Anti-proteinuric effect of an endothelin-1 receptor antagonist in puromycin
aminonucleoside induced nephrosis in rat.

Running title: Anti-proteinuric effect of ambrisentan

Jiro Kino, Shoji Tsuji, Tetsuya Kitao, Sohsaku Yamanouchi, Takahisa Kimata,*Kazunari Kaneko

Department of Pediatrics, Kansai Medical University, Osaka, Japan

Address Correspondence to:
Kazunari Kaneko, M.D, PhD.
Department of Pediatrics, Kansai Medical University
2-5-1 Shinmachi Hirakata, 573-1010, Osaka, Japan
Phone: +81-72-804-2517

Fax: +81-72- 804-2504

E-mail: kanekok@hirakata.kmu.ac.jp

Statement of Finantial Support:
This work was supported by Grants-in-Aid for Scientific Research (C) from the Japan
Society for the Promotion of Science (KAKENHI, Grant numbers 25461634)

Disclosure statement: The authors declare no conflict of interest.

Category of study: Basic science1Abstract
Background: The pathogenesis of minimal change nephrotic syndrome (MCNS)
remains unclear although recent studies suggest endothelin 1 (ET-1) and CD80 of
podocytes are involved. We investigated the potential of antagonist to ET-1 receptor
type A (ETRA) as therapeutic agent through suppression of CD80 in rat model of
MCNS.
Methods: Puromycin aminonucleoside (PAN) was injected to Wister rats to induce
proteinuria: some were treated with ETRA antagonist (ambrisentan) and others weretreated with 0.5% methylcellulose. Control rats were treated with phosphate bufferedsaline (PBS) without PAN. Blood and tissue samples were collected. Quantitative PCR
was used to determine the expression of Toll-like receptor-3 (TLR-3), nuclear factor-kB(NF-kB), CD80, talin, ETRA and ET-1 in the kidney.
Results: Amount of proteinuria in the ETRA antagonist treatment group was
significantly lower than other groups. The same-day body weight, serum creatinine
values and blood pressure were not significantly different. ETRA antagonist restores
podocyte foot process effacement as well as the aberrant expression of TLR-3, NF-kB
and CD80 in PAN-injured kidneys.
Conclusions: The ETRA antagonist may be promising drug for MCNS as it showed ananti-proteinuric effect independent of renal vasoconstriction. Its action was considered
to be through suppression of CD80 expression on podocytes.

2Introduction
Nephrotic syndrome (NS) is characterized by heavy proteinuria. Leakage of massive
amounts of serum proteins into the urine leads to a hypoalbuminemia, edema,
hypercoagulable state, higher rate of infectious disease, and dysregulation of fluidbalance. Childhood NS is classified into three types: idiopathic (INS, 90% of cases),secondary (10%), and congenital (1%). INS is further classified into two major
histological variants: minimal change nephrotic syndrome (MCNS, 85%) and focal
segmental glomerulosclerosis (FSGS, 10%) [1, 2].
The pathogenesis of massive proteinuria in MCNS remains unclear though the abnormal
expression of several podocyte-related molecules such as CD80 (also known as B7-1)
or c-mip has recently attracted attention for its relationship with MCNS pathogenesis[3-5].
Endothelin (ET), which has a potent vasoconstrictor effect, was shown to be expressed
not only in endothelium but also in podocytes, and the potential for ET to cause
structural changes of podocytes leading to proteinuria has been reported [6-8].
Therefore, the possibility of using a therapeutic drug that is an ET receptor type A(ETRA) antagonist has gained prominence. In fact, it has been reported to reduce
proteinuria and prevent glomerulosclerosis [9-11] though its mechanisms of action
remains to be clarified.
Taken together, we wonder whether ETRA antagonist (ambrisentan) ameliorates
proteinuria in INS through modification of podocyte-related molecules including CD80.
To test this hypothesis, we: i) determined the ability of ambrisentan to reduce
proteinuria in rats with puromycin aminonucleoside (PAN)-induced nephrosis, ananimal model of MCNS, and ii) analyzed the potential molecular mechanisms of action3

of ambrisentan on kidneys of rats with nephrosis induced by PAN.

Methods
Animal and protocol
The protocol was approved by Kansai Medical University (no.15-065). Female Wisterrats (6 weeks old) were purchased from CLEA Japan, Inc. (Tokyo, Japan). PAN
nephrosis was induced by a single intravenous injection of PAN (Sigma, St. Louis, MO,
USA) dissolved in PBS at a dose of 50 mg/kg body weight. Ambrisentan was purchasedfrom Glaxo Smith Kline (Brentford, UK). Systolic blood pressure was measured using a
rat manometer (Brain Science Idea, Tokyo, Japan). A group of PAN injected rats (n =15) were left untreated and received sham oral gavage vehicle solution (0.5%
methylcellulose), while the treatment group also received ambrisentan (10 mg/kg, n =15) dissolved in 0.5% methylcellulose orally once daily beginning two days before PAN
injection and until day 9. Five control rats also received 0.5% methylcellulose by oral
gavage daily with PBS injection. At day 9 or 12, the rats were anesthetized by isoflurane
inhalation. The kidneys were removed immediately, and the renal cortex was processed.
During the course of the experiments, 24-h urine specimens were obtained from all rats
in metabolic cages each day, and urinary protein was measured using the pyrogallol red
method. Serum creatinine was measured by standard laboratory methods.

Transmission electron microscopy
For ultrastructural analysis, the renal cortex was dissected after systemic perfusion with
PBS. Immediately, the tissues were fixed in 2% glutaraldehyde for overnight, post-fixedin 1% osmium tetroxide, dehydrated in a graded acetone series, and embedded in4Epon-Araldite. Ultra-thin sections, cut to 0.08 µm thick and stained with uranyl acetate
and lead citrate, were examined using a Hitachi H-7100 (Tokyo, Japan).

RNA extraction and quantitative reverse transcription-PCR
Total cellular RNA was extracted from the processed renal cortex using the RNeasy
Mini Kit® (QIAGEN Inc., Hilden, Germany). To quantify mRNA expression, thereal-time SYBR-Green assay was performed. For this, 1 µg of RNA was converted tosingle-strand DNA (TAKARA Inc., Shiga, Japan). Each cDNA was mixed with 10 µM
of forward and reverse primers and 12.5 µL of SYBR Premix Ex Taq II (TAKARA Inc.,
Shiga, Japan). Quantitative PCR was performed in an Applied Thermal Cycler Dice®
Real Time System II (TAKARA Inc., Shiga, Japan). The following primers were used:
TLR-3 forward, 5’- AGGTATTGAACCTGCAACACAATGA-3’;
TLR-3 reverse, 5’- CCCAAGTTCCCAACTTTGTAGATGA-3’;
NF-κB forward, 5’- GATGGGACGACACCTCTACACATA-3’;
NF-κB reverse, 5’- CCCAAGAGTCGTCCAGGTCA-3’;
CD80 forward, 5′-TCGTAGGTGAAACACCTGA-3′;
CD80 reverse, 5′-CCGGAAGCAAAGCAGGTAATC-3′;
Talin forward, 5′- TGTGCCAATGGCTACCTGGA -3′;
Talin reverse, 5′- GTCCTGTCAGCTGCTGCTTTAGTTC-3′;
Ednra forward, 5’- GCATTAACCTGGCAACCATGAAC-3’;
Ednra reverse, 5’- GGACTGGTGACAACAGCAACAGA-3’;
ET-1 forward, 5′-ACCTGGACATCATCTGGGTCAAC-3′;
ET-1 reverse, 5′-TTTGGTGAGCACACTGGCATC-3′;
GAPDH forward, 5′-GGCACAGTCAAGGCTGAGAATG-3′;
5

GAPDH reverse, 5′-ATGGTGGTGAAGACGCCAGTA-3′.
The threshold cycle (Ct) was used for determining the relative expression level of eachgene, by normalizing to the Ct of GAPDH. The ΔΔCT was used to calculate the relativechange.

Statistical analysis
Two-way analysis of variance was used for statistics among 3 experimental groups
followed by a Tukey's test for post-hoc analysis. Significance was defined as P < 0.05.
All error bars in the graphs represent SEM.

Results
Evaluation of body weight, and blood pressure.
At first, we evaluated whether ambrisentan have an impact on body weight andsystemic blood pressure using every three rats at day -1, day 1, day 4, day 7 and day 9.
As a result, blood pressure had no significant difference in all dates (Fig. 1A). There
was no significant difference in body weight in the same way (Fig. 1B).

Ambrisentan reduces PAN-induced proteinuria in rats.
As shown in Fig. 2, 10 female Wistar rats developed massive proteinuria after a single
intravenous injection of PAN (50 mg/kg), which peaked on day 9 (urinary total protein[U-TP] 747.7 mg/kg/day: dashed line). In contrast, 5 control rats receiving intravenous
PBS maintained baseline levels of urinary protein (U-TP at day 9: 4.7 mg/kg/day: dottedline). To determine the ability of ambrisentan to reduce PAN-induced proteinuria, 10
rats/group were also treated daily with oral administration of ambrisentan (10 mg/kg),
6

which significantly reduced proteinuria (U-TP at day 9: 272.6 mg/kg/day: solid line).
Thus, ambrisentan treatment ameliorated PAN-induced proteinuria. As shown in Fig. 3,
no statistically significant differences in serum creatinine were found among the threegroups of rats at day 9 (n = 5 per each groups, P = 0.46). These findings suggest that the
reduction of proteinuria by ambrisentan is independent of the effects on renal
hemodynamics.

Ambrisentan restores foot process effacement in PAN-injured glomeruli.
Evaluation of glomeruli by electron microscopy revealed that PAN-injured glomeruli
showed structural changes of podocytes (Fig. 3A), with their edema and foot processeffacement. In contrast, ambrisentan treatment after PAN injection ameliorated theseverity of foot process effacement compared to PAN injection alone (Fig. 3B).

Ambrisentan restores both the expression of ｍRNA of TLR-3, NF-κB and CD80
and talin in kidney tissues.
Enhanced glomerular mRNA expression of TLR-3 {encodes for Toll-like receptor-3(TLR-3)}, NF-κB {encodes for nuclear factor-kB (NF-kB)} and CD80 (encodes forCD80) was inhibited after treatment with ambrisentan (Fig. 5A-C). In contrast,
glomerular mRNA expression of talin was suppressed by PAN injection, which wasrestored by ambrisentan (Fig. 5D).
Regarding glomerular mRNA expression of ETRA (encodes for ETRA) and ET-1
(encodes for ET-1), they were not significantly affected by PAN injury. However,treatment with ambrisentan decreased glomerular mRNA expression of ETRA and7

increased that of ET-1 (Fig. 5E, F). *P < 0.05. All error bars in the graphs represent
SEM.

Discussion
ET, that was discovered in 1988, is a powerful 21-amino-acid vasoconstrictive peptidethat originates in the endothelium. There are three types of the ET peptide family (ET-1,
2, and 3), which are found in most mammals including humans; among them, ET-1
shows the strongest physiological effects [12, 13]. ET-1 production in endothelial cells
is promoted by bioactive substances including transforming growth factor-β,vasopressin, adrenalin, and other cytokines, whereas its production is inhibited by nitricoxide, prostacyclin, atrial natriuretic peptide, and others [14, 15]. ET-1 combines with
two receptors of type A and type B (ETRB). When these receptors are stimulated by
ET-1, various intracellular signaling systems such as the protein kinase C pathway or
mitogen-activated protein kinase activation pathway are activated [16, 17].
ET-1-mediated activation of these receptors is known to be involved in pulmonary
hypertension and hypertensive status [18, 19]. Therefore, various ET receptor
antagonists including selective ETRA antagonist and ETRB antagonist as promising
agents for pulmonary hypertension are currently under development [20, 21].
ET family peptides are found in endothelial cells as well as in all other organs,including the kidneys [22]: ET production occurs in the glomerular endothelial cells,
podocytes or renal tubular cells [23, 24]. Excessive or decreased production of ET-1 isthought to cause disequilibrium between the two types of receptors, leading toproteinuria and kidney diseases [6, 8]. It has been postulated that numerous
ET-dependent mechanisms contribute to proteinuria and kidney diseases [25, 26]: ET
8

promotes collagen production and stimulates glomerular fibronectin synthesis; ET
becomes activated under conditions associated with renal disease progression, such as
diabetes, insulin resistance, obesity, dyslipidemia, reactive oxygen species formationand inflammation[27, 28].

An involvement of ET-1 in the development of proteinuria in MCNS was suggested byan increase of ET-1 excreted into the urine of the patients [29]. An animal study using
PAN-treated nephrosis in rats further supported an important role of ET-1 in MCNS as
it demonstrated a significant decrease in proteinuria following treatment with an ETRA
antagonist though the contradicting results also existed [30, 31]. However, to the best of
our knowledge, there have been no studies to investigate the molecular mechanisms
within podocytes in the development of ET-1 induced proteinuria.
Regarding the mechanisms of proteinuria in MCNS, it was recently hypothesized thatstructural changes occur because of immunological abnormalities that activate
podocytes (two-hit theory) [32]. This theory is illustrated in Figure 6 and is summarized
as follows: 1) Some types of antigens (viral RNA or serum protein) bind to the TLRs
present on the podocytes (1st hit); 2) NF-κB of podocytes is activated by TLR
stimulation; 3) NF-κB activation causes overexpression of CD80 on podocytes leading
to suppression of β1 integrin by blocking the binding of talin to the β1 integrin; 5)
Because of these structural changes, serum proteins from glomerular capillaries leakinto the urine, and; 6) regulatory T-cells (Treg) which control the expression of CD80
circulating in the blood are functionally or quantitatively reduced (2nd hit). Continued
proteinuria sustains the abnormal condition described above 1) - 6) [33]. In this theory,
central player is a CD80, which is a 53 kD membrane associated protein. It had beenlong believed that CD80 was only expressed on B-lymphocytes and antigen presenting
9

cells where it acts as a costimulatory receptor with role in T-cell activation [34, 35]. The
role of CD80 in podocyte pathobiology was proposed by Reiser et al. in an experimental
model where lipopolycaccharide injection in mice, resulted in increased expression of
CD80 in podocytes and proteinuria, while no proteinuria occurred in CD80 knockout
mice [3]. Furthermore, Yu et al. have recently found that CD80 positive podocytes
have a reduced capacity to attach to the surrounding matrix through β1 integrin. They
found that CD80 positive podocytes change their morphologic characteristics, leading to
podocyte foot processes detachment from the glomerular basement membrane and thus
proteinuria [36]. Recent report that children with steroid-sensitive NS are characterized
by increased expressions of TLR-3, TLR-4 and CD80 mRNA in peripheral bloodmononuclear cells also lend support for this theory [37].
In this study, we evaluated this theory using an in vivo MCNS model animal to clarify
the molecular pathway within podocytes in ET-1 induced proteinuria and therapeuticpotential of the selective ETRA antagonist such as ambrisentan. We used PAN as anephrotoxin because it is commonly used to induce massive proteinuria in rats. In
addition, it has advantage than other nephrotoxins for the study of the pathogenesis ofproteinuria in MCNS because rats develop massive proteinuria within 10 days after its
single injection and is known to induce foot process effacement, a hallmark of MCNS
histology. Furthermore, the previous reports that PAN induced CD80 activation in
cultured podocytes [3] prompted us to choose it. As a result, an administration ofambrisentan to PAN rats showed effects against proteinuria without significant changes
in serum creatinine, urine volume and blood pressure. The suppression of effacement of
podocyte foot process was also confirmed by electron microscopy. Study by RT-PCR
using the renal tissue from PAN-induced nephrosis rats treated with ambrisentan
10

disclosed the following characteristics: 1) suppressed mRNA expression of TLR-3,
NF-kB and CD80, all of which were up-regulated by PAN-induced nephrosis; 2)
increased mRNA expression of talin-1, which plays an important role of attachment of
podocytes to the surrounding matrix through β1 integrin; 3) Although glomerularmRNA expression of ETRA and ET-1 were not significantly affected by PAN injury,
treatment with ambrisentan decreased glomerular mRNA expression of ETRA andincreased that of ET-1 in agreement with the previous report [38]. These findings
suggest the existence of feedback loops on production of ETRA and ET-1 within renal
glomeruli [6].
Based on these results, the following can be predicted regarding the appearance ofproteinuria in PAN-injected rats and the anti-proteinuric effects of ambrisentan (Fig. 6):
Stimulation by PAN administration caused activation of NF-kB intracellular signaling
pathway within podocytes through TLR-3; Activated podocytes express CD80 on
cellular surfaces; Overexpressed CD80 downregulated talin expression resulting in
detachment of podocyte from the glomerular basement membrane [39]; Uponadministration of the ambrisentan, these signaling pathway was suppressed bydownregulating the expression of TLR-3, NF-kB, and CD80 by several possible
cascades and these restored the talin expression and reduced the amount of proteinuria.
A limitation of this experiment was that we could examine the expression of only six
molecules in the podocytes. Some reports have suggested that ET-1 stimulation via
ETRA induces signal transmission by mitogen-activated protein kinase, matrix
metalloprotease-9, Rho-kinase, and other pathways, causing changes to the podocyte
cytoskeleton and structural changes to the slit membranes [10, 27, 40]. These signaling
pathways might be also involved.
11

In conclusion, our results suggest that ambrisentan acts on podocytes to exhibit ananti-proteinuric effect that is through NF-kB intracellular signaling pathway and isindependent of renal vasoconstriction in PAN-induced nephrosis in rats, an animalmodel of MCNS. Therefore, ETRA antagonist such as ambrisentan may have potential
as a therapeutic agent for MCNS.

Statement of Financial Support
This study was supported by Grants-in-Aid for Scientific Research (C) from the Japan
Society for the Promotion of Science (KAKENHI, Grant numbers 25461634).

Disclosure statement
The authors declare no conflict of interest.

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Acknowledgments
We thank Ms A. Yoshioka at Kansai Medical University for their expert technicalassistance, and also Ms. T. Katsuta in the Department of Pediatrics, Kansai Medical
University, for aid in the preparation of this manuscript.

17

Figure Legends
Fig.1. Blood pressure and body weight had no significant difference in all dates
There was no significant difference among groups in terms of systolic arterial pressure(SAP) by tail cuff measurement (Fig. 1A). Regularly measured body weights were notdifferent among three groups (Fig. 1B). Solid line: rats injected with PAN andambrisentan; broken line: rats injected with PAN; dotted line: rats received no treatment
except PBS injection. All error bars in the graphs represent standard error of the mean
(SEM).

Fig. 2. Detailed analyses of proteinuria in PAN-injected Wister rats.
PAN injection without ambrisentan in the Wister rats (n = 10) induced proteinuria which
gradually increased in its amount up to day 9 and gradually decreased thereafter (brokenline). In contrast, treatment group with 10 mg/kg of ambrisentan (n = 10) significantly
reduced the amount of proteinuria during day 7, 9 and 12 (solid line). The control rats
receiving intravenous PBS maintained baseline levels of urinary protein even at day 9(dotted line). During the course of the experiments, 24-h urine specimens were obtained
from all rats in metabolic cages each day, and urinary protein was measured using the
pyrogallol red method. *P < 0.05; **P < 0.01. All error bars in the graphs represent
SEM.

Fig. 3. Effect of ambrisentan on the serum creatinine in PAN-induced nephrosis inrats.
Nine days after PAN injection, the rat were sacrificed and the serum concentration ofcreatinine was measured (n = 5, in each group). No statistically significant differences18

in serum creatinine values were found among the three groups of rats at day 9. The
central horizontal line in the box represents the median value, and the bottom and top
edges of the box are located at the 25th and 75th percentiles, respectively. The central
vertical lines extend from the box to the 90th or 10th percentiles.

Fig. 4. Histological analyses of the kidneys of PAN-injected rat with or withoutambrisentan.
The panels show representative images of the electron microscopy in the glomeruli of
kidneys. Nine days after PAN injection, rats were sacrificed and kidney histology was
analyzed. Electron microscopic analysis revealed podocyte flattening and extensive
effacement of foot processes only in the glomeruli of the PAN-injected rat (Fig. 4A,
x2,000) but not in the glomeruli of the PAN-injected rat pre-treated with ambrisentan(Arrows in Fig. 4B, x1,800)

Fig. 5. Ambrisentan restores both the enhanced expression of TLR-3 and CD80
in kidney tissues of PAN-induced injury.
Total RNA was extracted from dissected renal cortex from control, PAN-injected, andPAN-injected rats treated with ambrisentan (n=5 per each groups). Fig. 5A-C:
Glomerular mRNA expression of TLR-3 (encodes for TLR-3) and CD80 (encodes forCD80) was significantly increased after PAN-induced injury. These enhanced
expressions were significantly inhibited by treatment with ambrisentan. GlomerularmRNA expression of NF-κB (encodes for NF-κB) indicated a similar tendency although
it did not reach statistical significance; Fig. 5D: In contrast, glomerular mRNA
expression of talin was suppressed by PAN injection, which was restored by19ambrisentan though it did not reach statistical significance either; Fig. 5E, F: Regarding
glomerular mRNA expression of ETRA (encodes for ETRA) and ET-1 (encodes forET-1), they were not significantly affected by PAN injection while treatment withambrisentan decreased glomerular mRNA expression of ETRA and increased glomerularmRNA expression of ET-1. *P < 0.05, **P<0.01. All error bars in the graphs represent
SEM.

Fig. 6. Hypothetical molecular mechanism of podocyte on the appearance ofproteinuria in PAN-injected rats
Stimulation by PAN administration caused activation of NF-kB intracellular signaling
pathway within podocytes through TLR-3. Activated podocytes express CD80 on
cellular surfaces; Overexpressed CD80 downregulated talin expression resulting in
detachment of podocyte from the glomerular basement membrane. Upon administrationof the ambrisentan, these signaling pathway was suppressed by downregulating theexpression of TLR-3, NF-kB, and CD80 by several possible cascades and these restored
the talin expression and reduced the amount of proteinuria. Solid arrow lines indicate
the stimulation or activation while dashed lines denote the inhibitory signals.

20