Effect of omega-3 food supplementation in ischemia induced acute kidney injury

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Aus der Klinik für Nieren- und Hochdruckerkrankungen der Medizinischen Hochschule Hannover

Effect of omega-3 food supplementation in ischemia induced acute kidney injury

INAUGURALDISSERTATION

zur Erlangung des Grades eines Doktors der Humanbiologie

-Doctor rerum biologicarum humanarum- (Dr.rer.biol.hum)

vorgelegt von Shu Peng aus Hubei, V.R. China

Hannover 2018

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Annahme durch den Senat: 11.12.2018

Präsident: Prof. Dr. med. Christopher Baum Wissenschaftliche Betreuung: Prof.‘in Dr. med. Faikah Güler

Wissenschaftliche Zweitbetreuung: Prof. Dr. med. Stephan Immenschuh

1. Referent: Prof. ‘in Dr. med. Faikah Güler 2. Referent: Prof. Dr. med. Stephan Immenschuh 3. Referent: Prof. Dr. med. Gregor Warnecke

Tag der mündlichen Prüfung: 11.12.2018

Prüfungsausschuss:

Vorsitz: Prof. Dr. rer. biol. hum. Roland Jacobs 1. Prüfer: Prof. ‘in Dr. med. Faikah Güler 2. Prüfer: Prof. Dr. med. Stephan Immenschuh 3. Prüfer: Prof. Dr. med. Gregor Warnecke

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3.2.3 Effect of omega-3 supplementation on renal morphology and tubular function after IRI 22 3.2.4 Effect of omega-3 on mediators of inflammation HO-1 and NRF2 after IRI 23 3.2.5 Effect of omega-3 supplemenation on inflammation after IRI 25 3.2.6 Effect of omega-3 on pro-inflammatory cytokines (IL-6, MCP1) after IRI 25

4. Discussion 27

5. Summary 31

6. Abbreviations 32

7. Acknowledgements 35

8. References 36

9. Abstracts for posters at national and international conferences 43

10. Curriculum vitae 44

11. Declaration 45

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1. Introduction

Acute kidney injury (AKI) is a severe complication after major surgery and solid organ transplantation. AKI increases morbidity and mortality of the patients, which is oftentimes caused by renal ischemia reperfusion injury (IRI). 1.1 Pathophysiology of renal ischemia-reperfusion injury

Severity of IRI induced renal damage presents differently in the different parts of the kidney because partial oxygen pressure (pO2) decreases continuously from cortex to medulla¹. The medulla has the lowest oxygen saturation with pO2:10 – 20 mmHg whereas the cortex has 50-60 mmHg. Therefore, the medulla is most sensitive to hypoxia due to IRI and exhibits cell damage first. AKI is characterized by loss of the tubular brush border, vacuolization, epithelial flattening and detachment, tubular cast formation, dilatation and loss of nuclei. In addition, local tubular necrosis and apoptosis along the nephron and loss of integrity of the basal membrane are also typical. Consequences are increased capillary permeability, interstitial edema, and leukocytes infiltration¹. Kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL)² are upregulated after AKI in the proximal and distal renal tubules, they can also be found in urine as AKI biomarkers³.

As pO2 decreases in the tissue, transition from aerobic to an anaerobic metabolism occurs, resulting in the accumulation of metabolic products and intracellular acidosis⁴. As metabolic demand exceeds adenosine triphosphate (ATP) production, ATP depletion occurs and ATP-dependent membrane pumps fail, which results in intracellular accumulation of sodium and calcium. Consequences of this are increased intracellular oncotic pressure, cell swelling, membrane rupture and cell death.

After restoring perfusion, ischemia-induced electron transport chain dysfunction in mitochondria⁵ results in excessive production of reactive oxygen species (ROS) beyond anti-oxidant scavenging capacity⁶. ROS, in turn, act on membrane permeability pore of mitochondria and augment mitochondrial rupture⁷. It is toxic to membrane lipids, proteins, and nucleic acids and can cause cell death⁸. In addition, ROS promote the formation of the inflammasome⁹ and activate caspases enhancing production of pro-inflammatory cytokines¹⁰. The renal microvasculature is sensitive to IRI: intravascular plugging by leukocytes, fibrin, or platelets and endothelial swelling may cause a non-reflow phenomenon or intravascular obstruction¹¹. Moreover, leukocyte adhesion and transmigration¹² are facilitated by upregulation of endothelial adhesion molecules on endothelial cells due to alteration in flow-mediated biomechanical forces. In addition, vasoconstrictors like endothelin-1, thromboxane A2 and angiotensin II pro-coagulation factors also released in IRI¹.

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necrotic and injured cells, and can lead to activation of the complement system and immune cells (leukocytes,

antigen presenting cells and T cells), stimulating epithelial and endothelial cells through pattern recognition receptors (PRRs) or toll-like receptors (TLRs). Consequently, inflammatory cytokines and chemokines like TNF-α, MCP-1, IL-6 and TGF-β are produced.

Innate and donor specific immune responses are promoted by IRI in transplantation¹³; Neutrophils, macrophages and monocytes, dendritic cells, and in the later phase lymphocytes, mediate IRI induced AKI and repair. Neutrophils are the first leukocytes invading the kidney in about 30 min after reperfusion, and bind to the activated endothelium especially in the outer medulla. They release neutrophil extracellular traps (NETs), proteases, ROS, myeloperoxidases and cytokines that are cytotoxic¹⁴. Monocytes and macrophages are the second type of leukocytes infiltrating the tissue from 1 h after reperfusion onwards. M1 macrophages have pro-inflammatory effects and contribute to fibrosis in the later phase of IRI¹⁵. M2 macrophages have anti-inflammatory effects and promote kidney repair¹⁶. T-lymphocytes arrive a few days later. CD4+ T cells enhance renal IRI¹⁷ and regulatory T cells (Tregs) reduce inflammation¹⁸. Interestingly, IL-6 was not only reported with pro- or anti-inflammatory function in AKI, but also as mediator of rejection in renal transplantation¹⁹. AKI can result in regeneration by replacing the damaged tubular epithelium and restoration of microvasculature²⁰. Tubular epithelial cells can dedifferentiate, migrate and proliferate after IRI²¹. On the other hand, AKI can also have incomplete tubular repair, ongoing inflammation and subsequent fibrosis. Maladaptation causes chronic kidney disease (CKD), which has been linked to peritubular capillary rarefaction¹², chronic hypoxia and ongoing macrophages activation²². Different treatment strategies aim to reduce AKI and CKD among them omega-3 food supplementation have been suggested.

1.2 Renal ischemia-reperfusion injury and delayed graft function

Following kidney transplantation (ktx), IRI usually manifests as delayed graft function (DGF)²³. IRI leads to an orchestra of cell death including apoptosis, regulated necrosis (RN)²⁴, necrosis and ferroptosis²⁵, which activate innate immunity through damage-associated molecule pattern (DAMP) mediated pathways²⁶ and promote adaptive immune response²⁷. In ktx, cold ischemia times (CIT) of >20 hours and higher donor age are relevant risk factors for DGF with prevalence rates of up to 60%²⁸.

1.3 Renal ischemia-reperfusion injury and acute kidney injury

IRI is characterized by decreasing renal blood flow, tubular epithelial damage, cell death and inflammation^29-31 due to impaired oxygen and nutrient supply and waste product accumulation³². Hypovolemia caused by diarrhea or vomiting, massive bleeding, sepsis or dehydration are the common reasons for renal perfusion impairment resulting in prerenal AKI.

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High incidence rates of AKI have been observed after major cardiac surgeries and also in solid organ transplantation, ranging from 5%-50% after ktx, 50-60% after lung transplantation, and 40-70% after liver transplantation^33,34. AKI is associated with long-term complications and has been correlated to enhanced post-surgical mortality³⁵. In a recent survey on patients of Intensive Care Units (ICU) in China ~22% of patients³⁶ suffered from AKI with morbidity and mortality rates of 30% to 70%³⁷

1.4 Omega-3 polyunsaturated fatty acids as food supplementation

Omega-3 polyunsaturated fatty acids (n3-PUFAs) are among the most popular food supplements, secondary to the vitamins, in the U.S. The intake of n3-PUFAs, especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), seems to have anti-inflammatory, triglyceride and blood pressure lowering effects. However, evidence on their protective effects in cardiovascular diseases in different studies remains controversial.

1.5 Aims

To investigate whether dietary n3-PUFAs has preventive effects on ischemia induced AKI in a mouse model of renal IRI and to study how dietary n-3 PUFAs modulate oxylipin patterns in the renal tissue.

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2. Material and methods

2.1 Animals

C57BL/6J^Han-ztm (B6, H^2b) adult male mice (11-13 weeks of age; bodyweight 23-28 g) were used in all experiments. Mice were housed and bred in the Institute of Laboratory Animal Sciences of Hannover Medical School. The light and dark cycle was 14/10 h. Mice had free access to domestic quality food and drinking water. After surgery, all mice were monitored daily for physical condition. Mice were treated in accordance with our institution’s guideline for experimental animal welfare. All experiments were approved by the local animal protection committee from Lower Saxony State department of animal welfare and food protection (LAVES; Aktenzeichen: 33.9-42502-04- 14/165 and 14/1569, surgeries were performed by Dr.

Song Rong, Dr. Rongjun Chen).

Termination of the study:

Mice were sacrificed under deep isoflurane anesthesia (5%) in combination with analgesic treatment followed by total body perfusion with cold PBS causing cardiovascular and respiratory arrest.

Termination criteria during clinical follow-up:

Criteria to terminate the study included signs of neurological disorders, passivity of mice, peripheral edema, refusal to eat and shaggy appearance. Studies were terminated prematurely if the mice had body weight reduction >20%, elevation of serum-creatinine >300 µmol/L or a clinical assessment score of 3 or less. Mice with a score of 4 were re-assessed within four hours.

The following scoring scheme was used for clinical assessment of mice:

Score Quality Characteristics

1 Moribund No activity, expected death

2 Lethargic No activities, decreased food intake

3 Quiet, reduced food intake Uninterested in the environment, rare activity, sleepy 4 Limited activity Responds to affection, frequent activity breaks 5 Active Curious, quick, sporadic activity breaks

6 Very active Strong, wide awake, quick movements

2.2 Equipment for animal surgeries

 Surgery microscope M690 (Leica Service, Bensheim, Germany)

 Univentor 400 anaesthesia unit (TSE systems, Bad Homburg, Germany)

 Heating circulator bath table C10-B3 (HAAKE GmbH, Karlsruhe, Germany)

 Syringe pump (TSE systems, Bad Homburg, Germany)

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2.6 Antibodies for immunohistochemistry IHC primary antibodies against mouse:

Antibody Marker for Company Clone Species Dilution

NGAL Tubular damage, neutrophils

Dianova 043-29 Rat 1:1000

A1M Proximal tubular dysfunction

Prof. Akerstrom University Lund

Rabbit 1:500

Gr-1 Neutrophils AbD serotec RB6-8C5 Rat 1:1000

NRF2 transcription factor (antioxidant genes)

GeneTex Ep1808 Rabbit 1:100

HO-1 hemoxygenase-1( t ubules, leukocytes)

Enzo life science

ADI-OSA-110 Mouse 1:75 IHC secondary antibodies:

Antibody Company Number Dilution

Alexa Fluor-555 goat anti-rat IgG Invitrogen A21434 1:500 Alexa Fluor-555 donkey anti-rabbit IgG Invitrogen A31572 1:500 Alexa Fluor-555 donkey anti-mouse IgG Invitrogen A31570 1:500 Alexa Fluor-555 donkey anti-goat IgG Invitrogen A21432 1:500

2.7 Chemicals and kits

Chemicals and kits were from the following companies:

Abcam (Cambridge, UK); Biochrom (Berlin, Germany); BD Bioscience (Heidelberg, Germany);

Cell Signaling Technology (Danvers, USA); Carl Roth GmbH (Karlsruhe, Germany) Dianova (Hamburg, Germany); ebioscience (Frankfurt am Main, Germany); Eppendorf (Hamburg, Germany);Fluka (Steinheim, Germany); Fresenius Kabi (Graz, Austria); Keyence GmbH (Erkrath, Germany); Lonza (Basel, Switzerland); New England Biolabs (Ipswich, UK);

QIAGEN (Düsseldorf, Germany); Roche Diagnostics GmbH (Mannheim, Germany); Takara Clontech (Saint- Germain-en-Laye, France);

2.8 Real-time polymerase chain reaction (rt-PCR)

2.8.1 Primers for real-time polymerase chain reaction (rt-PCR)

Name Company Product number Sequence

PAI-1 BioTez GmbH Berlin-Buch

26195-20 Forward:

5’-ATGTTTAGTGCAACCCTGGC-3’

26195-20 Reverse:

5’-CTGCTCTTGGTCGGAAAGAC-3’

IL-6 Qiagen QT00098875 Quantitec

MCP-1 Qiagen QT00167832 Quantitec

TNF-a Qiagen QT00104006 Quantitec

HPRT Qiagen QT00166768 Quantitec

Housekeeper

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2.8.2 Solutions for rt-PCR 70% ethanol:

 RNase-free water 15 ml

 Ethanol (100%) 35 ml

DNase I stock solution (Stored at –20 °C )

 Dissolve lyophilized DNase I (1500 Kunitz units) in 550 μl RNase-free water ( with RNase-free needle and syringe)

 Invert the vial gently (do not vortex)

 Divide into single-use aliquots(stored at –20 °C for maximum 9 months)

 Thawed aliquots can be stored at 2–8 °C for maximum 6 weeks

 The aliquots cannot be frozen again after thawing.

10×SYBR solution:

 SYBR Green I nucleic acid gel stain (10000×Concentrate) 5.0 μl

 Dimethyl sulfoxide (DMSO) 5.0 ml

 SYBR Green master mix (containing 2 mM MgCl2):

 10×PCR buffer with Magnesium chloride 2.0 μl

 PCR nucleotide mix 0.4 μl

 10×SYBR 1.0 μl

 0.1% Tween 20 2.0 μl

 Diethylpyrocarbonate (DEPC) water 4.6 μl

SYBR Green master mix (containing 5 mM MgCl2):

 10×PCR buffer (without Magnesium chloride) 2.0 μl

 25 mM Magnesium chloride (Roche Diagnostics GmbH) 4.0 μl

 PCR nucleotide mix 0.4 μl

 0.1% Tween 20 2.0 μl

 DEPC water 0.6 μl

 10×SYBR 1.0 μl

PCR reaction solution for one well:

 Forward primer 0.6 μl

 Reverse primer 0.6 μl

 SYR Green master mix 10 μl

 DEPC water 3.7 μl

 FastStart Taq DNA Polymerase (Roche Diagnostics GmbH) 0.1 μl 2.8.3 mRNA isolation

Procedure:

According RNeasy Mini Kit (Qiagen):

 Add 10 µl β-Mercaptoethanol (Sigma-Aldrich) to 1 ml buffer RLT.

 Clean forceps and homogenizer with RNAse AWAY (Thermo Fisher Scientific).



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 350 µl 70% ethanol was added and mixed by pipetting.

 Then total 700µl solution was transferred to a RNeasy spin column placed in a 2 ml collection tube

 Centrifuge for 15 s at 8000xg. The flow through was discarded.

 350 µl RW1 buffer was added

 RNeasy spin column was centrifuged 15 s at 8000g. The flow through was discarded.

 Add 10 µl DNase I stock solution to 70 µl RDD buffer and mix. 80 µl of the mix was transferred to the RNeasy spin column for 15 min incubation at room temperature.

 350 µl RW1 buffer was added to the RNeasy spin column

 15s centrifugation at 8000g. The liquid was discarded.

 500 µl RPE buffer was added to the RNeasy spin column

 15 s centrifugation at 8000xg. The liquid was discarded.

 500 µl RPE buffer was added to the RNeasy spin column

 15s centrifugation at 8000xg. The liquid was discarded.

 2min centrifugation at 8000xg. RNeasy spin column was transferred to a new 1.5 ml collection tube.

 30 µl RNase-free water was added into the spin column

 1min incubation. Then spin column was centrifuged at 8000xg for 1 min to elute the RNA.

 RNA concentration was measured with an Eppendorf Bio Photometer (at 260 nm).

2.8.4 Synthesis of cDNA from RNA

 Takara Prime Script RT reagent KIT RR037A was used for cDNA synthesis

 5x Prime Script buffer (2 µl) was mixed with Prime Script RT Enzyme Mix I(0.5 µl), Oligo dot Primer (0.5 µl, 50 µM) and with Random Hexamers (0.5 µl, 100µM) and added to 10 µl of 0.1g/µl RNA.

 The mix was incubated in a thermal cycler (MJ Research, BIO RAD) at 37°C (15 min) for reverse transcription followed by 5s incubation at 85°C to denature the enzyme.

2.8.5 Light cycler rt-PCR protocol

Light Cycler 96 PCR System (Roche) was used under following conditions:

20 µl PCR reaction mix was added to each 96-well plate well. Pre-incubation at 96°C for 10 min to activate the polymerase, 45 cycles for amplification was done. Denaturation with 95°C for 10 s, then 10 s at 60°C for annealing and 10 s at 72°C for extension.

Reaction per well:

 DNA (0.01 µg/µl) 5 µl

 DEPC-treated water (Sigma Aldrich) 3 µl

 Forward Primer (10 pmol/µl) 1 µl

 Reverse Primer (10 pmol/µl) 1 µl

 Takara SYBR qPCR: SYBR Premix Ex Taq 10 µl

 or Qantitect Primer assay (Qiagen) Primer (100pmol/µl) 2 µl

Each sample was measured as a triplicate. HPRT was used as house keeper for normalization. Quantification was performed by Light Cycler96 SW 1.1 software using the excel add-in. 5-7 mice were used per group for qPCR analysis.

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2.9 Histology and immunohistochemistry

Tissue fixation was done in 4% paraformaldehyde (PFA) with paraffin embedding or in isopentane on dry ice for cryosections. Paraffin embedded tissue was cut in 2 µm sections and staining was done according to the antibody protocol.

Deparaffinization, rehydration and dehydration as well as mounting after staining were conducted as follows:

Deparaffinization:

 5min x 3 times in Roti-Histol (Carl Roth) Rehydration:

 3min x 3 times in 100% ethanol

 2min x 2 times of 96% ethanol

 1 min in 70% ethanol

 1 min rinsed in 50% ethanol

 Shortly rinsed in distilled water Clearing:

 2min x 3times incubation in Roti-Histol Mounting:

 Roti-Histokit mounting medium (Carl Roth GmbH) was used.

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 In running tap water for 10 min.

 Counterstaining in hematoxylin for 1 min.

 In running tap water for 10 min.

2.9.2 Immunohistochemistry

Antigen retrieval was done according to the antibody supplier recommendations and optimization studies using either trypsin digestion or 15 min microwave incubation in citric acid was done as well.

Citric acid solution for microwave treatment

1g citric acid monohydrate (Sigma Aldrich) was dissolved in 500ml distilled water( 0.01 M pH 6.0)

Microwave Oven:

 Slides were incubated in citric acid solution and heated in microwave for 8 min on full power.

 Slides were kept warm in the microwave for 2 min and then heated again for another 8 min.

 Slides were cooled on ice for 30 min.

Trypsin solutions for tissue digest:

 1 mg trypsin tablet (Sigma Aldrich) was dissolved in 1000 µl distilled water.

Phosphate buffered saline (Th Geyer):

 10x Phosphate buffered saline (PBS) in distilled water for 1xTrypsin

 Samples were incubated trypsin solution in a humid chamber for 15 min at 37 °C.

Staining procedure:

 Slides were washed in PBS after antigen retrieval.

 Samples were incubated in primary antibody (diluted in PBS) in a humid chamber in darkness for 60 min at RT and washed in PBS afterwards.

 Incubation with fluorescence labeled secondary antibody in a humid chamber in darkness for 60 min at RT

 After washing in PBS, slides were embedded in DAPI containing mounting medium (Dianova) for nuclei staining.

Evaluatin and quantification:

Slides were quantified in a blinded manner using a Leica imaging microscope, mostly at a 200-fold magnification.

Gr-1⁺, CD3⁺ and F4/80⁺ leukocytes were scored semi-quantitatively:

0 = no leukocyte per view field, 0.5 = baseline expression, 1= local single layer infiltration of leukocyte, 2 = local double layer infiltration, 3 = multilayer infiltration per view field, 4 = dense infiltration per view field.

NGAL, A1M, NRF2, and HO-1 were scored by the percentage of positive renal tubules in each view field.

Fibronectin and CollagenⅣ were scored semi-quantitatively:

0.5 = baseline expression, 1= single layer of fibers, 2 = double layer of fibers, 3 = dense, multilayer of fibers, 4 =broad, multilayer of fibers.

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2.10 Fatty acid and oxylipin analysis

Analysis of fatty acid (FA) composition and oxylipin in plasma and renal tissue was done by gas liquid chromatography (GC) in a collaboration with Prof. Nils Schebb and was carried out by Katharina Rund M.Sc. (University of Vetinary Medicine, Hannover).

Fatty acid composition was analyzed in 50 μL plasma and renal tissues (30–35 mg) as described previously^38,39. First, blood and tissues were extracted with methanol/

methyl tert-butyl ether (1:2, v/v); Second, they are derivatized to FA methyl esters with methanolic hydrogen chloride (acetylchloride in methanol (1:10, v/v); Third, gas chromatography with flame ionization detection (GC-FID) was used for analysis. Fourth, calculate the relative pattern and absolute fatty acid concentrations with response factors.

Results are shown as mean ± standard error of the mean (SEM).

Extraction and analysis of oxylipins from plasma (200 μL) and kidney (50±5 mg) was carried out as described³⁹. Kidney (50±5 mg) was homogenized and centrifugation (20 000 x g, 5 min, 4°C); the organic phase was collected with sample extraction from 750 μL ethyl acetate. A vacuum centrifuge (Christ, Osterode am Harz, Germany) was used for evaporation of combined organic phases, and reconstitution of dried lipid extract was done in 300 μL methanol. The samples were diluted to 6 mL with water and acidification was done by acetic acid (<pH 3) before extraction on C18 cartridges (500 mg; Macherey-Nagel, Düren, Germany). Elution was done by methyl formate. Oxylipins were quantified using liquid chromatography-mass spectrometry (LC-MS) as described³⁹. Results are presented as mean

± SEM.

2.11 Organ preservation

At the endpoint mice were deeply anesthetized with 5% isoflurane. Midline laparotomy was performed followed by whole body perfusion via the left ventricle with ice-cold 0.9% PBS resulting in cardiovascular and pulmonary arrest. Kidneys were harvested and cut into 4 parts and fixed as follows:

- Isopentane on dry ice (-40°C) for immunohistochemistry;

- Liquid nitrogen for protein biochemistry (-80°C);

- RNA later for mRNA isolation,

- 4% paraformaldehyde (PFA) for paraffin embedding

If flow cytometry was planned 70% of the kidney was placed in ice-cold PBS and 25% was fixed in PFA for morphology.

2.12 Statistical analysis

GraphPad prism 5.0 version (GraphPad Software Inc., San Diego, CA) was used for statistical analysis. One-way ANOVA was used in multiple group analysis, students T-test was used when two groups were compared. Data are demonstrated as mean ± standard error (SEM). Significant differences were recognized as *p<0.05, **p<0.01 and ***p<0.001.

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3. Results

The impact of renal IRI on lipid mediator expression profiles was analyzed. In addition, a treatment study with omega-3 food supplementation was done to test the efficacy to prevent renal damage after IRI.

3.1 Lipid mediators in renal ischemia reperfusion injury

B6 mice undergoing 35min of renal IRI develop AKI with almost total recovery within 2-3 weeks. However, a longer ischemia time of 45min causes severe AKI with chronic inflammation and fibrosis⁴⁰. In this study, 35 and 45min unilateral IRI was correlated to the alterations of lipid mediators.

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Oxylipin composition in renal tissue was investigated by Katharina Rund M.Sc. (University of Vetinary Medicine Hannover). Arachidonic acids (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were investigated. Surprisingly, oxylipins did not show any differences between groups at d1 after IRI.

Figure 7: Oxylipin expression in renal tissue after sham surgery and IRI at d1.

Aarachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are similar in all groups (mean ± SEM, n=5 to 6 mice/group).

3.2 Food supplementation with omega-3 to attenuate AKI

In the next part of the study we investigated the role of a dietary omega-3 polyunsaturated fatty acid (PUFA) supplementation on the extent of renal damage after IRI. Since bilateral IRI was done, clinical chemistry and survival could be monitored. Kidney damage was analyzed at day 1 after IRI by histology, immunohistochemistry and mRNA expression of pro-inflammatory cytokines. A control group received standard chow (STD) and was compared to the omega-3 supplementation group (2% in the chow containing 10% fat, STD+n3). Feeding was done for two weeks prior to IRI. Five to seven mice per group were investigated for each experiment.

5 (R ,S )-F 2 t-Is o P 5 (R ,S )-F 3 t-Is o P 4 (R ,S )-F 4 t-Is o P 0

2 0 4 0 6 0

pmol/g tissue

IR I 3 5 m in IR I 4 5 m in s h a m 4 5 m in

( A R A ) ( E P A ) ( D H A )

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4. Discussion

Omega-3 and omega-6 polyunsaturated fatty acid (PUFA) are essential for mammals, because they do not have enzymes that synthesize double bonds at n-6 and n-3 positions in a carbon chain, therefore, humans are dependent on dietary n-3 sources⁴⁸.

Ratios between n6-PUFA and n3-PUFA have been reported to play an important role in pathogenesis of chronic inflammatory^49,50, cardiovascular disease^51,52 and also in cancer.

Beneficial effects are mediated by n-3 PUFA and detrimental effects by n-6 PUFA. The ideal ratio of n-3/n-6 PUFA for human beings was 1:1 before agricultural revolution, however, it has been changed to 1:20 over the last 150 years in the western diet. Then, cereals became the major source of calories which resulted in excessive n6-PUFA and insufficient n3-PUFA uptake⁵³.

Docosahexaenoic acid (C22:6 n3, DHA), eicosapentaenoic acid (C20:5 n3, EPA) and α-linolenic acid (C18:3 n3, ALA) are the main types of n3-PUFA with physiological significance. They act as structural components of phospholipids in cell membranes and are ligands to nuclear transcription factors, such as nuclear-factor kappa B (NFB), retinoid X receptor (RXR), peroxisome proliferator-activated receptor (PPAR), sterol regulatory element binding proteins (SREBPs) and substrates for endocannabinoids. DHA and EPA can be synthesized in humans from ALA. However, the conversion rate of ALA into EPA and then to DHA, which occurs primarily in the liver is less than 15%⁵⁴, resulting in low concentrations of DHA and EPA in the circulation. In the U.S, EPA and DHA are the most popular non-vitamin/mineral dietary supplementation products and are bought over the counter frequently.

In renal diseases, protective effects of n-3 PUFA were observed in AKI^55-57, diabetic nephropathy⁵⁸, chronic kidney disease^59-61, IgA nephropathy^62-74, nephrolithiasis⁷⁵, lupus nephritis⁷⁶, cyclosporine induced nephrotoxicity^77,78 and transplant nephropathy⁷⁹. However, the effect of n-3 PUFA was not always consistent among different studies, mainly arising from such variables as dose and duration of diet, proportion of EPA and DHA, ratio between n3 and n6-PUFA, quality and sample size of the study population, severity of injury and comorbidities of the patients^80,81.

Fat-1 transgenic mice are engineered with the gene encoding for n-3 FA desaturase on C57BL/6 background, which convert n-6 to n-3 PUFA. Therefore, without n-3 PUFA food supplementation they have a reduction of n-6 PUFA simultaneously with elevated n-3 PUFA levels in organs and tissues. These mice serve as a good model for investigating the role of n-3 PUFA and n-6/n-3 ratio in disease prevention and treatment⁸². Some researchers suggest,

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mice were reported to have reproductive abnormalities⁸³ and were more susceptible to tuberculosis⁸⁴. Moreover, food supplementation of n-3 PUFA showed greater effect on lipid mediator profiles and eicosanoid level in kidneys of wildtype mice than the genetic impact of the fat-1 transgene^38,85. In our study we wanted to investigate whether omega-3 food supplementation can attenuate AKI in a well-defined translational renal IRI mouse model.

In our study, n-3 PUFA pre-treatment significantly attenuated s-creatinine and BUN elevation at day 1 after 30min of bilateral renal IRI. A similar study with 35min of bilateral IRI on fat-1 transgenic mice showed that not only renal function was preserved but also injury of the tubuli was alleviated⁸⁶. In contrast, we did not observe reduced histological damage in n-3 feeding mice. There are three main variables leading to such differences: First, fat-1 transgenic mice produce n-3 PUFAs endogenously. Therefore, their n3-/n-6 ratio, DHA and EPA level would differ from that in our study with exogenous n-3 food supplementation⁸⁰. Unfortunately, the group did not report lipid mediator profiles which makes the comparison in oxylipins impossible. Second, the degree of renal IRI seems to be less severe in the fat-1 mouse IRI study. Even though, longer ischemia time (35min vs 30min in our study) was used serum creatinine and BUN elevation was less than 5-fold. In our model >6-fold increase was present in the control group. Consecutively, the morphological damage was less in the fat-1 study.

Third, difference in surgical procedures, body temperature^87-89 and anaesthesia may also influence the results of IRI studies but are difficult to compare since not all details are given in the previous publication. In pilot studies we have investigated the effect of the body temperature on IRI and optimized our model to 32°C during the surgery, however, many researchers use 37°C. There are many studies in the experimental setting as well as in patients pointing towards preconditioning of the kidney due to the choice of anaesthetics^90,91. Many researchers use a combination of Ketamine /Rumpon others prefer inhalation anaesthesia with isoflurane. The latter has been described to attenuate IRI^92,93. All the above-mentioned variables alter the outcome of renal IRI and make direct comparison of different studies difficult.

As n-3 PUFA consists of alpha-ALA, DHA and EPA, each of them and their proportions in combination may have different influence on the results. According to the literature, DHA⁹⁴, EPA⁹⁵ and EPA+DHA (DHA: 80 mg/kg/day + EPA:120 mg/kg)⁹⁶were relevant to ameliorate renal function in another renal IRI model in mice. Intraperitoneal injection of DHA coppeled to bovine serum albumin (BSA) was done 4 hours after 20min bilateral renal IRI and resulted in improved renal function and morphology. However, salutary effects of DHA were dose dependent: decreasing doses of DHA from 4 to 2 mg/kg body weight were less effective, DHA dose of more than 5 mg /kg or less than 1 mg/kg did not show any effects. Overall, DHA treated mice had much better survival during 7 days of follow-up. TNF-alpha induced iNOS mRNA abundance was inhibited by levels of DHA⁹⁴.

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In our study, pro-inflammatory cytokine elevation of IL-6 and MCP-1 as well as leukocyte infiltration was not affected by n-3 PUFA supplementation. However, we observed beneficial effects on tubular transport function with enhanced uptake of A1M. Gronert’s group⁵⁵ reported that 4-week acute increase of dietary n-3 PUFA with concomitant decrease of n-6 PUFA improved renal function and reduced renal inflammation in mice after 30min bilateral IRI, and decreased mortality of mice after 45min bilateral IRI: Their feeding period was with four weeks longer than the two weeks in our study. Their control mice received n-6 rich diet (n-6 PUFA 30g/kg, n-3/n-6 ratio=1/30) and the treatment group received n-3 rich diet (5g% fat in which 7%

is n-3 PUFA, n-3 PUFA 14g/kg, n3/n-6 ratio=7/3).Our control mice received a sunflower oil diet and our treatment mice received diet enriched with 1% EPA and 1% DHA. Difference in baseline FA state had been linked to difference in individual responses to n-3 PUFA, in simple words, the lower the baseline n-3 PUFA level, the higher the increase in n-3 PUFA derived oxylipins ⁴². Unfortunately, the study of Gronert⁵⁵ did not report baseline serum creatinine, thus we cannot compare the severity of renal injury.

In conclusion, effect of omega-3 on tissue damage might depend on severity of the injury. In our model protection was seen on renal function but not in the tissue. Since in the clinical settings, severity of IRI in patients are unpredictable, omega-3 food supplementation might not be sufficient to overcome AKI. Many clinical trials revealed beneficial effect of n-3 PUFAs on chronic inflammatory diseases⁹⁷, coronary heart disease⁹⁸, IRI and delayed graft function⁹⁹, whereas others did not ^100,101. It might be more robust to correlate beneficial effect with oxylipin levels; however, this is oftentimes not reported. Schebb et al ⁴² has reviewed patterns of oxylipins in eleven studies which changed in response to their precursor n-3 PUFAs, especially for EPA derived oxylipins. We observed in our IRI study both DHA and EPA elevation in omega-3 fed mice; similar trends were seen in their respective anti-inflammatory metabolites. Consecutively, pro-inflammatory oxylipins from n-6 PUFA metabolism were reduced, as n3-PUFA and n6-PUFA compete for the same enzymes in their metabolic pathway (Fig 16. gives and overview about PUFA metabolic pathways and oxylipins biosynthesis). Also, some authors have reported the beneficial effects of omega-3 on mitochondrial dynamics and function¹⁰². For instance, the mitochondrial membrane phospholipid fatty acid composition in patients has been successfully altered by omega-3 dietary supplementation, which delayed the opening of mitochondrial permeability transition (MPT) in cardiomyocytes¹⁰³. In the recent years, regulated necrosis (RN) was reported to predominate apoptosis and have vital function in the pathophysiology of IRI induced AKI¹⁰⁴, which may be regulated by cyclophilin (Cyp) D-mediated MPT^105,106. However, it remains unclear whether omega-3 can affect RN in renal IRI^24,104, even though MPT is recognized as

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5. Summary

The effect of dietary omega-3 supplementation on renal ischemia reperfusion injury (IRI) were investigated. The mice received standard diet (STD) with low levels of omega-3 fatty acid (n-3 PUFAs) for two weeks and or omega-3 enriched diet (STD+n3). Both groups were sacrificed at day 1 after bilateral IRI of 30 min. Kidney damage was analyzed by histology, immunohistochemistry and mRNA expression of pro-inflammatory cytokines. Clinical chemistry was measured and was correlated with severity of renal injury. Analytic chemistry was performed to study lipid mediator profiles of n-3 PUFAs and their metabolites. Omega-3 food supplementation significantly attenuated creatinine and BUN increase. In addition, elevation of liver enzyme and lactate dehydrogenase was inhibited as well. Impairment of renal tubular re-absorption was detected after IRI whereas omega-3 treatment resulted in protection of tubular function. Acute kidney injury (AKI) and inflammation was more severe in outer medulla than in cortex but did not reveal differences between groups. Pro-inflammatory cytokines were also comparable. The effect of dietary omega-3 on tissue damage might depend on severity of the injury and was not strong enough to protect tissue damage in this model. We have shown that omega-3 food supplementation had beneficial effect on renal function but did not overcome inflammation and acute kidney injury. In clinical settings, severity of organ injury in patients is unpredictable, therefore n-3 PUFA food supplementation might not be sufficient to prevent AKI.

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6. Abbreviations

α alpha

β beta

ARA arachidonic acid

AV allograft vasculopathy

ASL arterial spin labeling

A1M alpha 1 microglobulin

AKI acute kidney injury

ATP adenosine triphosphate

BSA bovine serum albumin

BUN blood urea nitrogen

CD4 cluster of differentiation 4

CIT cold ischemia time

CKD chronic kidney disease

COX cyclo-oxygenase

DAMP damage-associated molecular patterns

DAPI 4’-6’diamino-2-phenylindole

DEPC diethylpyrocarbonate

DGF delayed graft function

DNase I deoxy ribonuclease I

DHA docosahexaenoic acid

DMSO dimethyl sulfoxide

DTT dithiothreitol

EPA eicosapentaenoic acid

ESRD end-stage renal disease

FACS fluorescence-activated cell sorting

FCS fetal calf serum

Fig. figure

GAPDH glyceraldehyde 3-phosphate dehydrogenase

GFR glomerular filtration rate

HMGB1 high mobility group box-1

HO-1 heme oxygenase-1

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HPETE hydroperoxyeicosatetraenoic acid

HPEPE hydroperoxyeicosapentaenoic acid

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid

ICU intensive care unit

IFTA interstitial fibrosis and tubular atrophy

IHC immunohistochemistry

IL-1β interleukin-1β

IL-6 interleukin-6

IgG immunoglobulin G

IgM immunoglobulin M

iNOS inducible nitric oxide synthase

IRI ischemia reperfusion injury

KIM-1 kidney injury molecule-1

L/D live/dead

LDH lactate dehydrogenase

LL living leukocytes

LOX lipoxygenase

M mol/L

MPT mitochondrial permeability transition

MCP-1 monocyte chemotactic protein-1

MRI functional magnetic resonance imaging

mRNA messenger ribonucleic acid

MT masson trichrome

n3-PUFA omega-3 polyunsaturated fatty acid

NADP nicotinamide adenine dinucleotide phosphate

NF-κB nuclear factor-κB

NGAL neutrophil gelatinase-associated lipocalin

Nrf2 nuclear respiratory factor 2

NO nitric oxide

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PCR polymerase chain reaction

PFA paraformaldehyde

PMA phorbol 12-myristate 13-acetate

PMN polymorphonuclear cells

P-selectin CD62 antigen-like family member P

PTC peritubular capillaries

pTEC primary proximal tubular epithelial cell

qPCR quantitative polymerase chain reaction

RBC red blood cell

RIPA radio immuno precipitation assay buffer

ROS reactive oxygen species

STD standard diet

TBS tris buffered saline

TGF-β transforming growth factor-beta

TLR toll-like receptor

TNF-α tumor necrosis factors-alpha

VCAM-1 vascular cell adhesion molecule 1

vs versus

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7. Acknowledgements

The accomplishment of this presented work and my study would not have been possibly achieved without the generous helps and guidance of the following people:

First of all, I appreciate Prof. Dr. med. Faikah Güler for accepting me as her student. She has great patience and a kind heart. She not only taught me how to plan and perform a study, but also the principle of academic medicine. She taught me how to think, talk, write and question results in an academic way. Without her, I am not the current myself.

I also appreciate Dr. med. Song Rong for his great help in my career and in my daily life. He was always taking care of me and tried to guide me to the correct way. He helped me a lot in organizing my life in Hannover.

I appreciate Herle Chlebusch, she trained me and supported me with her friendly and positive manner during the experiments. She was an incredible teacher and technical supervisor.

I appreciate the support of Michaela Beese and Dr. Anja Thorenz, who trained me in mRNA isolation and qPCR.

I am thankful to Dr. Vijith Vijayan for his support in practical experiments and to Professor Stephan Immenschuh for being my co-supervisor.

I appreciate the excellent collaboration with Professor Dr. Nils Helge Schebb and Katerina Rund in the lipid mediator studies.

I really appreciate the constant exchange with my fellow students Li Wang, Dr. Rongjun Chen, and Dr. Beina Teng, who helped me constantly in the lab and in my daily life.

I appreciate the support of Dr.Chichung Chen and Patricia Bolanos-Palmieri who helped me correcting spelling errors in the thesis.

I am deeply grateful to my parents, grandfather and aunts for their love and support.

I am deeply grateful to my grandmother who died from lung cancer; she raised me and helped me to mature. Thinking of her, I am filled with peace and courage.

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9. Abstracts for posters at national and international conferences

Shu Peng, Katharina Rund, Song Rong, Rongjun Chen, Nils Helge Schebb, Faikah Gueler.

Dietary Omega-3 food supplementation to attenuate renal ischemia reperfusion injury. 27^th International congress of the transplantation society (TTS), Madrid, Spain, 30.06-05.07.2018.

Shu Peng, Katharina Rund, Song Rong, Rongjun Chen, Nils Helge Schebb, Faikah Gueler.

Dietary food supplementation with omega-3 in renal ischemia reperfusion injury attenuates acute kidney injury (AKI). International Meeting on Ischemia reperfusion Injury in Transplantation, Poitiers, France, 19-20.04.2018.

Faikah Gueler, Vijayan Vijith, Jan Hinrich Bräsen, Shu Peng, Song Rong, Rongjun Chen, Hermann Haller, Anja Thorenz, Stephan Immenschuh. Alloantibodies in a mixed cellular and antibody mediated rejection model in mice. German Transplant Society 26^th Annual Meeting (DTG 2017), Bonn, Germany, 25-28.10.2018

Effect of omega-3 food supplementation in ischemia induced acute kidney injury

Aus der Klinik für Nieren- und Hochdruckerkrankungen der Medizinischen Hochschule Hannover