Background
Sepsis is a systemic response of the body to host infections. Septic conditions are often clinically difficult to diagnose and contribute to higher morbidity and mortality rates in ICUs world-wide [
1]. Increased mortality due to sepsis is oft caused by bacterial infections and the lipid A-containing lipopolysaccharide (LPS), a component of the bacterial wall of the gram-negative bacteria, is a major player in the pathogenesis the of gram-negative sepsis [
2]. The application of animal models have demonstrated that LPS-induced endotoxemia provokes tissue inflammation by a massive release of the inflammatory mediators as inflammatory cytokines and can lead to acute kidney injury in mice [
3]. LPS treatment induced in animal models caused a systemic hypotension and organ dysfunction similar to what occurs in clinical sepsis [
4], thus providing insight into the understanding of the pathogenesis of the acute renal injury. Recent studies have shown that LPS can activate the hypoxia-inducible transcription factor-1 alpha (HIF-1α) under normoxic conditions [
5]. The pharmacological activation of HIF-1α through inhibition of HIF-prolyl-hydroxylases (PHDs) [
6] or administration of erythropoietin, a HIFs target gene, improved renal function and reduced acute kidney injury in endotoxemic mice [
7] as well as in mice suffering from polymicrobial sepsis [
8]. Recently, we reported that preconditional suppression of PHDs by application of 3,4-dihydroxybezoate (3,4-DHB), a non-specific PHDs inhibitor, was renoprotective in two murine septic models [
9]. However, this effect was mainly localised to the kidney, we did not observe a protective systemic effect in the survival study [
10]. The discovery of the MAPK-organizer 1 (MORG1), a molecular scaffold for multiple proteins of the MAPK cascade [
11] and a binding partner of the HIF-prolyl hydroxylase domain containing protein 3 (PHD3) [
11] opens a new opportunity to study the role of pre-elevated HIFs level and its function in AKI due to sepsis and inflammation. Genetic manipulation of
MORG1 expression has revealed that
MORG1
−/−
mice exhibit embryonic lethality, whereas
MORG1
+/−
mice did not show phenotypic differences with the wild-type mice. Moreover, we found that
MORG1
+/−
animals are protected from renal injury in a murine model of ischemia/reperfusion due to increased HIF-1,2α expression and stabilisation [
12]. In normoxia HIFs are hydroxylated by PHDs followed by ubiquitination and proteasomal degradation [
13]. Reduced oxygen supply in the cells is detected by PHDs and this inhibits their HIF-prolylhydroxylase activity, resulting in HIF stabilisation and transcriptional activation. Recent research from our lab has also shown that reduced expression of MORG1 could contribute to cellular adaptation to ischemic/ hypoxic conditions through the cellular binding partner(s) PHD3/HIFs. In a murine hypoxia model
MORG1
+/−
mice were protected from systemic hypoxia- dependent renal injury due to an enhanced stability of HIF-1,2α and/or a reduced TNF-α expression in a PHD3/MORG1 dependent manner [
14] compared to the
MORG1
+/+
mice which developed overt renal damage and inflammation in an animal model of systemic hypoxia [
14]. Thus, we hypothesised that
MORG1 heterozygosity could attenuate kidney damage and inflammation, thus representing an important tool to gain insight into the cellular mechanisms of renal injury and inflammation related hypoxia in a well-established murine model of LPS-induced endotoxemia.
Methods
Animal treatment and endotoxemia induction
The animal experiments were performed according to the guidelines set by the local Animal Committee of the State of Thuringia application (file numbers 02–023/10 and 02–023/11). The animal experiments were approved by the Animal Committee of the State of Thuringia (Thueringer Landesamt fuer Lebensmittelsicherheits und Verbraucherschutz Abt. 2, Gesundheitlicher Verbraucherschutz, Veterinaerwesen, Pharmazie, Bad Langensalza, Germany) with files numbers 02–023/10 and 02–023/11 and were carried out in accordance to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals (8th edition; available online:
https://www.ncbi.nlm.nih.gov/books/NBK54050/) and to the European Community Council Directive for the Care and Use of Laboratory Animals (Directive 2010/63/EU;
http://ec.europa.eu/environment/chemicals/lab_animals/legislation_en.htm). The study was performed on wild-type mice C57BL/6 J (Jackson Laboratories, Main, USA obtained from Charles River Laboratories, Sulzfeld, Germany) and
MORG1 heterozygous animals. The
MORG1
+/−
mice were generated as described in [
12] and were backcrossed for more than 12 generation to mice with C57BL/6 J genetic background. 12–16 weeks old
MORG1
+/+
and
MORG1
+/−
male mice, weighting 20–25 g were used in the study and received a standard diet and free access to tap water. 18 wild-type mice (
MORG1
+/+
) and 18 heterozygous
MORG1 (
MORG1
+/−
) animals were used for the study and were randomly separated into 4 groups (
n = 9 per group) see below. All animals were bred and maintained at the “Central Experimental Animal Facilities (ZET)” at Friedrich Schiller University Hospital, Jena, Germany with regular 12/12 h light/ dark cycles and 23 ± 1 °C room temperature. The mice used in the experimental procedure were age and sex matched and were randomly separated into the experimental groups and either received intraperitoneal injection (i.p.) of 0.9% NaCl (
MORG1
+/+
and
MORG1
+/−
control group) for 24 h or an i.p. application of 5 mg/kg BW lipopolysaccharides from
E. coli O111:B4 (LPS) to induce endotoxemia, purchased from Sigma-Aldrich Chemie GmbH, (Taufkirchen, Germany) for 24 h (
MORG1
+/+
and
MORG1
+/−
LPS group). The experimental treatments were performed according to the approved experimental LPS dose and procedure from the Animal Committee of the State of Thuringia, Bad Langensalza, Germany (file 02–023/10). Briefly, the LPS dose of 5 mg/kg BW was freshly prepared and injected once by i.p. application + a vehicle solution of 25 μl/ g BW of 0.9% NaCl, which was applied by subcutaneous injection. The treatments were performed under a short isofluran anaesthesia using a standard procedure described elsewhere [
15‐
17]. The clinical status of the animals was evaluated every 4 h by applying a Clinical Severity Score (CSS) as described previously [
18]. The score uses the following parameters and ranges from 1 to 4 for each of them: spontaneous activity; reaction to exogenous stimuli and posture [
18]. 24 h after treatments the mice were deep anesthetised with isofluran before being sacrificed by cervical dislocation as approved by the Animal Committee of the State of Thuringia and described in the European Community Council Directive for the Care and Use of Laboratory Animals (Directive 2010/63/EU; ANNEX IV (3.Table).The kidneys were collected and paraffin embedded or kept frozen at − 80 °C for further analysis. Blood samples were collected for blood plasma isolation and analysis. The collected urine samples were also stored at − 80 °C until analysed.
Survival analysis
The systemic effect of endotoxemia was investigated by survival studies as described previously [
15]. The survival study was approved from the Animal Committee of the State of Thuringia (file number 02–023/11). The survival analyses were carried out because there are so far no data about the
MORG1
+/−
mice sensitivity to lipopolysaccharide exposure. The mice were randomly separated into experimental groups as described above. Each group contained 10 animals and received a single dose of 0.9% NaCl or LPS. The LPS application was performed as described above. During the 72 h survival analyses the mice were weighed once daily and the clinical well-being of the animals was estimated by the application of CSS [
18], which was evaluated every 4 h. The CSS was approved by the Animal Committee of the State of Thuringia. Subsequently the animal’s survival was monitored and recorded every 6 h over a 72 h period and the mice had a free access to water and standard rodent food. The animals who survived the analyses were deep anesthetised with isofluran before being sacrificed by cervical dislocation, a standard procedure, in accordance to the approved protocol by the Animal Committee of the State of Thuringia (file number 02–023/11).
Evaluation of kidney function and morphology
The kidney functional parameter or renal morphology and tubular injury were performed on kidney harvested 24 h post induction of endotoxemia by LPS administration or application of saline solution in the corresponding experimental groups. In order to calculate the urinary albumin-creatinine-ratio (ACR) during the corresponding treatment, the animals were kept in metabolic cages (Techniplast, Hohenpeißenberg, Germany) and 24 h urine was collected. The urinary levels of creatinine and albumin were determined by enzyme-linked immunosorbent assay (ELISA), respectively from Cayman Chemical Company (Tallinn, Estland) and CellTrend GmbH (Luckenwalde, Germany). The urinary concentration of neutrophil gelatinase-associated lipocalin (NGAL) of the corresponding treatment, we also measured in 24 h collected urine. The urinary and plasma levels of NGAL were detected by mouse NGAL ELISA kit, purchased from BioPorto Diagnostics, Gentofte, Denmark. The assay was performed according to manufacturer instructions. The limit of the sample detection was 75 pg/ml and the assay range was 10–1000 pg/ml NGAL. The plasma levels of creatinine (Cre) and blood urea nitrogen (BUN) were measured in blood plasma 24 h post LPS application or saline injection in all experimental groups by colorimetric chip assay on clinical chemical analyzer (Fuji DRI-CHEM 3500i, Fujifilm, Dusseldorf, Germany). The Cre and BUN concentrations are expressed in mg/dl. Tubular damage was estimated by periodic acid Schiff-reaction (PAS) performed on 2 μm paraffin kidney sections using a PAS staining kit (Baacklab, Schwerin, Germany). The staining was evaluated using an Axioplan microscope and AxioVision Rel. 4.6. software (Zeiss, Jena, Germany). The tubular damage was estimated by scoring system where 0 represent no damage and 5 corresponded to more than 90% injured proximal tubule. We also investigated the proximal tubular cells injury based on the positivity of immunohistological detection of Kidney injury molecule 1 (KIM1) in renal tissue in all experimental groups. The number of damaged (positively stained) tubuli per kidney section was counted and the average number is graphically presented.
Assessment of protein expression in renal tissue by immunohistochemistry
The kidneys were fixed in 10% neutral-buffered formalin and were paraffin embedded. For immunohistological analyses, we used 4 μm paraffin kidney sections with heat-induced antigen retrieval, as described elsewhere [
19]. For immunohistochemistry the kidney sections were blocked with 5% BSA for 1 h at room temperature, followed by incubation with the primary antibodies overnight at 4 °C. The following primary antibodies were used: goat-polyclonal antibody anti-HIF-2α purchased from R&D Systems (Wiesbaden, Germany) and used in 1:200 dilution, an anti-CD3 antibody (1:100 dilution) was obtained from Dianova GmbH (Hamburg, Germany), an anti-PHD3 antibody (1:100 dilution) was from Santa Cruz Biotechnology (Heidelberg, Germany), and anti- KIM1 antibody (1:1000 dilution) was purchased from Cloud One Corp. (Houston, TX, USA). The corresponding secondary antibodies, HRP-conjugated (1:500 dilution), were purchased from KPL Inc. (Gaithersburg, MD, USA). Peroxidase substrates were either 3-amino-9-ethylcarbazole (AEC) or diaminobenzidine (DAB) as appropriate, both purchased from (Vector Laboratories Inc., Burlingame, CA, USA). When appropriate the nuclei were counter-stained with hematoxylin (Vector Laboratories Inc.) for 2 min. The stains were mounted and microscopic analyses were performed by Axioplan microscope and AxioVision Rel. 4.6. software (Zeiss, Jena, Germany). A minimum of 4–6 animals per experimental group were investigated. Routinely, the staining was analysed “blind” from a person unaware of the experimental protocol via a semi-quantitative scoring method (for detection of CD3 positive cells) to estimate the staining intensity as previously reported [
20] or the number of detectable positively stained cells per field were counted by Image software using a cell counting analyses and presented graphically as mean ± SEM.
Assessment of protein expression in renal tissue by western blot analyses
For protein analyses the kidneys were extracted 24 h following LPS i.p. application or saline solution and the protein expression was evaluated. Assessment of protein was performed in kidneys homogenised in complete lysis M supplemented with inhibitors (Roche, Mannheim, Germany) and 1 mM sodium-orthovanadate. Routinely 3 to 4 animals per group were randomly selected for Western blot analyses. The lysates were vortexed, kept on ice for 15 min and centrifuged at 14,000 rpm for 20 min at 4 °C. The supernatant was then used for the further analysis. To evaluate the protein expression 12% or 15% SDS-PAGE were performed, followed by transfer of the gels onto a PVDF membrane. The detection of the protein was performed by ImageQuantTM LAS 4000 biomolecular imager system (GE Healthcare, Upsala, Sweden) via ECL- visualisation. The following antibodies were used for protein detection: anti-phospho Iκ-Bα (1: 200 dilution), anti- Iκ-Bα (1: 200 dilution), anti-phospho-IKKα,β (Ser 180, Ser181) (1: 200), anti-IKKα (1: 200), anti-vinculin (1: 2000 dilution), anti-TNFα (1: 200 dilution), anti-PCNA (proliferating cell nuclear antigen) (1: 500 dilution), anti-NF-κB (1:200). All antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The expression of caspase-3 was detected by anti-caspase-3 antibody (1:1000 dilution) purchased from abcam, Cambridge, UK. The assessment of the nuclear NF-κB levels was performed by preparation of kidney nuclear extracts by the use of ProteoExtract® Subcellular Proteome Extraction kit (Merck, Darmstadt, Germany). The expression of the proteins was subjected to densitometry measurement using ImageJ software.
RNA isolation, reverse transcription and real - time PCR
Renal expression of the genes of interest was analysed 24 h post endotoxin administration or saline injection. Renal tissue was homogenized using a SpeedMill P12 homogenizer (Analytik Jena Bio Solutions, Jena, Germany) and total RNA was isolated using the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The synthesis of cDNA was performed routinely from 1 μg of total RNA using the M-MLV Reverse Transcription system (Invitrogen Life Technologies, Darmstadt, Germany). The gene specific primers for the real – time PCR (RT-PCR) are as follows:
tnf-α - forward: 5’-GGCAGGTCTACTTTGGAGTCATTGC-3′, reverse: 5’ ACATTCGAGGCTCCAGTGAATTCGG 3′,
hprt (hypoxanthine phosphoribosyltransferase) forward: 5’-ATCAGTCAACGGGGGACATA-3′, reverse: 5’-AGAGGTCCTTTTCACCAGCA-3′,
hif-2α - forward: 5’-AAGCTCCTGTCCTCAGTCTG-3′,
hif-2α reverse: 5’-CATCCTCATGAAGAAGTCAC-3′,
pdh3 forward: 5’-GCTATCCAGGAAATGGGACA-3′,
pdh3 reverse: 5’-GGCTGGACTTCATGTGGATT-3′
ngal (lipocalin-2) forward: 5’-CACCACGGACTACAAGTTCGC-3′, 3′
ngal (lipocalin-2) reverse: 5’-TCAGTTGTCAATGCATTGGTCGGTG-3′,
Epo forward: 5-CCACCCTGCTGCTTTTACTC-3′,
Epo reverse: 5’-CTTGAAGAGAACCTGGGAGT-3′,
iNOS (inducible nitric oxide synthase, also known as NOS2) forward: 5’ AGCTGGCTCGCTTTGCCACG 3′,
iNOS reverse: 5’ GCCTCCTTTGAGCCCTTTGT 3′;
Kim1 (Kidney injury molecule 1) forward: 5’ ATGAATCAGATTCAAGTCTTC 3′,
Kim1 reverse: 5’ TCTGGTTTGTGAGTCCATGTG 3′. All primers were purchased from Invitrogen, Darmstadt, Germany. The relative expression of the gene of interest was quantified by RT-PCR using a Q-tower thermocycler (Analytik Jena Bio Solutions, Jena, Germany). The quantitative real-time PCR was done as previously described The expression of the gene of interest was normalized to the expression of
hprt and the relative expression ratio was quantified by ΔΔCT method, where
R = 2
-ΔΔCT [
21]. The mRNA levels in saline treated
MORG1
+/+
mice was set as 1.
Statistical analysis
All values are presented as mean ± standard error of mean (SEM). Statistical analyses were performed with the statistical package SigmaPlot 13 software (SYSTAT Software, San Jose, CA, USA). Results were evaluated by the Kruskal-Wallis One way Analyses of Variances on Ranks test, followed by the Mann-Whitney-Rank Sum-Test to analyse the differences between two groups. All Pairwise Multiple Comparison Procedures were performed with Student-Newman-Keuls Method. Survival was analysed by the Kaplan-Meier test with log-rank statistic. Differences were considered significant when p < 0.05.
Discussion
Suppression of inflammation and improvement of tissue hypoxia are important factors to reduce organ injury [
30]. Accelerated renal hypoxia is a key factor in the renal pathogenesis of AKI. Bacterial infection and local hypoxia oft co-exist in acute and chronic clinical conditions resulting in adverse clinical outcomes [
31]. Hypoxia-inducible factors (HIFs) are the main transcription factors that regulate adaptive responses against hypoxia by activation of the expression of several target genes [
32]. HIFs are rapidly eliminated in normoxic conditions due to the prolyl-hydroxylation activity of the PHDs. Yet, during hypoxia the enzymatic activity of PHDs is suppressed due to low oxygen levels leading to subsequent stabilisation and activation of HIFs [
13]. There is a growing body of research demonstrating the beneficial effects of pharmacological HIF activation [
7,
8,
33‐
35] or administration of the proteins which are generated due to HIF target gene expression such as EPO [
9,
36] and VEGF. Yet, studies have consistently shown the complexity in determining the most optimal timing for application of HIF inhibitors. HIF inhibitors have also been associated with elevated mortality rates and are known to have severe side-effects, despite reducing renal inflammation and improving renal function in sepsis [
8]. We and others have previously shown that the MAPK organizer protein 1 (MORG1) plays a scaffold function by accommodation and coordination of multiple proteins from different cellular networks, related to the MAPK [
10] and/or PHD3/HIF axes [
11]. The exact mechanism by which MORG1 interacts with all these molecules and coordinates their function is currently incompletely understood. Recent research in our laboratory has shown that MORG1 heterozygous deficiency is renoprotective in a model of renal ischemia/reperfusion due to an elevated expression of HIF [
12], or partially attenuated ischemic brain injury [
37] in a model of focal cerebral ischemia. Moreover,
MORG1
+/−
mice have attenuated renal damage in a model of short-time induced hypoxia in mice [
14]. In this regard in the present study we focussed on the question whether a reduced MORG1 expression/ increased HIF stabilisation could potentially aid in preventing inflammation related renal injury. We utilised the well-established murine model of LPS-induced endotoxemia. In agreement with previous reports, we found that LPS administration in wild-type mice induced renal damage, mainly localized in the cortex and manifested by an increased tubular dilatation [
9,
38,
39]. Although the application of 5 mg/kg BW LPS induced a mild, but not severe damage, as higher doses of LPS administration, we detected nephrotoxicity (tubular damage characterised with an induced KIM1 expression in the injured proximal tubili), although it was not so obvious as in other models [
8]. On the other hand, LPS-treated
MORG1
+/−
heterozygous mice were clearly protected from tubular damage, showed a lower KIM1 tubular immunoreactivity, revealed less proteinuria and NGAL renal expression. We have to admit that although, our experimental data showed a significant histological improvement of the renal tissues in endotoxemic
MORG1
+/−
mice relatively to the wild-type
MORG1
+/+
LPS treated animals, there were still increased levels of plasma BUN in both genotypes. This discrepancy could be due to the lower sensitivity of the heterozygous mice to LPS induced nephrotoxicity, which also reflected the lower renal inflammation (a conclusion that seems not likely because
MORG1
+/−
mice are not totally protected from damage). We believe that other extrarenal factors such as volume status, catabolism may have affected the plasma BUN levels. More detailed studies including more parameters for assessment of the renal function at different time-points are necessary to proof whether the clearly observed improvement in histological injury may ultimately be also seen functionally.
The improved histological renal injury could be also related to an elevated HIF-2α protein expression in tubules (not only in nuclei, but also in the cytoplasm), which was associated with a reduced basal PHD3 protein expression in the MORG1
+/−
compared to MORG1
+/+
mice. LPS application further attenuated the PHD3 expression in renal tissue 24 h post endotoxemia induction in both genotypes.
Several studies have reported that PHD3 is a HIF-2α target gene, therefore one could expect an up-regulation of PHD3 in the renal tissues when HIF-2α levels are elevated, it is possible that we did not detect this because it will happen in a later time in the renal tissue. Or it is possible that in those conditions PHD3 is regulated via HIF-α independent mechanism. Furthermore, our data confirmed that endotoxemia increased the plasma levels of the pro-inflammatory cytokine IL-6 in plasma and the TNF-α renal expression in wild type
MORG1
+/+
mice [
29,
40] while in contrast IL-6 and TNF-α induction were significantly reduced in
MORG1
+/−
mice. This finding open the question whether MORG1 heterozygosity could play a role not only in the HIFs pathway, but as well in the NF-κB signalling path, that is activated through LPS/ TLR4 signalling. Upon LPS or TNF-α stimulation NF-kB is activated in an IKKα,β, depended manner, which phosphorylates IκB-α leading to proteasomal degradation of IκB-α and subsequent liberation, nuclear translocation and activation of the NF-κB complex [
41‐
43]. Indeed, we were able to detect an intact NF-κB signalling path in wild-type LPS treated
MORG1
+/+
mice, characterised with an increased phosphorylation levels of IκB-α and IKKα,β, as well as elevated NF-κB nuclear translocation and increased
iNOS expression. Intriguingly, in endotoxemic
MORG1
+/−
mice the NF-κB signalling was impaired as the phosphorylation levels of IκB-α and IKKα,β were significantly inhibited in endotoxemic
MORG1
+/−
mice, compared with the wild-type LPS treated mice, similarly NF-κB activity was inhibited as revealed by the reduced
iNOS mRNA production. What causes these effects in endotoxin treated
MORG1
+/−
mice? An alternative interpretation of the data could be that MORG1 heterozygous mice are less sensitive to the LPS induced nephrotoxicity, which could be associated with lower levels of TLR4 in those animals leading to reduced inflammation and an inhibition of the NF-κB signalling path compared with the wild-type endotoxemic mice. WE are currently further study this possibility, but have not answer yet. Furthermore, a recent finding of the Huh, H. et al. [
44] reporting a scaffolding role of STRAP (Serine-threonine kinase receptor associated protein) for NF-κB [
44]. Interestingly, STRAP is a scaffold protein, which as MORG1 belongs to the WD-40 repeats proteins and its depletion shows striking similarities with our data in regard to NF-κB regulation.
In addition, the observed anti-inflammatory effect of MORG1 down-regulation could be also related to a reduced vasodilataion and hypotension in MORG1 endotoxemic mice. Although we did not performed experiments to test this opportunity it was shown in LPS treated rats that a reduction of NF-κB signalling path was associated with an increased vasoconstriction in mice [
45,
46].
One the other hand, a role of PHD3 in controlling the NF-κB signalling through inhibition of IKKβ phosphorylation, independent of PHD3 hydroxylation activity was suggested [
47]. Our data also confirm this novel molecular link. These mechanisms most likely play a role in fine-tuning the regulation and coordination of HIFs (hypoxia) and NF-κB (inflammation) in the cell, involving MORG1 in a scaffolding function in the PHD3 and NF-kB complexes. As the LPS-treated mice in our experiments demonstrated a reduced expression of MORG1 (only) in the heterozygous
MORG1
+/−
mice, perhaps this strongly suggests that MORG1 may be a new piece of the NF-κB puzzle. Reduced inflammation in
MORG1
+/−
endotoxemic mice is/may also related to an inhibition of the NF-κB signalling complex. These
MORG1
+/−
anti-inflammatory effects observed in endotexemia are uncoupled from the plasma accumulation of NGAL or INFγ, where no differences were found between both genotypes after administration of LPS. On the other hand, we found that the renal expression of
ngal and the urinary levels of NGAL were reduced in
MORG1 heterozygous mice that underwent endotoxemia treatment. Thus, the plasma NGAL protein could be a result of other organ damage and a response to a systemic effect due to inflammation, than an early marker of the renal injury. Moreover, it is difficult to use as an early marker, protein which is almost not produced in the healthy mice and humans, thus there is not a good comparison start point for the analyses.
Thus, modulation of MORG1 scaffolding function or expression, according to our data, may offer a very promising therapeutic target to help prevent acute renal injury.