Targeting brain death-induced injury van Erp, Anne Cornelie

Targeting brain death-induced injury van Erp, Anne Cornelie

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van Erp, A. C. (2018). Targeting brain death-induced injury. Rijksuniversiteit Groningen.

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Autophagy is reduced in the liver and kidney following brain death independent of mTOR activation

Anne C. van Erp Rolando A. Rebolledo Petra J. Ottens Johan G.M. Burgerhof Ina Jochmans Diethard Monbaliu Jacques Pirenne Henri G.D. Leuvenink Jean-Paul Decuypere

In preparation

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CHAPTER

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ABBREVIATIONS

ALT Alanine Transaminase AST Aspartate Transaminase AMPK AMP-Activated Kinase BAX Bcl-2-associated X Bcl-2 B-cell lymphoma 2

BD Brain Death

cC3 Cleaved Caspase 3 CT Threshold Cycle DGF Delayed Graft Function ECD Extended Criteria Donation GFR Glomerular Filtration Rate HAES Polyhydroxyethyl Starch HMP Hypothermic Machine Perfusion IR Ischemia-Reperfusion

IRR Intrarenal Vascular Resistance

LC3-PE Phosphatidylethanolamine-Conjugated LC3 LC3-II See LC3-PE

LDH Lactate Dehydrogenase MAP Mean Arterial Pressure

mTOR Mammalian Target of Rapamycin mTORC1 Rapamycin (mTOR) Complex 1 NA Noradrenalin

ROS Reactive Oxygen Species RR Respiratory Rate

T3 3,3’,5-Triiodo-L-thyronine

UF Ultrafiltrate

8 ABSTRACT

Introduction

Donor brain death (BD) increases tissue injury and apoptosis. Apoptosis is counteracted by autophagy, a protective, stress-adaptation mechanism inhibited by mammalian target of rapamycin (mTOR). Whether mTOR-dependent autophagy is affected after BD is unknown.

Therefore, this study investigated if and how BD modulated autophagy and mTOR activity in the liver and kidney of brain-dead rats.

Methods

BD was induced in mechanically-ventilated rats by inflation of an epidurally-placed balloon catheter. Vehicle (EtOH) or rapamycin (1 mg/kg) was administered intraperitoneally 2 h prior. After 4 h of BD, plasma and tissue were collected and the left kidney normothermically perfused for 90 min in an isolated perfused kidney model. Tissue injury and function were assessed with routine biochemistry. Autophagy (p62, Beclin 1, LC3I/II), apoptosis (Bax, Bcl2, cC3), and mTOR activity (phospho-S6) markers were analyzed with Western blot and qPCR.

Results

Most prominently in the liver, brain-dead animals displayed increased mTOR activity and reduced levels of autophagic marker LC3-II concomitant with increased autophagy degradation substrate p62 and cC3. Autophagy stimulation by rapamycin reduced mTOR activity, but did not induce autophagy (Beclin 1, LC3, p62), or attenuate BD-induced injury (ALT, AST, creatinine, urea, LDH) or apoptosis (cC3, Bax, Bcl2), or improve renal function after reperfusion.

Conclusion

BD reduced autophagy in the liver and to a lesser extent in the kidney. Inhibition of mTOR

with rapamycin did not induce autophagy or attenuate injury or apoptosis in the liver or

kidney, nor improve renal function after reperfusion. Thus, BD results in an attenuation of

autophagy in an mTOR-independent fashion.

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INTRODUCTION

Organ transplantation has become the treatment of choice for patients with end-stage organ disease. However, the success of transplantation is limited by a global shortage of suitable organs as well as graft loss following transplantation. These problems can be addressed by increasing the use of suboptimal organs, while simultaneously improving graft longevity. However, increasing the use of suboptimal organ grafts from brain death (BD), cardiac death, or extended-criteria donors requires preservation of organ quality prior to transplantation. Most organs worldwide are currently obtained from brain-dead donors

. However, transplantation of these grafts results in higher rejection and lower survival rates after transplantation compared to living donor grafts

. BD induces derailments in body homeostasis including inflammation, hemodynamic instability, hormonal impairment, and metabolic changes

. In the liver and kidneys, this results in tissue injury, metabolic alterations, oxidative stress, and apoptosis

.

Autophagy is a protective, stress-adaptation pathway that can counter apoptosis

. Several types of autophagy exist, all of which involve the transportation of intracellular compounds to the lysosomes for degradation

. The most-studied type of autophagy is macro-autophagy (referred to as autophagy for the remainder of this manuscript), which involves the formation of double-membranous vesicles called autophagosomes, which can engulf entire organelles and other intracellular products (see Fig 1). Normally, autophagy occurs at a basal level in the cell and serves as a housekeeping mechanism that removes and recycles damaged or old cellular constituents

. In the presence of stressors, such as hypoxia, energy depletion or cellular damage, autophagy becomes stimulated and serves as a protective mechanism removing damaged and toxic cellular products, thereby preventing oxidative stress and apoptosis

. In addition, during nutritional stress the autophagic recycling of cellular macromolecules into their molecular building blocks addresses an intrinsic pool of nutrients, thereby promoting survival. However, excessive activation of autophagy could evoke cellular death, which implies that there is a fine balance between protective, pro-survival effects of autophagy on the one hand, and detrimental, pro-death effects of autophagy on the other

. Autophagy dysregulation has been linked to human diseases such as aging, degeneration, inflammation, and cancer

. During the transplantation process, autophagy (dys)regulation is closely linked to oxidative stress during each of the steps of the transplantation process (reviewed in detail elsewhere)

. Amongst others, the extent of oxidative stress, cold ischemia duration, gender and donor age are each suggested to influence autophagy regulation

. However, the effects of donor BD on autophagy remain unknown.

Autophagy regulation occurs largely through the mammalian target of rapamycin (mTOR)

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complex 1 (mTORC1, see Fig 1). mTORC1 receives input from several intracellular pathways sensing changes in energy status, growth factors, oxygen levels, hormonal perturbations, and metabolic stress

. mTORC1 integrates these signals and controls the activity of many downstream processes such as protein and lipid synthesis, cellular metabolism, ATP production, and autophagy. mTORC1 is the target of mTOR inhibitors rapamycin (or sirolimus), everolimus and deferolimus. These immunosuppressive drugs have each gained applicability in field of organ transplantation for their ability to modulate the B- and T-cell immune response as well their anti-proliferative properties following kidney and liver transplantation

. However, effects of rapamycin treatment have shown to be both detrimental and beneficial in ischemia-reperfusion (IR) injury and transplantation studies

. Thus far, how donor BD affects mTOR remains unexplored.

Figure 1. Overview of the autophagy process. Autophagy is initiated by the ULK1 and class III PI3K complex. ULK1 complex is negatively regulated by mTOR and positively by AMPK, thereby responding to nutrient or energy deprivation. Rapamycin in turn inhibits mTOR complex, thereby stimulating ULK1 complex and thus autophagy initation, as well as the phosphorylation of protein S6. The Class III PI3K complex requires Beclin 1, which is inhibited by Bcl-2. For elongation of the isolation membrane formation, Atg5-Atg12 and LC3-II are required. LC3-II is attached to the membrane of the autophagose. In the presence of Atg4, LC3-II will become delipidated (a process inhibited by H2O2).

Otherwise it remains on the inner membrane and to be degraded inside the autophagolysosomes.

Mitochondria can be also degraded (mitophagy) via recruitment of Sqstm1/p62. The latter protein also recruits Keap1 for degradation, thereby enabling Nrf-2-dependent anti-oxidant transcription.

Eventually autophagosomes fuse with lysosomes to form autophagolysosomes in which degradation of cellular components occurs. The figure is adapted from a paper by Van Erp et al

In this study, we hypothesized that BD-induced apoptosis and hepatic and renal injury are

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related to autophagy dysregulation, which could be counteracted by autophagy stimulation using rapamycin. Therefore, we investigated the effects of BD on autophagy in the liver and kidney using a rodent BD model. We studied how autophagy stimulation using mTOR inhibitor rapamycin affected autophagy, organ quality and apoptosis. Kidney function was assessed with an ex vivo renal reperfusion model following BD.

MATERIALS AND METHODS

Animals and treatment

Male adult Fisher F344 rats (250–300 g) were used. Animals were cared for according to the guidelines of the Institutional Animal Care and Use Committee of the Rijksuniversiteit Groningen (IACUC-RUG), in line with the Experiments on Animals Act (1996) issued by the Ministry of Public Health, Welfare and Sports of the Netherlands. The study was approved by the IACUC-RUG.

Rats were randomly assigned to the sham or BD group (n = 8 per group). Sham-operated rats served as controls and were ventilated for 0.5 h under anesthesia before the experiment was ended. One mg/kg bodyweight of rapamycin (LC Laboratories, cat. nr. AY-22989) or an equivalent volume of vehicle (25% EtOH in NaCl 0.9%) was administered intraperitoneally 2 h prior to the start of BD induction, followed by a 4 h BD period after confirmation of BD in the brain-dead animals, or a 30 min period under anesthesia in the sham-operated animals.

Animals were randomly assigned to one of four experimental groups:

• Sham-operated animals receiving vehicle (n=8)

• Sham-operated animals receiving rapamycin (n=8)

• Brain-dead animals receiving vehicle (n=8)

• Brain-dead animals receiving rapamycin (n=8)

Brain death model

The BD model used in this experiment was based on the gradual onset brain death model

developed by Kolkert et al

. Briefly, animals were anaesthetized with 5% isoflurane in a

mixture with oxygen (1L/min). Animals were intubated via a tracheostomy and mechanically

ventilated with 2% isoflurane in 100% oxygen with the following ventilation settings: tidal

volume of 2.5 ml per stroke, positive end-expiratory pressure of 2 cm of H

0, and initial

respiratory rate (RR) of 80 cycles per min, corrected based on end tidal CO

levels which

were kept between 20-22 mmHg. Cannulas were placed in the femoral artery and vein to

monitor blood pressure and provide intravenous access. A frontolateral hole in the skull was

drilled to allow for a 4F Fogarty balloon catheter to be inserted into the extradural space,

after which BD was gradually induced by slow inflation using a syringe pump (0.16 ml/min)

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(Terufusion, Termo Co, Tokyo, Japan). This procedure was identical for sham-operated rats, expect for inflation of the balloon. Sham-operated rats were ventilated for a total of 0.5 h under anesthesia before the experiment was ended. In the BD group, balloon inflation was stopped once the mean arterial pressure (MAP) rose above 80 mmHg, representing the autonomic storm that is characteristic of BD. BD was confirmed by the absence of corneal reflexes and a positive apnea test. The MAP was kept above 80 mmHg throughout the experiment. If necessary, colloid infusion with polyhydroxyethyl starch (HAES, 100 g/L in saline) was administered to maintain a normotensive MAP. In the case of an inadequate response to HAES, treatment with intravenous noradrenaline (NA) (1mg/mL) was initiated.

A blanket control system was used to maintain a body temperature of 37°C. After 4 h of experimental BD duration, the abdomen was opened, urine collected from the bladder and 5 mL of blood collected from the abdominal aorta. The abdominal organs were subsequently flushed via the aorta with 50 mL cold saline. After the flush-out, the liver and right kidney were harvested and tissue samples were snap-frozen in liquid nitrogen and stored at -80°C or fixated in 4% paraformaldehyde. Tissue plasma en urine were also snap-frozen.

Reperfusion model

After 4 h of BD, the renal artery, vein, and ureter of the left kidney were cannulated. The left kidney was then flushed with ice cold saline before the perirenal fat was removed and the kidney extracted from the body. The kidney was then normothermically perfused for 90 min in an isolated perfused kidney (IPK) model. The IPK device is a kidney perfusion system that is pressure- (100 mmHg) and temperature- (37°C) controlled and consists of a membrane oxygenator, a roller pump (Ismatec MS-2/6-160; IDEX Health and Science), a heat exchanger (Radnoti Heating coil, 5.5 mL) connected to a water bath (Julabo Labortechnik), a temperature probe connected to a heater (Tristar KA-5038) via a control switch, pressure, flow, and oxygen sensors, an organ chamber, and a perfusion fluid reservoir, all of which are placed in a plexiglass box for insulation (Fig S1). The kidney was perfused with oxygenated (95% O

, 95% CO

) perfusion medium (perfusate), with a partial oxygen pressure of at least 60kPA and a pH between 7.35-7.45, via de arterial and venous cannulas. Ultra-filtrate (UF) (urine) produced was collected via the ureter cannula. The perfusion medium consisted of 100 mL William’s Medium E GlutaMAX (Life technologies, USA), 0.08 g/dL creatinine (Sigma- Aldrich, the Netherlands), 0.715 g/dL HEPES (Sigma-Aldrich, the Netherlands), and 5 g/dL Bovine Serum Albumin (GE-healthcare, United Kingdom). Flow and (pre- and post-renal) oxygenation were recorded at 10 min intervals. Sampling of perfusate and UF was done at the beginning and after 15, 30, 60 and 90 min of perfusion, after which samples were stored at -80°C for further analyses. At 90 minutes of perfusion the kidney was disconnected, snap-frozen in liquid nitrogen and stored at -80°C or fixated in 4% paraformaldehyde.

To determine renal function, intrarenal vascular resistance (IRR), oxygen consumption, UF

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production, and glomerular filtration rate (GFR) were calculated. IRR was calculated by dividing the pressure (mmHg) by the flow (mL/min). The amount of oxygen consumption was calculated by subtraction of the venous from the arterial oxygen consumption (µmol/L), divided by the flow times the weight of the kidney (grams). The UF production was calculated in mL/min. To determine the GFR, creatinine levels in perfusate and UF samples were determined as described below. GFR was calculated multiplying the UF creatinine (µmol/L) times the UF volume, divided by perfusate creatinine levels (µmol/L).

Hepatic and renal biomarkers

Alanine transaminase (ALT), aspartate transaminase (AST), creatinine, urea, lactate dehydrogenase (LDH), and bilirubin in plasma and/or perfusate and/or UF were determined at the clinical chemistry laboratory of the University Medical Centre Groningen according to standard procedures.

Western Blot

Liver and kidney tissues were homogenized in RIPA buffer (10 mM sodium phosphate (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 0.5 mM DTT, 1% Triton X-100 and Complete EDTA- free Protease Inhibitor Tablets (Roche Diagnostics). After centrifugation, the lysate was stored at -80°C. Determination of protein concentration was performed with the Bradford assay; 50 µg of protein was eventually loaded per well for protein gel electrophoresis.

Electrophoresis was performed with Any kD Mini-PROTEAN TGX Precast gels (Bio-Rad).

After electrophoresis, proteins were transferred onto a PVDF membrane using the Trans- Blot Turbo Transfer system (Bio-Rad). Membranes were then blocked with 5% milk powder in PBS-Tween (0.1%) and incubated overnight with primary antibody in PBS-Tween with 2% milk powder. Primary antibodies were: anti-LC3 (Nanotools, 0231-100/LC3-5F10) anti-Bax (Santa Cruz Biotechnology Inc., sc-493), anti-Sqstm1/p62 (Sigma-Aldrich, P0067) and anti-rpS6 (Cell Signaling Technology, 2217), anti-phospho-rpS6 (4858, Cell Signaling Technologies), anti-Beclin 1 (LS-B3203, LifeSpan Biosciences), anti-cleaved caspase 3 (9664, Cell Signaling Technologies) and Anti-GAPDH (G8795, Sigma-Aldrich NV). After three subsequent wash steps of five min, secondary antibody was added in PBS-Tween with 2% milk powder. Secondary antibodies were HRP-linked (Cell Signaling Technology).

Then, after three subsequent wash steps, the antibodies were detected using ECL reagent (Thermo Scientific) and the Chemidoc MP system (Bio-Rad). Bands were quantified using the Chemidoc Imaging software.

Real-Time quantitative PCR

Total RNA was isolated from whole liver and kidney sections and cDNA synthetized using

methods described in detail elsewhere

. In short: samples were first checked for DNA

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contamination, after which cDNA was synthesized by incubation of 1 μl T11VN Oligo-dT (0,5 μg/μl) and 1 μg mRNA at 70°C followed by cooling. Afterwards, 0.5 μl RnaseOUT Ribonuclease inhibitor (Invitrogen, Carlsbad, USA), 0.5 μl RNase water (Promega), 4 μl 5x first strand buffer (Invitrogen), 2 μl DTT (Invitrogen), 1 μl dNTP’s, and 1 μl M-MLV reverse transcriptase (Invitrogen, 200U) were added and the mixture incubated first at 37°C and then at 70°C.

Fragments of genes involved in apoptosis (B-cell lymphoma 2 (BCL2) and Bcl2-associated X (BAX)) and autophagy (MAP1LC3B, BECN1, SQSTM1/p62) were amplified with the primer sets outlined in Table 1. Real-Time PCR methods used were described previously

. Briefly, pooled cDNA from brain-dead rats was used as an internal reference, and gene expression normalized with the mean of household gene β-actin. Real-Time PCR was performed using 10 μl of SYBR Green mastermix (Applied biosystems, Foster City, USA), 0.4 μl of primer (50 μM), 4.2 μl of nuclease free water, and 10 ng of cDNA, and performed using the Taqman Applied Biosystems 7900HT Real-Time PCR System. Results were expressed as 2−△△CT (CT:

Threshold Cycle).

Table 1. Primer sequences used for Real-Time PCR.

Gene Primers Amplicon size (bp)

BAX 5’-GCGTGGTTGCCCTCTTCTAC-3’

5’-TGATCAGCTCGGGCACTTTAGT-3’ 74

BCL2 5’-CTGGGATGCCTTTGTGGAA-3’

5’-TCAGAGACAGCCAGGAGAAATCA-3’ 70

BECN-1 5’-CAAGTTCATGCTGACGAATCTCAA-3’

5’-CCCCTAAGGAGCAAGTCACTTGTT-3’ 81

β-actin 5’-GGAAATCGTGCGTGACATTAAA-3’

5’-GCGGCAGTGGCCATCTC-3’ 74

MAP1LC3B 5’-GTAATGCTTATCCTGCATCAAGTTTCT-3’

5’-CCACTGGGCGATCAGCTTT-3’ 79

SQSTM1/p62 5’-AGAACTTAGGTGAAGCCAACTGAAAG-3’

5’-TTAGACACAAAAACTAAACTGGCCATT-3’ 84

Statistical analysis

To detect differences in AST/ALT levels between groups, we estimated that with an absolute difference of 50%, σ of 0.3, α of 0.9, and a two-tailed test, eight animals per group were needed.

Due to the two-factorial design of the experiment, the two-way ANOVA test was used to

analyze the plasma and qPCR analyses (SPSS Statistics 20, IBM Software, NY, USA). Data

were checked for a normal distribution and equality of variances of the dependent variable

across groups using Levene’s test of equality of error variances (indicated by p > 0.05), and

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the absence of any outliers. Data transformations were applied if these assumptions were not met, after which data were normally distributed, variances of the dependent variable equal across groups, and there were no outliers. This allowed us to use the two-way ANOVA test despite small group sizes. Results were then checked for a significant interaction term.

If the interaction term was not significant, the p-values of the main effects were recorded.

If the interaction term was significant, the main effect between individual groups was evaluated using a student T-test in the case of a normal distribution, or a Mann Whitney tests if the data was not normally distributed (IBM SPSS Statistics 23). For data that did not meet the assumptions for the two-way ANOVA test (i.e. not normally distributed and unequal distribution of variances), the Mann-Whitney was performed to compare between two groups individually (IBM SPSS Statistics 23).

For the WB analyses, if data was normally distributed, variances of the dependent variable were equal across groups, and there were no outliers, a pooled student T-test was done in the case of equal variances, whereas a separated student T-test was done in the case of unequal variances of the dependent variable. If data was not normally distributed, a Mann- Whitney test was run to compare between two groups (IBM SPSS Statistics 23).

For IPK data, descriptive statistics confirmed the assumption of equal distributions of residuals. A linear mixed model was used with repeated measures over time to analyze the impact of the treatment (BD or sham) on IPK parameters in the kidney, with fixed effects of time, treatment group, and the interaction of treatment and time (IBM SPSS Statistics 23). This model was chosen because it takes the dependency of the measurements across time into consideration and prevented list-wise deletion caused by missing data points.

The model selection for covariance parameters was chosen based on the best fit according to the Bayesian Information Criterion. All statistical tests were 2-tailed and p < 0.05 was regarded as significant. Results are presented as mean ± SD (standard deviation).

RESULTS

Brain death model

We used a mixed effects model to determine whether blood pressure profiles were different

between groups over time. We showed that rapamycin treatment resulted in a significantly

lower blood pressure profile of on average 15.9 mmHg compared to vehicle-treated animals

(112 ± 3.56 vs. 128 ± 5.03, p = 0.006, Fig S2A). Furthermore, rapamycin treatment resulted

in a significantly higher use of HAES in brain-dead compared to sham animals (p = 0.008, Fig

S2B). The use of NA was not different between groups (Fig S2C).

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Hepatic and renal biomarkers

Several hepatic (AST, ALT, and LDH) and renal injury markers (creatinine and urea) were assessed to evaluate the effect of rapamycin treatment on tissue injury in brain- dead and sham animals. No significant interaction terms were found for any of the injury markers. Comparing brain-dead to sham animals, all injury markers were significantly higher following BD (ALT, AST, creatinine, urea: p < 0.001, LDH: p = 0.032, Fig 2). Regarding urea, rapamycin treatment significantly increased plasma urea levels (p = 0.006, Fig 2), suggesting rapamycin might have nephrotoxic side effects.

However, rapamycin treatment did not alter any of the other plasma injury markers.

Figure 2. Brain death-induced liver and kidney failure was not affected by rapamycin treatment.

Plasma levels of A. aspartate transaminase (AST), B. alanine transaminase (ALT), C. lactate dehydrogenase (LDH), D. creatinine, and E. urea after 4 h of experimental time in sham and brain-dead animals treated with vehicle (EtOH) or rapamycin (1mg/kg) 2h prior to brain death induction. Results are presented as the mean ± SD, n = 8 per group (*p < 0.05, **p < 0.01, ***p < 0.001, where brackets indicate non-parametric test were used, and a straight line parametric tests).

Reperfusion model

Renal function was assessed in an isolated IPK model following BD through assessment of the flow, UF production, IRR, and GFR. We used a linear mixed model to evaluate whether these parameters were significantly different between groups over time. For O

consumption and GFR, no significant effects of time, treatment, or group were found (Fig 3A,D). Regarding UF production, there was no significant effects of rapamycin treatment.

We did find a significant interaction term between time and group (BD vs. sham), indicating

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a significantly higher UF production in brain-dead compared to sham animals over time (p

= 0.011, Fig 3B). Estimated effects were: UF (BD) = 0.124 – 0.0003 * time; UF (sham): 0.055 + 0.0001 * time. Rapamycin also did not alter IRR. However, a significant interaction term was found, indicating regarding IRR over time between brain-dead and sham animals (p = 0.023, Fig 3C). Estimated effects for IRR were: IRR (BD): 11.98 + 0.036 * time and IRR (sham):

17.96 – 0.059 * time.

Figure 3. Brain death-induced increase in ultrafiltrate production and intrarenal vascular resistance was unaffected by rapamycin treatment. Renal function parameters A. Oxygen consumption ((mmol*min)/(gram/L)), B. ultrafiltrate (urine) production (mL/min) C.

intrarenal vascular resistance (mmHg*min/mL), and D. Glomerular Filtration Rate (GFR, creatinine clearance in mL/min) during 90 min of warm oxygenated perfusion that followed 4 h of experimental time in sham and brain-dead animals treated with vehicle (EtOH) or Rapamycin (1mg/kg) 2h prior to brain death induction. Results are presented as the average mean ± SD, n = 8 per group (*p < 0.05).

Western blot expression of autophagy- and apoptosis-related proteins

We used Western blot to identify several autophagy- (BECN1, p62, pS6, S6, LC3-I/II) and

apoptosis-related (cC3, Bax) proteins. In the liver, BD resulted in a significant reduction in

protein expression BECN1, which is part of autophagy-initiator class III PI3K complex, as well

as increased pS6/S6 levels, suggesting increased mTOR activity following BD. Finally, levels

of autophagy markers LC3-II were increased, whereas levels of apoptosis-effector

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Figure 4. Reduction of autophagy and induction of apoptosis in the liver and kidney following brain death, which was unaffected by mTOR inhibition. Western blot expression of autophagy-related proteins Beclin 1, SQSTM1/p62, pS6/S6, LC3-1, and LC3-II, and pro-apoptotic protein Bax and cleaved caspase 3 after 4 h of experimental time in sham and brain-dead animals treated with vehicle (EtOH) or rapamycin (1mg/kg) 2 h prior to brain death induction. Results are presented as the fold induction of the mean ± SD (/average mean of control group), n = 8 per group (*p < 0.05, **p < 0.01, ***p

< 0.001).

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cC3 were increased (LC-3 and cc3: p = 0.001, Fig 4A), suggesting lower autophagic flux yet increased apoptosis following BD in the liver. In the kidney of brain-dead animals, we observed significantly higher levels of autophagy degradatation protein p62, suggesting decreased autophagic flux, and increased levels of cC3 (p62: p = 0.011, cC3: p = 0.008, Fig 4D). In the liver of sham animals, rapamycin treatment resulted in decreased pS6/S6 levels, yet increased LC3-II and LC3-I/II levels, suggesting autophagy was able to inhibit mTOR and induce autophagy in sham animals (pS6/S6: p < 0.001, LC3-II: p = 0.003, LC3-I/II: p = 0.001, Fig 4B). In the kidney, rapamycin treatment was unable to inhibit mTOR or induce autophagy in sham animals (Fig 4E). Interestingly, rapamycin treatment resulted in increased p62 and cC3 levels in the kidney, suggesting a possible induction of apoptosis in rapamycin-treated sham animals (p62: p < 0.001, cC3: p = 0.045, Fig 4E). In brain-dead animals, rapamycin treatment resulted in mTOR inhibition in both the liver (p = 0.002, Fig 4C) and the kidney (p < 0.001, Fig 4F). Besides a reduction in p62 levels in both organs (liver: p = 0.003, kidney:

p = 0.017), and a reduction in LC3-I levels in the kidney (p = 0.007), no other autophagy- or apoptosis-related protein expression was altered in rapamycin-treatment brain-dead animals (Fig 4C,F). Pictures of the individual blots can be found in Fig S3.

Real-Time quantitative PCR evaluation of autophagy- and apoptosis-related gene expression

We assessed mRNA expression of several autophagy- (LC3, SQSTM/p62, Beclin 1) and apoptosis-related (Bax, Bcl2, Bax/Bcl2) proteins to evaluate the effects of brain death and rapamycin treatment on autophagy and apoptosis. BD resulted in a significant increase in autophagy-related gene expression in both the liver and kidney, as seen by a reduction in LC3, yet increase in Beclin 1 and SQSTM/p62 expression in the liver (LC3: p < 0.001, Beclin 1: p = 0.021, SQSTM/p62: p < 0.001: Fig 5A-C), and reduction in LC3 and increase in SQSTM/

p62 in the kidney (LC3: p = 0.036, SQSTM/p62: p = 0.001, Fig 5D-F). Except for a reduction LC3 expression in the liver of brain-dead animals (p = 0.003), rapamycin treatment did not result in any significant changes in autophagy-related gene expression (Fig 5).

In the liver and kidney, BD resulted in a significant increase in the expression of pro-apoptotic

gene Bax, which was unaffected by rapamycin treatment (liver: p = 0.001, kidney: p = 0.040,

Fig 6A,D). Gene expression of anti-apoptotic gene Bcl2 and the Bax/Bcl2 ratio were not

significantly different in any of the groups (Fig 6B,C,E,F).

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Figure 5. Autophagy gene expression in the liver and kidney following brain death is unaffected by mTOR inhibition. Relative mRNA expression of autophagy-related genes LC3, Beclin 1, and SQSTM1/

p62 after 4 h of experimental time in sham and brain-dead animals treated with vehicle (EtOH) or

rapamycin (1mg/kg) 2 h prior to brain death induction. Results are presented as the mean ± SD, n = 8

per group (*p < 0.05, ** p < 0.01, *** p < 0.001, where brackets indicate non-parametric tests were

used, and straight lines parametric tests).

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Figure 6. Apoptosis gene expression in the liver and kidney following brain death is unaffected by

mTOR inhibition. Relative mRNA expression of apoptosis-related genes B, E) B-cell lymphoma 2 (Bcl-

2) and A,D) Bcl2-associated X (Bax)), and C, F) their ratio after 4 h of experimental time in sham

and brain-dead animals treated with vehicle (EtOH) or rapamycin (1mg/kg) 2 h prior to brain death

induction. Results are presented as the mean ± SD, n = 8 per group (*p < 0.05, ** p < 0.01, where

brackets indicate non-parametric test were used, and a straight line parametric tests).

8 DISCUSSION

The primary focus of this study was to investigate how BD affects autophagy in the liver and the kidney. Secondly, we assessed whether autophagy stimulation with mTOR- inhibitor rapamycin affected organ quality and apoptosis following BD. Our results show that autophagy is reduced in both organs following BD. This reduction was accompanied by increased levels of autophagy inhibitor mTOR in the liver but not in the kidney. Inhibition of mTOR with rapamycin effectively reduced mTOR activity but was unable to reverse the BD- induced reduction in autophagy, nor attenuate BD-induced injury and apoptosis in the liver and kidney. These data, therefore, suggest that autophagy regulation during BD occurs via mTOR-independent pathways.

The novel, main finding that autophagy is inhibited in both the liver and the kidney following BD is supported by reduced protein levels and mRNA expression of several autophagy markers (see Figure 1 for an overview of the autophagy process). We observed reduced protein levels of LC3, the most widely used marker for autophagy

. Increased LC3-II suggests more autophagosomes, but this can reflect either a stimulation of autophagy or an accumulation of autophagosomes caused by inhibition of autophagosome-lysosome fusion (and thus inhibition of the so-called autophagic flux)

. However, as we also observed increased levels of SQSTM1/p62, a substrate for autophagic degradation, our data suggest inhibited autophagic flux following BD in both organs. As SQSTM1/p62 levels are strongly dependent of cell type and context, we additionally examined expression of SQSTM1/p62 and LC3 genes. In support of our Western blot analyses, mRNA expression levels of LC3 and SQSTM1/p62 further support a decrease in autophagic flux in both organs following BD, possibly in part due to transcriptional changes.

The reduction in autophagic flux was accompanied by increased hepatic phospho-S6 (PS6)/

S6 protein levels in the liver. As phosphorylation of protein S6 is (indirectly) dependent

on mTORC1 activity

, this suggests increased mTOR activity. Interestingly, in the kidney

mTOR activity was unaffected. However, to further clarify the exact role of mTOR in the

(dys)regulation of autophagy during BD in the liver, rats were pretreated with mTOR-

inhibitor rapamycin. Although rapamycin treatment did not affect PS6/S6 in the kidney of

sham-operated rats, it reduced mTOR-activity in both the liver and kidney following BD,

as evidenced by reduced PS6/S6 levels. Surprisingly, this mTOR inhibition did not reverse

autophagy-related changes in protein levels or mRNA expression, suggesting no effect of

rapamycin on autophagic flux. Rapamycin solely decreased Sqstm1/p62 protein levels, but it

is unclear whether this change was related to the autophagic flux, as this protein is involved

in other intracellular pathways besides autophagy.

This inability to induce autophagy is

further supported by our data showing no effect of rapamycin treatment on BD-induced

injury (increased plasma AST, ALT, creatinine, urea, and LDH), kidney function (IVR, UF

8

production) or apoptosis (protein expression of apoptosis effector cC3 and pro-apoptotic Bax protein and mRNA levels). Altogether, our data suggests that autophagy dysregulation during BD occurs via pathways independent of mTOR activity.

Dysregulation of autophagy can be of clinical importance, as defective autophagy regulation has been linked to many diseases including aging, cancer, infectious diseases, and tissue degeneration

. Even though the effects of BD on autophagy were not previously investigated, several BD-related stressors including hypoxia, ATP deprivation, and metabolic stress are known to increase upstream autophagy signaling

. Therefore, it was interesting to observe decreased autophagic activity during BD, suggesting autophagy attenuation.

When looking at disease models related to organ transplantation, results on autophagy are often conflicting. Experimental studies on ischemia-reperfusion (IR) injury show that autophagy is both increased and decreased in the liver

, and mostly increased in the kidney

. Interestingly, in the context of hepatic IR injury, modulation of autophagy, whether it be inhibition in the case of increased autophagy, or stimulation in the case of decreased autophagy, proved largely protective in the liver

. This suggested that stimulation of autophagy in the context of BD might be beneficial in the liver. In studies on renal IR injury, however, autophagy modulation was both beneficial and detrimental

. One reason for these conflicting results regarding autophagy regulation is that the activation of autophagy is dependent on the environmental context. The level or duration of (e.g. oxidative, IR) injury might determine whether autophagy modulation proves beneficial or detrimental

. In this respect, it would be interesting to analyse autophagy fluctuations over shorter and longer periods of BD. Furthermore, most autophagy modulators are non-specific and target not only the autophagic machinery but also other cellular processes, making interpretation of their effects difficult

. Even though the proposed role for autophagy in the kidney during the transplantation process is not yet clear, autophagy dysregulation likely influences organ quality prior to transplantation.

Down-regulation of autophagy could in part be responsible for the detrimental effects of

BD in the liver and kidney, exemplified by increased apoptosis in both organs. Even though

apoptosis and autophagy exhibit a complex, intertwined relationship, these processes

generally co-exist in a mutually exclusive manner

. Autophagy can inhibit apoptosis by

degrading extrinsic pro-apoptotic effector caspases and by removing reactive oxygen species

ROS, cell death triggers, and damaged organelles

. Conversely, when a certain threshold

is reached and apoptosis becomes activated, autophagy is frequently inhibited in part by

the cleavage of several autophagy proteins

. Indeed, we observed increased apoptosis yet

reduced autophagy in both the liver and kidney. Given their mutual exclusive relationship,

autophagy stimulation during BD might reduce apoptosis and improve organ function.

8

Modulation of autophagy using rapamycin in our model was unable to stimulate autophagy, despite effectively inhibiting mTOR activity. However, mTOR inhibition does not preclude autophagy induction as there are also mTOR-independent pathways, including the phophoinositol signaling pathway, the cAMP-related and Ca

-related pathways, which can be modulated with compounds like lithium, carbamazepine, and Ca

-channel antagonists

. Other autophagy regulators include trehalose, sucrose and raffinose, which function as chemical chaperons to induce autophagy outside of mTOR-regulation

. Interestingly, we showed that thyroid hormone T3 was able to induce autophagy and reduce apoptosis in the liver but not the kidney of brain-dead rats

. Similar results were observed in a mice study on hepatic IR injury, where T3 preconditioning attenuated hepatic injury, even though this likely occurred via the mTOR pathway

. Further research into different (mTOR-independent) autophagy modulators in the BD setting are needed to elucidate regulatory mechanisms of autophagy in a brain-dead donor.

Several limitations apply to this study. Firstly, as this study served as a proof-of-principle study in the brain-dead donor only, no long-term effects of mTOR inhibition by rapamycin were studied. Furthermore, the dynamic nature of autophagy means that measuring autophagic flux can be a challenging endeavour particularly in in vivo models

. Ideally, we would have liked to add additional immunohistochemical analyses, but unfortunately these techniques for autophagy analysis are difficult to perform in rodent samples

. To account for this, we have analysed multiple autophagy markers using both qPCR and western blot.

Finally, as mTOR inhibition was unable to induce autophagy, future studies will need to be conducted to further investigate mTOR-independent pathways in order to decipher the role of autophagy during BD.

In conclusion, we have identified autophagy as a novel player in the pathophysiology of

the brain-dead donors, as autophagy in the liver and kidney was inhibited already within

the brain-dead organ donor. The pro-survival and pro-death properties of autophagy and

its close relationship with cellular stress and cellular death pathways makes autophagy an

interesting new pathway to assess organ quality and therapeutic target for improving organ

quality prior to transplantation. As mTOR inhibition was unable to alter autophagic activity,

therapeutic strategies that target mTOR-independent pathways should be considered when

modulating autophagy in brain-dead donors.

8

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SUPPLEMENTARY FIGURES

Figure S1. Experimental setup of Isolated Perfused Kidney (IPK) device.

8

Figure S2 Rapamycin treatment resulted in a significantly lower blood pressure and increased use of colloid polyhydroxyl starch (HAES) compared to vehicle-treated animals during 4h of brain death.

A. The graph represents the mean arterial pressures (MAP) in mmHg, measured by intravenous cannulation of the left femoral artery. The record started with the BD induction, considering time “0”

as the end of BD induction and the start of the BD period. The blood pressure profile of rapamycin- treated rats (red graph) was on average 15,9 mmHg lower than vehicle-treated animals (blue graph). B.

Amounts of HAES and C. noradrenaline (1 mg/ml) given to brain-dead rats during the 4h experimental

procedure to maintain a MAP above 80 mmHg. Results are presented as the mean ± SD (n = 8 per

group, **p < 0.01).

8 Fig S3. Western blots of liver (A-C) and kidney (D-F).