.05; **p < 0.001; ***p < 0.000. < LOQ = below limit of quantification (i.e., detectable but not quantifiable).

Analytical standards PVNFKLLSH (Hemopressin, pepcan-9), RVDPVNFKLLSH (Pepcan-12, PC-12, RVD-Hemopressin), HKLRVDPVNFKLLSH (Pepcan-15, PC-15), HAHKLRVDPVNFKLLSH (Pepcan-17, PC-17), SDLHAHKLRVDPVNFKLLSH (Pepcan-20, PC-20) and SALSDLHAHKLRVDPVNFKLLSH (Pepcan-23, PC-23) were synthesized by Genecust Europe. The internal standard RVDPVNFKFL (5,5,5-D3) SH (PC-12-d₃) was synthesized by SHBC. The proteases inhibitor cocktail was obtained from Roche applied science (Complete Ultra Tablets Mini). HPLC-grade methanol, acetonitrile and formic acid were obtained from Sigma-Aldrich, Steinheim, Germany. Deionized water (18.2 MΩ x cm) was obtained from an ELGA Purelab Ultra Genetic system (VWS (UK) Ltd, ELGA LabWater, UK).

Assays were performed as previously described⁴⁷. Clean membranes expressing hCB2 were diluted in silanized plastic tubes with GTPγS binding buffer (50 mM Tris-HCl, 3 mM MgCl₂, 0.2 mM EGTA and 100 mM NaCl at pH 7.4 supplemented with 0.5% fatty acid-free BSA) in the presence of 10 μM of GDP and 0.1 nM of [³⁵S]GTPγS. The mixture was kept on ice until the binding reaction was started by adding the vehicle or compounds. Non-specific binding was measured in presence of 10 μM GTPγS. The tubes were incubated at 30 °C for 90 min under shaking and then put on ice to stop the reaction. An aliquot (185 μL) of the reaction mixture was rapidly filtered through a 96-well microplate bonded with GF/B glass fibre filters previously pre-soaked with ice-cold washing buffer (50 mM of Tris-HCl pH 7.4 plus 0.1% fatty acid-free BSA). The filters were washed six times with 180 µL of washing buffer under vacuum. The radioactivity was measured after the addition of 45 µL of MicroScint20 scintillation cocktail. Specific binding was calculated by subtracting the residual radioactivity signal obtained in presence of an excess of GTPγS and the results were expressed as % of vehicle control.

cAMP assays were performed in CHO-K1-hCB2 stably transfected with the pGloSensorTM 22-F plasmid (Promega) as previously described⁴⁸. Briefly, cells were seeded in 96-well plate and after attachment the medium was replaced by 40 µL of equilibration medium (GloSensor™ cAMP Reagent diluted in 5% v v⁻¹ of CO₂-independent medium plus 10% FBS). The plates were incubated for 2 h at RT in the dark. Compounds or vehicle were diluted in 10 µL assay medium containing 1 µM of forskolin and 250 µM of 3-isobutyl-1-methylxanthine (IBMX). Chemiluminescence was monitored continuously starting from immediately after the addition and for 30 min (keeping the plate at RT and in the dark). Basal levels of chemiluminescence (cells plus IBMX and vehicle without forskolin) were subtracted to the measured values. Results were normalized by subtracting the residual cAMP production and expressed as % of vehicle control.

The PathHunter^® β-arrestin cell line (CHO-K1-HOMSA-CNR2) was maintained in culture in F-12 medium supplemented with 10% FBS, 200 μg/mL hygromycin and 800 μg mL⁻¹ Geneticin (G418). For the experiments, 100 μL well⁻¹ of cell suspension (2 × 10⁵ cell/mL) was plated in 96-well plate and left overnight in the incubator. The day after, tested compounds or vehicle were diluted in 40 μL of CO₂-independent medium plus 10% FBS, added to the cells and incubated for 90 min at 37 °C. Following stimulation, signal was detected using the PathHunter® detection Kit according to the instruction protocol and the chemiluminescence was measured. Background levels were subtracted and results expressed as % of vehicle control.

Maxisorp microtiter 96 wells plates (Nunc) were coated by incubation with 100 μL of 1 µg/mL synthetic pepcan-12 (Genscript) in PBS pH 7.2 at 4 °C O/N. Washing of peptide excess and blocking of non-specific sites was performed with PBS-T 0.05% pH 7.2 and PBS-T 0.05% + 1% BSA fatty acid free respectively. Calibration concentration points (0.01-0.1-1-3-10-30-100-1000 nM) or sample were pre-incubated with 0.0125 nM of 1A12 mAb for 30 min at room temperature and added to the wells and incubated for 2 h at room temperature. After washing, plates were incubated at room temperature for 60 min with 50 μL HRP- conjugated secondary antibody goat anti-mouse IgG (Bio-Rad). Plates were finally developed with 50 μL of 3,3′,5,5′-tetramethylbenzidine substrate (Pierce) after washing. This reaction was stopped with 50 μL of 2 M H₂SO₄ after sufficient colour development (usually after 20 min). The OD of the reaction product was then recorded at 450 nm. Sample preparation: mouse tissues were weighted, homogenized in PBS + Protease inhibitor cocktail (Roche) and two times their volume of acetonitrile were added. After centrifugation at maximum speed for ten min, the supernatant was collected, dried in a speed vacuum and resuspended in 200 μL of PBS.

Radioligand binding assays using [³H]CP55,940 and [³H]WIN55,212-2 were performed with CB2 receptor membrane preparations as reported previously²⁷. Briefly, 20 μg of membranes of CHO-K1-hCB2 cells were incubated in 500 μL of binding buffer (50 mM Tris-HCl, 2.5 mM EDTA, 5 mM MgCl₂, 0.5 mg/mL fatty acid-free BSA, pH 7.4) in silanized glass tubes in presence of different concentrations of pepcans and 0.5 nM [³H]CP55,940 (168 Ci/mmol) or 2.5 nM [³H]WIN55,212-2 (40 Ci/mmol) for 60 min at 30 °C. Nonspecific binding of the radioligand was determined in the presence of 10 μM WIN55,512-2 or CP55,940, respectively. After the incubation time, membrane suspensions were rapidly filtered through a 0.1% polyethyleneimine presoaked 96-well microplate bonded with GF/B glass fiber filters (UniFilter-96 GF/B, PerkinElmer Life Sciences) under vacuum and washed 12 times with 150 μL of ice-cold binding buffer Filters were added with 40 μL of MicroScint20 scintillation liquid, and radioactivity was measured with the Trilux top counter (PerkinElmer Life Sciences). Data were collected from 3–10 independent experiments, each performed in triplicate, and the nonspecific binding was subtracted. The results are expressed as a percentage of vehicle-treated samples.

Dissociation kinetics experiments were performed as described before²⁷. Briefly, 20 μg of membranes of CHO-K1-hCB2 cells were incubated in 500 μL of binding buffer in silanized glass tubes and incubated with 0.5 nM [³H]CP55,940 for 60 min at 30 °C. Radioligand dissociation was initiated by the addition of 1 μM WIN55,212-2 in the presence of 300 nM pepcan-12 or solvent (DMSO) and incubated for 1–120 min, at 30 °C. Nonspecific binding of the radioligand was determined in the presence of 10 μM CP55,940. After the incubation time, membrane suspensions were rapidly filtered through a 0.1% polyethyleneimine presoaked 96-well microplate bonded with GF/B glass fiber filters and washed as described above.

The equilibrium dissociation constant (Kd value) of the fluorescein-labeled pepcan-12 derivative pepcan-F4 was determined using CHO-K1-hCB2 cells as previously described²⁷. Equilibrium saturation binding was determined experimentally, and maximal specific binding (saturation) was reached already at 5 min at 37 °C. Different concentrations of pepcans and 100 nM pepcan-F4 were incubated together with 105 cells suspended in 200 μL of binding buffer in silanized plastic tubes, under shaking for 60 min at 37 °C. After centrifugation at 4 °C and washing with ice-cold binding buffer, the cells were resuspended in ice-cold PBS and transferred into 96-well black plates, where the fluorescence intensities were measured using a Tecan Farcyte reader (485 nm excitation/535 nm emission). The specific binding was determined after washing and subtracting the nonspecific fluorescence calculated in parallel experiments carried out in non-transfected CHO cells.

The investigation conforms to the Guide for the care and use of laboratory animals published by the US National Institutes of Health (NIH, publication No. 85–23, revised 1996) and was approved by the Institutional Animal Care and Use Committee of the National Institute on Alcohol Abuse and Alcoholism (NIAAA). Animal experiments complied with Swiss regulations and were carried out in accordance with relevant Swiss guidelines and regulations and approved by the Kantonale Amt für Landwirtschaft und Natur, Veterinärdienst. Male C57Bl6/J mice (weighting between 25–30 g) and female Swiss albino mice (8 weeks) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA and Janvier Labs) and were housed in a room maintained at 12 h light-dark cycles and at constant temperature and humidity. Mice had free access to standard chow and drinking water. Adrenalectomized (ADX) mice were purchased from The Jackson Laboratory and male C57BL6/J mice were subjected to bilateral adrenalectomy by the vendor. When surgery was completed, mice were held by the vendor for approximately one week to allow wound healing before they delivered. ADX mice showed neither signs of distress nor unusual behavior. The only diet adjustment that had to be arranged was to provide 0.9% NaCl in drinking water ad libitum instead of normal tap water.

Swiss albino female mice were housed in groups of five per cage in a selected pathogen-free unit under controlled 12-h light/12-h dark cycle, ambient temperature 21 °C ± 1 °C and 40% to 50% humidity with free access to standard rodent chow and water. The mice were acclimatized for at least 3 days before the experiment. Swiss albino mice (females, 8 weeks old) were injected intraperitoneally (i.p.) with LPS (5 mg/kg) and sacrificed after 2 h. LPS was serotype 055:B5 (Sigma Aldrich, Switzerland).

Mice underwent unilateral kidney ischemia reperfusion as described elsewhere⁴⁹ (Hamar P., Proc Natl Acad Sci USA. 2004). Briefly, anesthesia was induced by the intraperitoneal administration of ketamine (100 mg/kg) and xylazine (10 mg/kg). After midline laparotomy, right kidney was removed in all cases to prevent compensation. The left renal pedicle was isolated and clamped for 25 min followed by reperfusion for 6 h, 12 h or 24 h according to the experimental setting. After the intervention, abdominal cavity was closed by using 6–0 vicryl suture (Ethicon, Somerville, NJ, USA) and mice were transferred into clean cages and allowed to recover. As post-operative care, buprenorphine was administered subcutaneously (Buprenorphine SR ZooPharm, Wildlife Pharmaceuticals, Fort Collins, CO, USA). Sham operated animals underwent the above-mentioned procedures except of left kidney clamping. At the end of reperfusion, mice were sacrificed by cervical dislocation, Blood was collected via cardiac puncture, tissues and post-ischemic kidneys were snap frozen in liquid nitrogen and kept at −80 °C until further use. Tissue samples were collected and fixed in 10% buffered formalin for histopathological evaluation as well. The function of the injured left kidney was assessed by measuring blood urea nitrogen (BUN) level in serum samples (IDEXX VetTest® Chemistry Analyzer and BUN test strips; Westbrook, ME, USA).

Partial hepatic ischemia reperfusion was performed as described by Batkai et al.¹³. Briefly, following anesthesia, the liver was exposed by midline laparotomy. The vascular pedicle common to the median and left-lateral lobes was clamped. In this model of partial hepatic ischemia, mesenteric venous congestion was prevented by allowing portal decompression throughout the right and caudate lobes of liver. The duration of hepatic ischemia was 1 h followed by 2 h, 6 h or 24 h reperfusion as indicated. Sham surgeries were completely identical except of hepatic vascular pedicle clamping. At the end of reperfusion, blood was collected and liver samples were removed and snap-frozen in liquid nitrogen as described above. Serum aspartate amino-transferase (AST) and alanine amino-transferase (ALT) levels were determined in serum samples (IDEXX VetTest® Chemistry Analyzer and AST and ALT test strips) in order to assess hepatic injury.

Tissue samples were fixed in 10% buffered formalin. After embedding and sectioning 5 µm slices, samples were stained with the conventional haematoxylin/eosin staining method.

All data generated or analysed during this study are included in this published article (and its Supplementary Information files).

Supplementary Figures