Tumor necrosis factor superfamily ligand mRNA expression profiles differ between humans and mice during homeostasis and between various murine kidney injuries
Devarapu et al. Journal of Biomedical Science
Tumor necrosis factor superfamily ligand mRNA expression profiles differ between humans and mice during homeostasis and between various murine kidney injuries
Satish Kumar Devarapu 1 4
Julia Felicitas Grill 1 4
Junhui Xie 1 3 4
Marc Weidenbusch 1 4
Mohsen Honarpisheh 1 4
Volker Vielhauer 1 4
Hans-Joachim Anders 1 4
Shrikant R. Mulay 0 1 2 4
0 Nephrologisches Zentrum, Medizinische Klinik und Poliklinik IV, Klinikum der Universität München , Schillerstr. 42, D-80336 Munich , Germany
1 Medizinische Klinik und Poliklinik IV, Klinikum der Universität München , Munich , Germany
2 Nephrologisches Zentrum, Medizinische Klinik und Poliklinik IV, Klinikum der Universität München , Schillerstr. 42, D-80336 Munich , Germany
3 Department of Endocrinology, Tongji Hospital, Tongji Medical College , Huazhong , University of Science and Technology , Wuhan , China
4 Medizinische Klinik und Poliklinik IV, Klinikum der Universität München , Munich , Germany
Background: Several tumour necrosis factor (TNF) based therapeutics have already been approved for human use and several others are emerging. Therefore, we determined the mRNA expression levels of the TNF superfamily ligands (TNFSF) - e.g. TNF-α, lymphotoxin (LT)-α, LT-β, Fas-L (CD95-L), TNF-related apoptosis-inducing ligand (TRAIL), TNF-related weak inducer of apoptosis (TWEAK), 4-1BBL, OX40-L (CD252) and amyloid precursor protein (APP) in healthy human and mouse solid organs. Methods: We used quantitative real time-PCR to analyse mRNA expression levels of TNFSF ligands. Murine models of acute ischemic renal injury, chronic oxalate nephropathy, and immune complex glomerulonephritis were used. Renal injury was assessed by PAS staining, and infiltrating immune cells were analysed by immunohistochemistry. Data was analysed using non-parametric ANOVA (non-parametric; Kruskal-Wallis test). Results: We observed significant differences in the mRNA expression levels of TNFSF ligands in human and mouse solid organs. Furthermore, we determined their mRNA expressions during acute and chronic kidney injuries in mice. Our data demonstrate that the mRNA expression levels of TNFSF vary depending on the type of tissue injury - for example, acute ischemic renal injury, chronic crystalline nephropathy, and immune complex glomerulonephritis. In addition, we observed that mRNA expressions of TNFSF ligands are differentially regulated during the course of a transient ischemic renal injury (IRI) and chronic kidney modelling. We observed that TNF-α, LT-β, and 4-1BBL were significantly upregulated during the progression of IRI and crystal-induced chronic kidney disease (CKD), whereas only 4-1BBL and TNF-α were significantly upregulated and LT-β was significantly downregulated during the progression of immune complex glomerulonephritis. The mRNA expression of Fas-L was higher during IRI whereas it decreased in a time dependent manner during the progression of crystal-induced CKD. Conclusion: We conclude that the injury- and species-specific differences of TNFSF ligands must be considered in order to avoid the misinterpretation and wrong conclusions during data extrapolation between species.
Crystal nephropathy; Ischemia reperfusion injury; Chronic kidney injury; Anti-GBM; Tumor necrosis factor; Tumor necrosis factor ligands; TNF superfamily
The potential of tumor necrosis factor (TNF) as a
therapeutic target was exploited and has been well characterized
in various diseases soon after its discovery in 1975 [
Until today, research in this field unveiled the existence of
19 TNF superfamily (TNFSF) proteins that signal through
29 receptors . The TNFSF members are expressed widely
and play major roles in immune responses, inflammation,
cell homeostasis, and tissue repair [
]. In addition, they
also contribute to disease pathogenesis, and therefore, are
also referred as “double-edged swords” . Most of the
TNFSF members are type II transmembrane proteins, while
some can be secreted proteins with biological activity [
Originally, macrophages were reported as a source of TNF
. However, later on, a cytokine lymphotoxin (LT), which
has similar cytotoxic effects like TNF, was reported to be
secreted by B lymphocytes [
]. In 1984, the homology
between TNF and LT was unveiled when they were purified
to homogeneity and their amino acid sequences were
]. This initiated the hunt for more
TNFlike proteins leading to the discovery of 19 TNFSF ligands
until today .
The TNFSF ligands have C-terminal TNF homolog
domain facilitating self-trimerization and receptor binding.
The TNF-receptor superfamily has two domains
consisting of an extracellular domain for ligand binding and a
cytoplasmic tail domain with adaptor proteins for signal
cascade activation [
]. After binding to the respective
receptors, all members of the TNFSF activate pathways
that involve NF-ĸB, JUN N-terminal kinase (JNK), p42/44
mitogen-activated protein kinase (MAPK), p38 MAPK
and generation of reactive oxygen species (ROS) that
orchestrate the pleiotropic effects of TNFSF ligands .
A huge amount of research on TNFSF ligands is
conducted in mice and the observations are often
extrapolated to human conditions. However, several discrepancies
in organ-specific expression levels of pattern recognition
receptors (PRRs), C-type lectin receptors (CLRs), TLR
accessory molecules as well as regulated necrosis-related
molecules, between species has already been reported
]. Therefore, we hypothesized that similar
organand species-specific differences might exist in TNFSF
ligands expressions, and hence, determined their mRNA
expression profiles in human and mice organs.
Furthermore, we checked their expression profiles in murine
acute tissue injury and chronic (progressive and immune
complex-mediated) tissue remodeling.
Human solid organ cDNA preparation
Total RNA from healthy human solid organ were
purchased from Clontech, Mountain View, CA. From each
individual sample, an equal amount of total RNA was used
as a template in cDNA preparation using Superscript II
(Invitrogen). As only a single pool was available for each
organ, no studies on biological replicates allowing
statistics could be performed. However, we used technical
triplicates of each pooled sample. Usage of human solid organ
RNA samples obeys the purchase and import of Clontech
local laws and regulations.
Mouse solid organ cDNA preparation for qRT-PCR experiments
Ten-twelve weeks old, adult C57BL/6 N male mice were
purchased from Charles River, Sulzfeld, Germany. Group of
five mice in a cage were housed in specific pathogen-free
conditions with ad libitum food and water. Mice were
sacrificed under general anesthesia by cervical dislocation. From
freshly collected tissues, RNA was isolated as described
]. Briefly, all organs immediately after harvest were
placed in RNA later solution and RNA was isolated with an
equal amount of tissue mass using Pure Link RNA Mini
Kit (Ambion, Germany) according to the manufacture
instructions. All RNA samples were subjected to DNAse
enzyme treatment and additional washing steps were
performed to remove traces of DNAse. A NanoDrop 1000
Spectrophotometer was used to estimate the RNA
concentrations, only samples with absorbance 260/280 between
1.95 and 2.05 were considered as pure RNA. The integrity
of the total RNA was determined by electrophoresis on 2%
(w/v) agarose gels as described. Further, cDNA was
transcribed using 1 μg of RNA as described before [
1 μg RNA was mixed with cDNA master mix (including
DTT, dNTPS, Rnasin, Acrylamide, Hexanucleotide and
Superscript II), and incubated for 1 h 30 min at 42 °C.
Supercript was inactivated by incubating at 90 °C for 5 min
and cDNA was stored at −20 °C until further use.
Renal Ischemic injury model: Groups of seven to eight
week old C57BL/6 mice (n = 5–10) under anesthesia
underwent unilateral renal pedicle clamping for 35 min
followed by reperfusion for 24 h, 5 days and 10 days as a
model of ischemia-reperfusion as described [
Throughout the procedure, body temperatures were
maintained at 37 °C using a heating plate (Kleintier-OP-Tisch
M12511, Medax GmbH, Germany) and an egg breeding
device (Octagon 20 Advance, Brinsea products Ltd., UK).
Injured and contralateral kidneys were harvested for RNA
isolation and histology analysis. Contralateral kidneys
served as internal control kidneys. Chronic oxalate
nephropathy model was developed by feeding mice with an
oxalate-rich diet that was prepared by adding 50 μmol/g
sodium oxalate to a calcium-free standard diet (Ssniff, Soest,
Germany) as previously described [
]. Mice were
sacrificed at day 7, 14 and 21 after exposure to oxalate-rich diet.
Autologous anti-GBM glomerulonephritis model was
developed by immunizing mice subcutaneously in both flanks
with 20 μg rabbit IgG (Jackson ImmunoResearch
Laboratories, West Grove, PA) in Freund’s complete adjuvant
(Sigma-Aldrich, Deisenhofen, Germany). Three days later,
mice received 150 μl nephrotoxic rabbit serum
intravenously . Animals were sacrificed on day 14, 21 and
42 days after anti-GBM serum challenge. Kidney samples
were harvested for RNA isolation and histology analysis.
All experimental procedures on animals were approved by
the Regierung von Oberbayern, München, Germany, and
were performed in accordance with their guidelines and
Quantitative real time-PCR
An equal amount of RNA (1 μg) was used to prepare
]. Complementary DNA was performed with
Superscript II reverse transcriptase (ThermoFisher,
Germany), 5× first-strand buffer (Thermo Fisher Germany),
DTT (Invitrogen, Germany), dNTPs (GE Healthcare,
Germany), linear acrylamide (Ambion, Germany),
hexanucleotide (Roche, Germany) and RNasin (Promega,
Germany). Reverse transcriptase reaction was performed
for 90 min at 42 °C then the reaction was heated at 85 °C
for 5 min using a Mastercycler pro (Eppendorf, Germany).
RN-related molecule mRNA expression in human and
mouse solid organs, as well as diseases model, were
quantified by RT-PCR using GAPDH as housekeeper gene for
human samples and 18 s for mouse samples as described
]. Each PCR reaction (20 μl) involved
10 × Taq Polymerase Buffer, Taq Polymerase, dNTPs, BSA,
PCR Optimizer, SYBR green dye, MgCl2, gene specific
primers and 0.2 μl of synthesized cDNA. SYBR Green Dye
fast-two step detection protocol from Light Cycler 480
(Roche, Mannheim, Germany) running program was used
for amplification. Each amplification step included initiation
step 95 °C, annealing step 60 °C and amplification step 72 °
C and was repeated 40 times. Gene-specific primers
(300 nM, Metabion, Martinsried, Germany) were used as
listed in Table 1. DdH2O was used as negative control for
target and housekeeper genes. A specific primer for each
target was designed using the primer designing tool (NCBI)
and In silico specificity screen (BLAST) was performed.
The lengths of amplicons were between 90 and 120 bp.
The kinetics of the PCR amplification (primer efficiency)
was calculated for each set of primers. The
efficiencycorrected quantification was performed automatically by
the Light Cycler 480 based on extern standard curves
describing the PCR efficiencies of the target and the reference
gene [ratio = Etarget ΔCPref (control − sample)].
ΔCPtarget (control − sample)/Eref
The high confidence algorithm was used to reduce the risk
of the false positive crossing point. All the samples which
rise above the background fluorescence (crossing point Cp
or quantification cycle Cq) between 5 and 40 cycles during
the amplification reaction were considered as detectable.
The melting curves were analyzed for every sample to
detect unspecific products and primer dimers. To visualize
the similarity and differences in gene expression profiles
among the samples, hierarchical cluster analysis were
performed using algorithms incorporated in the open-source
software MultiExperiment Viewer (MeV) version 4.9 [
Differentially expressed mRNAs were screened by Volcano
Plot between log(2)(fold change) gene expression
[unstandardized signal] against -log(10)(p-value) from the t-test
[noise-adjusted/standardized signal] [
After sacrifice, portions of harvested kidney tissues were
fixed in 4% neutral-buffered formalin, followed by
dehydration in graded alcohols and embedded in paraffin.
For further analysis such as periodic acid-Schiff (PAS)
staining or immunostaining 4 μm sections were
deparaffinized, rehydrated, transferred into citrate buffer, and
either autoclaved or microwave treated for antigen
retrieval and processed as described [
immunostaining following primary antibodies were used:
Collagen1α1 (Dako, Hamburg, Germany), anti-mouse
CD3, anti-mouse F4/80 (both Serotec, Oxford, UK).
Data were represented as mean ± standard error of the
mean (SEM). Comparisons between groups were
performed using non-parametric ANOVA (non-parametric;
Kruskal-Wallis test). A p-value less than 0.05 was chosen
as statistical significance.
TNF superfamily ligands mRNA expressions in adult human tissues
We quantified the mRNA expression levels of TNFSF
ligands e.g. TNF-α, LT-α, LT-β, Fas-L (CD95-L), TNF-related
apoptosis-inducing ligand (TRAIL), TNF-related weak
inducer of apoptosis (TWEAK), 4-1BBL, OX40-L (CD252)
and amyloid precursor protein (APP) using qRT-PCR in
healthy human organs. Human spleen constitutively
expressed all of these ligands with higher expression of
TRAIL compared to other ligands (Fig. 1a). However, the
expression of LT-α, Fas-L, and AAP remained low (Fig. 1a).
In general, all of these ligands were expressed at higher
levels in lungs and lower levels in brain, heart, and liver.
OX40L and LT-α were expressed at higher levels in Testis.
Thymus showed very high mRNA expression levels of
LTα, TRAIL, and 4-1BBL whereas those of LT-β and OX40-L
were only modestly increased. All these ligand expressions
in kidney were similar to spleen except for Fas-L, which
was modestly high in the kidney and 4-1BBL, which was
lower. The mRNA expression levels of most of the other
molecules were lower in the solid organs compared to
spleen. Thus, mRNA expressions of TNFSF ligands are
variable in healthy human solid organs compared to spleen.
TNF superfamily ligands mRNA expressions in adult murine tissues
Next, we quantified the mRNA expression of the
abovementioned TNFSF ligands in the same organs from
healthy, 10-12 weeks old, male C57BL/6 N mice. We
found that mouse spleen constitutively expressed all of
these ligands. However, the mRNA expression levels of
LT-β and AAP were higher whereas those of Fas-L,
TRAIL, and OX40L were lower in the mouse spleen
(Fig. 1b). In sharp contrast to human organs, all mouse
organs, except thymus, expressed higher levels of AAP
(Fig. 1b). In addition, mouse spleen expressed very low
mRNA expression levels of TNFSF ligands compared to
human spleen. However, like human liver, mouse liver
also expressed very low levels of TNFSF ligands. The
mRNA expression of Fas-L was higher in thymus, lung,
heart, colon, and kidney; lower in the liver, and remained
unchanged in brain and testis compared to spleen. Lung
also expressed higher levels of TWEAK. TRAIL was
expressed at modestly high levels in lung and testis. The
latter also showed modestly higher levels of OX40-L.
The mRNA expression of other TNFSF ligands was
markedly lower in the mouse solid organs compared to
the spleen (Fig. 1b). Furthermore, we also compared the
organ-specific mRNA expression of TNFSF ligands
between humans and mice and observed obvious
differences between them (Fig. 2). For example, mouse brain
expressed very high mRNA levels of AAP (about 12 fold)
compared to human brain. AAP was also expressed at
higher levels in mouse kidney and lungs compared to
the human kidney and lungs, respectively. TRAIL,
41BBL, and LT-α were expressed at higher levels in
human thymus whereas OX40-L and LT-α were expressed
at higher levels in human testis compared to mouse
testis. Mouse heart expressed higher levels of Fas-L,
TWEAK, and AAP than the human heart. Thus, humans
and mice express different relative mRNA levels of
TNF superfamily ligands mRNA expressions in murine acute kidney injury
We further studied the changes in the mRNA expression
of TNFSF ligands during transient injury using a murine
model of ischemic renal injury (IRI). The IRI model is
characterized by acute tubular necrosis and
inflammation in an injury phase (day 1), which is followed by
tubular regeneration in a healing phase (day 5 and 10)
(Fig. 3a) [
]. We observed an increase in the
infiltration of F4/80+ macrophages and CD3+ T cells in the
injured kidneys, which is associated with increased renal
mRNA expression levels of TNFSF ligands at day 5 and
10 (Fig. 3a). The mRNA expression levels of TRAIL,
TWEAK, as well as OX-40 L, remained unchanged at
day 1, 5 and 10 (Fig. 3b-c). The mRNA expression levels
of APP were significantly downregulated whereas that of
Fas-L and LT-α was significantly upregulated at all time
points (Fig. 3b-c). Moreover, the mRNA expression
levels of TNF-α, LT-β, and 4-1BBL were increased only
in the healing phase, at day 5 and 10 (Fig. 3b-c).
Therefore, we conclude that mRNA expressions of TNFSF
ligands are differentially regulated during the course of a
transient injury – for example, ischemic renal injury.
TNF superfamily ligands mRNA expressions in murine chronic kidney disease
We observed higher levels of mRNA expressions of
TNFSF ligands in healing phase after a transient renal
injury. Therefore, we next studied the changes in their
expression during chronic tissue remodeling. We induced
chronic kidney disease (CKD) in mice by feeding them an
oxalate-rich diet for 21 days [
CKD was associated with a progressive increase of F4/80+
macrophages and CD3+ T cells infiltrating the renal
interstitial compartment as well as interstitial fibrosis from day
7 to 21 (Fig. 4a). Similar to IRI, the mRNA expression of
APP was significantly downregulated at all time points. At
day 7, mRNA expressions of TWEAK, LT-α, and OX-40 L
remained low. The later was only induced significantly at
day 14 whereas LT-α was significantly high at day 14 and
21. There were no significant differences observed in the
mRNA expression of TRAIL in oxalate-induced CKD.
However, mRNA expression levels of 4-1BBL, TNF-α, and
LT-β were significantly higher at day 7, 14, and 21. In
contrast, the mRNA expression of Fas-L significantly
decreased in a time-dependent manner during the
progression of oxalate-induced CKD (Fig. 4b-c). Thus, the
mRNA expressions of TNFSF ligands are also
differentially regulated during chronic kidney remodeling in
TNF superfamily ligands mRNA expressions in chronic immune complex glomerulonephritis
TNFSF ligands play an important role in the
pathogenesis of immune complex organ injuries [
we studied the changes in their mRNA expression levels
during an immune complex glomerulonephritis. We
used an animal model of autologous anti-GBM nephritis
and checked the expression of TNFSF ligands at day 14,
21, and 42 [
]. Immune complex glomerulonephritis
was associated with a progressive increase in glomeruli
with global lesions, as well as renal interstitial infiltration
of F4/80+ macrophages and CD3+ T cells (Fig. 5a).
Unlike, IRI and oxalate-induced CKD, the mRNA
expression of LT-β remained low at all time points during the
progression of immune complex glomerulonephritis
(Fig. 5b-c). We observed a transient increase in the
mRNA expression levels of TRAIL at day 21 (Fig. 5b-c).
On the contrary, expression of 4-1BBL and TNF-α were
significantly higher whereas APP expression was
significantly lower at day 14, 21 and 42 (Fig. 5b-c). The mRNA
expression of all other ligands was significantly higher
during immune complex glomerulonephritis
progression; however, TWEAK and LT-α induction was not
statistically significant in the later phase at day 42
(Fig. 5b-c). Together, we observed sharp differences in
kidney remodeling induced by persistent crystal injury
and immune complex mediated injury.
The TNFSF ligands exert pleiotropic effects [
Although they evolved mainly for the regulation of
immune cells in our body, over the years they have been
reported to be involved in several pathogenic disorders
as well. Here, our data demonstrate that alike PRRs [
], TLR accessory molecules [
], and regulated
necrosis-related molecules [
], the relative mRNA
expression profiles of TNFSF ligands differ between
physiological conditions in humans and mice, as well as
between various pathophysiological conditions in mice.
All TNFSF ligands activate NF-ĸB signalling, albeit to
different extent, therefore, the pathologies related to them
mostly involve NF-ĸB signalling-mediated inflammation,
for example – acute IRI. Several studies have reported the
contribution of TNFSF ligands e.g. Fas-L, TWEAK and
TNF-α, which are secreted by infiltrating immune cells, to
intrarenal inflammation during IRI [
we observed a robust increase in the mRNA expression
levels of TNFSF ligands after IRI that was also associated
with increased intrarenal immune cell infiltration. Recently,
TWEAK mRNA expressions were also reported to be
increased in various mouse models of CKD such as lupus
nephritis, immune complex glomerulonephritis, as well as
rat and human diabetic nephropathy . On the contrary,
although TRAIL mRNA expression was increased at day 1
after IRI [
], we did not observe changes in the TRAIL
mRNA expression at later time points in our study.
Moreover, we observed that the expression levels of TNFSF
ligands were highest during the healing phase after IRI,
suggesting their putative involvement during tubule
regeneration after an injury [
]. It may therefore interesting to
explore whether TNFSF ligands contribute to regeneration/
healing process upon acute kidney injury IRI.
Different functional states of immune cells regulate the
process of regeneration upon kidney injury. Persistent
inflammation is deleterious since it promote chronic tissue
remodelling (fibrosis) [
]. We studied progressive kidney
fibrosis using a murine model of oxalate-induced CKD.
Although, we observed a robust increase in the mRNA
expression levels of most TNFSF ligands in association
with infiltrating immune cells, the expression patterns in
progressive kidney injury were different from transient
kidney injury observed in the IRI model. This suggests
that different pathologies are driven by different TNFSF
ligands. Interestingly, the injection of recombinant murine
TNF-α in mice increased the expressions of TNF
receptors (TNFRs) in the kidney [
]. Accordingly, the
increased mRNA expression of TNF-α in kidneys of mice
exposed to oxalate-rich diet can be linked to the already
reported increased expression of TNFRs on tubular
epithelium during the progression of oxalate-induced CKD
]. TNFR induction supports the adhesion of calcium
oxalate crystals to tubular epithelium and hence
nephrocalcinosis eventually leading to CKD [
TNF-α has also been reported to contribute to the
pathogenesis of other forms of CKD – for example, diabetic
nephropathy, Alport glomerulosclerosis, and chronic
ischemic renal injury [
]. Therefore, it will be
interesting to study the functional contribution of each of the
other induced TNFSF ligands, as well as their receptors,
during the progression of CKD.
In addition to the persistent trigger, chronic
inflammation can also arise from tissue deposition of immune
]. Surprisingly, unlike transient and
persistent injuries, immune complex injury showed sharp
differences in the mRNA expression patterns of TNFSF
ligands, which did not at all correlate with the
increased renal infiltration of immune cells. This suggests
that the different pathomechanisms alter the mRNA
expression of TNFSF ligands. Nevertheless, we observed a
robust increase in the mRNA expression of TWEAK
and TNF-α in the early phase, which was already shown
to contribute to the disease pathology in mice, as well
as human, kidney disease [
]. Therefore, it
would be interesting to study the specific contribution
of all the induced genes in immune complex mediated
Furthermore, as TNFSF receptors mediate the effects
of TNFSF ligands, the contribution of TNFSF ligands to
disease pathologies also depend on TNFSF receptor
3, 23, 42
]. Hence, studying the organ- and
species differences in expression of TNFSF receptors
along with TNFSF ligands during health and disease
conditions deserves careful consideration.
We identified several TNFSF ligands that are induced
during ischemic CKD, crystalline CKD, as well as
immune complex glomerulonephritis. In addition, we have
identified significant differences in the mRNA expression
profiles of the TNFSF ligands in humans and murine
solid organs during physiological conditions. Therefore,
our data give robust reasons to consider these
speciesspecific differences of TNFSF ligands in order to avoid
the misinterpretation and wrong conclusions during data
extrapolation between species. Since TNF-based
therapeutics are already approved for human use, and several
more are likely to be found in the future based on
TNFSF ligands, the findings of our study recommend
validating results of rodent studies, not only kidney
diseases but also other pathogenic disorders, in human
We gratefully acknowledge the expert technical support of Dan Draganovici,
The work was supported by the Deutsche Forschungsgemeinschaft (DFG)
Availability of data and materials
All data and materials are available.
SKD and SRM designed the study concept and experiments. SKD, JFG, JX,
MW, MH, VV, SRM performed the experiments. SKD and SRM wrote the
manuscript. SKD, HJA and SRM read and finalized the manuscript. All authors
read and approved the final manuscript.
SKD is the first author and SRM is the corresponding author.
Ethics approval and consent to participate
All experimental procedures on animals were approved by the Regierung
von Oberbayern, München, Germany, and were performed in accordance
with their guidelines and regulations. Usage of human solid organ RNA
samples obeys the purchase and import of Clontech local laws and
Consent for publication
All the authors have consented for publication.
The authors declare that they have no competing interests.
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