http://www.kidney-international.

org original article

& 2011 International Society of Nephrology

Bradykinin receptor 1 activation exacerbates

experimental focal and segmental glomerulosclerosis
Rafael L. Pereira1, Bruna N. Buscariollo1, Matheus Corrêa-Costa2, Patricia Semedo1, Cassiano D. Oliveira1,
Vanessa O. Reis1, Edgar Maquigussa3, Ronaldo C. Araújo4, Tárcio T. Braga2, Maria F. Soares5,
Ivan C. Moura6, Denise M.A.C. Malheiros7, Alvaro Pacheco-Silva Filho1,8, Alexandre C. Keller1,9
and Niels O.S. Câmara1,2
1
Departamento de Medicina, Laboratório de Imunologia Clı´nica e Experimental, Disciplina de Nefrologia, Universidade Federal de
São Paulo, São Paulo, Brazil; 2Laboratório de Imunobiologia de Transplante, Departamento de Imunologia, Universidade de São Paulo,
São Paulo, Brazil; 3Departamento de Medicina, Laboratório de Biologia Molecular, Disciplina de Nefrologia, Universidade Federal de São
Paulo, São Paulo, Brazil; 4Departamento de Biofı´sica, Universidade Federal de São Paulo, São Paulo, Brazil; 5Departamento de
Patologia, Universidade Federal de São Paulo, São Paulo, Brazil; 6INSERM U699, Immunopathologie rénale, récepteurs et inflammation,
Faculté de Médecine Xavier Bichat, Paris, France; 7Departamento de Patologia, Universidade de São Paulo, São Paulo, Brazil; 8Hospital
Israelita Albert Einstein, São Paulo, Brazil and 9Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de
São Paulo, São Paulo, Brazil

Focal and segmental glomerulosclerosis (FSGS) is one of Focal and segmental glomerulosclerosis (FSGS) is one of the
the most important causes of end-stage renal failure. The most important causes of end-stage renal failure.1,2 FSGS is
bradykinin B1 receptor has been associated with tissue characterized by areas of glomerular sclerosis associated with
inflammation and renal fibrosis. To test for a role of the tubular atrophy and interstitial fibrosis with commitment
bradykinin B1 receptor in podocyte injury, we of the podocytes that lead to proteinuria.3 Podocytes are
pharmacologically modulated its activity at different time polarized cells that possess a cytoskeleton that modulates
points in an adriamycin-induced mouse model of FSGS. their foot processes that adhere to the glomerular basement
Estimated albuminuria and urinary protein to creatinine membrane.4,5 The foot processes are linked laterally by
ratios correlated with podocytopathy. Adriamycin injection negative charge structures named slit diaphragms,5 that are
led to loss of body weight, proteinuria, and upregulation an important filtration barrier composed of many proteins
of B1 receptor mRNA. Early treatment with a B1 antagonist like nephrin (NPHS-1), NEPH-1, podocin (NPHS-2),
reduced albuminuria and glomerulosclerosis, and inhibited CD2AP, ZO-1, and a-actinin-4.4,6,7
the adriamycin-induced downregulation of podocin, nephrin, Recently, some experimental data demonstrated a protec-
and a-actinin-4 expression. Moreover, delayed treatment tive role of bradykinin blockade in acute and chronic kidney
with antagonist also induced podocyte protection. injury models.8 Bradykinin signals through two G-protein-
Conversely, a B1 agonist aggravated renal dysfunction and coupled receptors, the B1 (B1RBK) and B2 (B2RBK)
even further suppressed the levels of podocyte-related receptors. B2RBK is constitutively expressed in most tissues
molecules. Thus, we propose that kinin has a crucial role in and mediates the majority of the physiological actions
the pathogenesis of FSGS operating through bradykinin of kinins. On the other hand, B1RBK is overexpressed in
B1 receptor signaling. inflammatory conditions.9,10 The absence or blockade of
Kidney International advance online publication, 16 March 2011; B1RBK is generally protective in renal disease models.8,11–13
doi:10.1038/ki.2011.14 Nevertheless, the role of B1RBK in FSGS is still unclear. Here,
KEYWORDS: bradykinin B1 receptor; focal and segmental glomerulosclero- we hypothesize that B1RBK also plays an important role in
sis; kinin; podocyte podocytopathy, which is a hallmark of FSGS. Blocking
B1RBK signaling could be a new strategy to halt the
progression of FSGS and prevent end-stage renal disease.
Correspondence: Niels O. S. Câmara, Department of Immunology, Institute RESULTS
of Biomedical Science IV, Universidade de São Paulo, Rua Prof Lineu Prestes,
Bradykinin receptors are upregulated in an
1730, 05508-900 São Paulo, São Paulo, Brazil or Disciplina de Nefrologia,
adriamycin-induced FSGS model
Universidade Federal de São Paulo, Rua Pedro de Toledo 720, Vila
Clementino 04023-900, São Paulo, São Paulo, Brazil. E-mail: niels@icb.usp.br FSGS was induced in BALB/c mice14,15 by a single
or niels@nefro.epm.br intravenous injection of adriamycin. The adriamycin-in-
Received 10 May 2010; revised 9 December 2010; accepted 21 duced nephropathy model is a model of FSGS that
December 2010 mimics many features of human disease. The animals

Kidney International 1
original article RL Pereira et al.: Bradykinin B1 receptor in FSGS

with adriamycin nephropathy lost weight (Supplementary Early treatment of animals with DALBK protects animals from
Figure S1A online). Serum creatinine was significantly higher adriamycin-induced nephropathy
in adriamycin-treated animals at day 28 after injection First, to address whether B1RBK is involved in the initiation
(Supplementary Figure S1B online). We analyzed the urinary of podocytopathy, animals were treated with des-Arg9-
protein/creatinine ratio (Supplementary Figure S1C online), [Leu8]-bradykinin (DALBK) on days 1–3 after adriamycin
estimated the amount of albuminuria (Supplementary Figure administration and killed on day 4.
S1D online), and quantified the glomerulosclerosis and DALBK-treated animals were fully protected from albu-
tubular degeneration at different time points (Supplementary minuria (Figure 2a) and presented less prominent weight
Figure S1E online). The adriamycin-treated animals showed a loss (Supplementary Figure S2A online) and proteinuria
progressive and significant augmentation in the amount of (Supplementary Figure S2B online).
proteinuria and albuminuria (Supplementary Figure S1C and The DALBK treatment augmented the expression of
D online) up to day 14. These animals also presented a NPHS-2 mRNA (Figure 2b); however, there was no difference
progressive increase in glomerulosclerosis and tubular in the renal protein levels of this marker (Figure 2c). Another
damage (Supplementary Figure S1E online). podocyte marker for slit diaphragm selectivity, NPHS-1,
To study the participation of kinin receptors during appeared to be restored by DALBK treatment (Supplemen-
adriamycin nephropathy, we first evaluated the expression of tary Figure S2C online). On the other hand, the mRNA levels
B1RBK and B2RBK mRNAs using real-time PCR (Figure 1). of a-actinin-4 showed no difference (Supplementary Figure
Treatment with adriamycin induced the expression of B1RBK S2D online); furthermore, mRNA levels of transforming
at 24 h (Figure 1a), and this expression progressively growth factor-b (TGF-b), plasminogen activator inhibitor-1
increased thereafter. The relative expression of B2RBK mRNA (PAI-1), and vimentin were upregulated in FSGS. The
increased progressively from day 7 until day 14, when its DALBK treatment downregulated mRNA levels of PAI-1,
expression stabilized (Figure 1b). The relative expression of vimentin (Supplementary Figure S2E and F online), and
B2RBK mRNA increased progressively from day 7 until day mRNA and protein levels of tumor necrosis factor-a (TNF-a;
14 (Figure 1b). As previous studies13,16 have shown a possible Figure 2d and e).
compensatory relationship between B1RBK and B2RBK, We found no difference in TGF-b levels among the groups
characterized by higher B1RBK expression in B2-knockout (Figure 2f). To investigate the effect of early DALBK
mice, we evaluated the ratio of B1RBK to B2RBK that was treatment on podocyte cell structure, we analyzed the
significantly higher in the adriamycin-treated animals from glomerular structure by electron microscopy. We observed
day 1 to day 10 after injection compared with other days that DALBK treatment protected against adriamycin-induced
(Figure 1c). podocyte foot process effacement (Figure 2g). The degrees of
glomerulosclerosis and tubular damage showed no differ-
ences, although the DALBK-treated groups presented no
mesangial hypercellularity observed in the adriamycin group
4 4
Control Control (Supplementary Figure S2G online).
*
B2R/HPRT 2–ΔΔCt
B1R/HPRT 2–ΔΔCt

ADM ADM
3 3 * *
*
* *
2
* *
2 * * Early blockade of B1RBK induced a sustained protection
1 1 against adriamycin-induced nephropathy
Here, we evaluated whether B1RBK blockade could lead to
0 0
0 1 4 7 10 14 28 0 1 4 7 10 14 28 long-term renoprotection. Animals were treated with DALBK
Days after adriamycin injection Days after adriamycin injection
on days 1–3 and were followed for up to 28 days. Indeed,
2.25 * * Control animals treated with DALBK showed lower albuminuria
ADM
1.80 (Figure 3a), body weight loss, and proteinuria (Supplemen-
* *
B1/B2 Ratio

1.35
tary Figure S3A and B online).
0.90
The mRNA levels of NPHS-2, which was downregulated
with adriamycin, were restored to basal levels with the
0.45
DALBK treatment (Figure 3b). NPHS-2 protein (Figure 3c),
0.00
0 1 3 7 10 14 28 NPHS-1, and ACTN-4 (Supplementary Figure S3C and D
Days after adriamycin injection
online) presented similar results.
Figure 1 | Bradykinin receptor mRNA expression kinetics The DALBK treatment diminished the adriamycin-in-
during adriamycin nephropathy. B1RBK mRNA expression starts duced upregulation of mRNA and renal tissue levels of
rapidly after adriamycin (ADM) injection (24 h) and increases
progressively until day 14 (a). B2R mRNA expression starts only TGF-b (Figure 3d and e) and PAI-1 and vimentin mRNA
7 days after ADM injection and continues until day 28 (b). In (c), (Supplementary Figure S3E and F online).
the B1R/B2R ratio demonstrates that B1R is more highly expressed The mRNA expression of TNF-a was reduced with
than B2R in the first 10 days of the disease. B1R, bradykinin 1
DALBK treatment (Figure 3f), and renal a-smooth muscle
receptor; B2R, bradykinin 2 receptor; HPRT, hypoxanthine
phosphoribosyltransferase 1. *Po0.05; n ¼ 5 animals per group. actin protein expression was less evident in DALBK-treated
Bars ¼ mean and s.e.m. animals (Figure 4). Furthermore, there was a redistribution

2 Kidney International
RL Pereira et al.: Bradykinin B1 receptor in FSGS original article

Urine albumin/creatinine ratio

0.90 * 2.5 1.0
#

NPHS-2/HPRT 2–ΔΔCt

LB +
DA DM
2.0

K
l
0.72

tro
0.8

NPHS-2/GAPDH
M
A
on

AD
C
0.54 1.5 0.6
NPHS-2
GAPDH
0.36 1.0 0.4

0.18 0.5 0.2

#
0.00 0.0 0.0

LB +

LB +

LB +
M
l
l

M
l

DA DM

DA DM

DA DM
tro
tro
tro

K
AD
AD
AD

on
on
on

A
C
C
C

Control
2.25 * 85 2.5
* *
–ΔΔCt

–ΔΔCt
1.80 68 2.0
TNF-α pg/ml
TNF-α/HPRT 2

TGF-β/HPRT 2
1.35 51 1.5
ADM
0.90 34 1.0

0.45 # 17 *# 0.5

0.00 0 0.0
ADM+DALBK

LB +
LB +
LB +

M
M
l
M

tro

DA DM
DA M
tro

DA DM

tro

K
K
K

AD
AD
AD

AD

on
on

on

A
A

C
C

Figure 2 | Early treatment with the bradykinin 1 receptor (B1R) antagonist des-Arg9-[Leu8]-bradykinin (DALBK) protects mice from
adriamycin (ADM)-induced nephropathy. The administration of DALBK on days 1–3 after ADM injection protects mice from the disease
symptoms. On day 4 after injection, the ADM þ DALBK-treated animals lost less albumin in the urine (a). DALBK treatment augments the
mRNA (b) and protein levels (c) of podocin. Serum protein (d) and mRNA levels of tumor necrosis factor-a (TNF-a; e) are downregulated in
the ADM þ DALBK group. The treatment did not affect the levels of transforming growth factor-b (TGF-b; f). After DALBK treatment on
day 7, animals were protected from podocyte foot process effacement (arrows), as shown by electron microscopy (g). The pictures
were taken with an original magnification of  10,000. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HPRT, hypoxanthine
phosphoribosyltransferase 1; NPHS-2, nephrosis 2, idiopathic, steroid-resistant (podocin). *Po0.05 vs control, #Po0.05 vs ADM. Bars ¼ mean
and s.e.m.

1.0
Urine albumin/creatinine ratio

5
* * ADM+
#
4 * 0.8
NPHS-2/GAPDH

Control ADM DALBK

3
* NPHS-2 0.6
Control
GAPDH
ADM
2 ADM+DALBK 0.4

1 0.2
# # # #
0 0.0 *
0 7 14 21 28
LB +
l

DA DM
tro

K
AD

Days after adriamycin injection

on

A
C

1.20
# 1.75 * 550 * 1.70
*
NPHS-2/HPRT 2–ΔΔCt

TGF-β/HPRT 2–ΔΔCt

0.96 1.40 440 1.36

TGF-α pg/ml
TGF-β pg/ml

0.72 1.05 # 330 1.02

0.48 * 0.70 220 # 0.68

#
0.24 0.35 110 0.34

0.00 0.00 0 0.00

LB +
LB +

LB +

LB +
l
l

M
l

M
DA DM
DA DM

DA DM

tro

DA DM
tro
tro

tro
K
K

K
AD

AD

K
AD

AD
on
on
on

on
A
A

A
C
C
C

Figure 3 | Bradykinin 1 receptor (B1R) antagonist des-Arg9-[Leu8]-bradykinin (DALBK) treatment promotes sustained protection of
mice from the progression of adriamycin (ADM)-induced nephropathy after 28 days. The administration of DALBK on days 1–3 after
ADM injection protects mice from albuminuria (a). The mRNA (b) and protein (c) levels of podocin, which were downregulated with ADM
injection, are at basal levels with DALBK treatment. The mRNA (d) and renal tissue (e) levels of transforming growth factor-b (TGF-b) were at
basal levels in the DALBK-treated group. The mRNA level of tumor necrosis factor-a (TNF-a; f) is downregulated in the ADM þ DALBK-
treated group. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HPRT, hypoxanthine phosphoribosyltransferase 1; NPHS-2, nephrosis
2, idiopathic, steroid-resistant (podocin). *Po0.05 vs control, #Po0.05 vs ADM; n ¼ 5 animals per group. Bars ¼ mean and s.e.m.

Kidney International 3
original article RL Pereira et al.: Bradykinin B1 receptor in FSGS

Control ADM ADM+DALBK

α-SMA

Podocin

Podoplanin

Figure 4 | Bradykinin 1 receptor (B1R) antagonist des-Arg9-[Leu8]-bradykinin (DALBK) treatment protects against proliferating cell
nuclear antigen (PCNA) deposition and also maintains the structure of podocyte-related proteins. The administration of DALBK
on days 1–3 after adriamycin (ADM) injection protects renal tissue from PCNA deposition (arrows) and also prevented the redistribution of
podocin (arrows) and podoplanin (arrows); n ¼ 5 animals per group. The pictures were taken with an original magnification of  40.

of podocin and podoplanin (Figure 4) in the glomeruli. The in renal mRNA and serum protein levels of TNF-a
DALBK-treated animals showed no glomerulosclerosis and a (Figure 5f and g).
minimal tubular degeneration. Furthermore, the animals The animals treated with DALBK showed no glomerulo-
showed no mesangial hypercellularity or tubular atrophy sclerosis. This result differs from those animals treated with
(Supplementary Figure S3G–L online), and low levels of only adriamycin (Supplementary Figure S5G–K online),
serum urea (Supplementary Figure S4 online). which on day 7 had a sclerosis index of 5%. We observed
lower degrees of tubular degeneration and mesangial
Delayed DALBK treatment reverses podocyte dysfunction hypercellularity and no tubular atrophy in the DALBK-
in adriamycin nephropathy treated group (Supplementary Figure S5G–K online).
Next, we evaluated whether B1RBK blockade is able to reverse
FSGS. To analyze this question, animals were treated on days Delayed blockade of B1RBK induces long-term protection
4–6 after adriamycin injection and were killed at day 7. against adriamycin-induced nephropathy
Mice treated with DALBK lost significantly less We next evaluated whether delayed treatment with DALBK
albuminuria (Figure 5a), and presented a lower body could halt the progression of adriamycin nephropathy
weight loss (Supplementary Figure S5A online), proteinuria by treating animals on days 4–6 after adriamycin injection
(Supplementary Figure S5B online), and serum urea levels and followed them until day 28.
(Supplementary Figure S4 online) compared with only In contrast to the first delayed treatment, the health of
adriamycin-treated animals. Postponed treatment with the animals improved with time, and on day 28 they showed
DALBK also restored the protein and mRNA levels of no signs of albuminuria (Figure 6a) and showed decreases in
podocin (Figure 5a–c) and mRNA level of nephrin (Supple- proteinuria (Supplementary Figure S6A online) and serum
mentary Figure S5C online), but no difference was found in urea (Supplementary Figure S4 online).
a-actinin-4 (Supplementary Figure S5D online). Furthermore, the levels of podocyte and fibrotic-related
The renal tissue active protein and mRNA concentrations proteins were returned to basal levels with delayed DALBK
of TGF-b (Figure 5d and e), which were upregulated after treatment (Supplementary Figure S6A–E online). Interest-
adriamycin treatment, were significantly reduced in the ingly, renal mRNA and protein levels of NPHS-2 were
DALBK-treated group. DALBK treatment also diminished restored to basal levels in the DALBK-treated group (Figure
the expression of the PAI-1 and vimentin (Supplementary 6b and c). The same was observed for renal mRNA levels of
Figure S5E and F online), and prevented the increase TGF-b and TNF-a (Figure 6d and e).

4 Kidney International
RL Pereira et al.: Bradykinin B1 receptor in FSGS original article

4.5 17
*# 1.0

Urime albumin/creatinine ratio

*

LB +
3.6

DA DM
0.8 #

NPHS-2/HPRT 2–ΔΔCt
14

K
l
tro

NPHS-2/GAPDH
A
on

AD
C
2.7 10 NPHS-2 0.6
GAPDH
1.8 7 0.4
*#
0.9 3 0.2

0.0 0 * 0.0 *

LB +
LB +

LB +
l

M
DA DM
DA DM
tro

tro

DA DM
tro
K
K
AD

AD

AD

K
on

on

on
A
A

A
C

C
2.0 * 600
*
160
*
1.70
*
480 128 1.36
TNF-β/HPRT 2–ΔΔCt

TNF-α/HPRT 2–ΔΔCt
1.5
*# *#

TNF-α pg/ml
TGF-β pg/ml

360 96 1.02
1.0
240 64 # 0.68

0.5 #
120 32 0.34

0.0 0 0 0.00
LB +

LB +
LB +

LB +
l

M
l

M
l
DA M

DA DM
DA M

tro
tro

tro

tro

DA DM
K

K
K

K
AD
AD

AD

AD
AD

AD

on
on

on

on

A
C
C

Figure 5 | Delayed treatment with the bradykinin 1 receptor (B1R) antagonist des-Arg9-[Leu8]-bradykinin (DALBK) reverses the C
progression of adriamycin (ADM)-induced nephropathy. The administration of DALBK on days 4–6 after ADM injection attenuates the
progression of ADM-induced nephropathy on day 7. The ADM þ DALBK-treated animals showed less albuminuria (a) than animals treated with
only ADM. DALBK administration augments the mRNA (b) and prevents the downregulation of protein levels of podocin (c), which were
downregulated by ADM injection. The DALBK þ ADM-treated group shows lower renal transforming growth factor-b (TGF-b) mRNA (d) and
protein levels by enzyme-linked immunosorbent assay (ELISA; e). The mRNA and serum levels of tumor necrosis factor-a (TNF-a) are also
downregulated after DALBK treatment (f, g). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HPRT, hypoxanthine phosphoribosyltransferase
1; NPHS-2, nephrosis 2, idiopathic, steroid-resistant (podocin). *Po0.05 vs control, #Po0.05 vs ADM. Bars ¼ mean and s.e.m.

Renal histology analysis showed no signs of adriamycin tubular atrophy that was not observed in the other groups
nephropathy (Supplementary Figure S6F–L online). (Supplementary Figure S8G and H online).

Effect of B1RBK antagonism on heme oxygenase-1 Effect of kinin B1 modulation on macrophage infiltration
expression induced by adriamycin-induced nephropathy
We observed that neither early nor delayed treatment of the Macrophage infiltration was analyzed by flow cytometry and
animals with DALBK induced heme oxygenase-1 mRNA by quantification of chemokine mRNAs associated with this
expression; however, this molecule was upregulated in process 17–19 (Figure 8). By day 4 after adriamycin injection,
adriamycin-treated mice (Supplementary Figure S7 online). B1RBK agonist, DABK, markedly augmented the level
of macrophages in the kidney (Figure 8d and g). However,
Treatment of animals with DABK aggravates at day 7, the group treated with the antagonist, DALBK,
adriamycin-induced nephropathy had a diminished level of macrophages within the kidneys
B1RBK can also be positively regulated using an agonist, induced by adriamycin injection (Figure 8e, f, and h).
des-Arg9-bradykinin (DABK). The early activation of Furthermore, we observed that adriamycin increased the
B1RBK accelerated the progression of FSGS (Figure 7 and renal levels of MCP-1 (monocyte chemotactic protein-1),
Supplementary Figure S8 online). DABK-treated animals at day 4 (Supplementary Figure S9A online) and levels of
exhibited augmented albuminuria compared with animals MCP-1, MIP-1 (macrophage inflammatory protein 1) and
subjected to only adriamycin treatment (Figure 7a). However, RANTES at day 7 (Supplementary Figure S9B, D, F, and H
total proteinuria and body weight were not significantly different online) after its injection, whereas DALBK diminished them.
from this latter group (Supplementary Figure S8A and B online). Interestingly, DABK enhanced the expression of MCP-1 and
Despite the clinical signs, DABK administration signifi- MIP-1 (Supplementary Figure S 9A and E online).
cantly diminished mRNA and protein expression of NPHS-2
(Figure 7b and c), and no differences were found in the B1RBK modulates metalloproteinases MMP-9 and TIMP-1
other molecules (Figure 7d–f and Supplementary Figure Matrix metalloproteinase-9 (MMP-9) is a molecule asso-
S8C–E online). Furthermore, the B1RBK agonist significantly ciated with extracellular matrix degradation and decrease
augmented vimentin mRNA expression (Supplementary of fibrosis.20 Here, we observed that DALBK augmented
Figure S8F online). Moreover, DABK treatment induced MMP-9 level (Supplementary Figure S10A and C online);

Kidney International 5
original article RL Pereira et al.: Bradykinin B1 receptor in FSGS

Control ADM ADM+DALBK

NPHS-2
GAPDH
Control
ADM 1.5 1.5

Urine albumin/creatinine ratio

ADM+DALBK
5

NPHS-2/HPRT 2–ΔΔCt
* * 1.2

NPHS-2/GAPDH
4 * 1.0
3
* 0.9

2 # 0.6 * 0.5
1 * # # # 0.3
0
0 4 7 14 21 28
0.0 0.0
*
Days after adriamycin injection

LB +
LB +

M
l

DA DM
tro
tro

DA DM

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K

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AD

on
on

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C
C
2.0
2.25
* *
TNF-β/HPRT 2 –ΔΔCt

TNF-α/HPRT 2–ΔΔCt
1.80 1.6

1.35 1.2

0.90 0.8
#
#
0.45 0.4

0.00 0.0
LB +

LB +
l

M
DA DM

DA M
tro

tro
K

K
AD

AD

AD
on

on
A
C

Figure 6 | The bradykinin 1 receptor (B1R) antagonist des-Arg9-[Leu8]-bradykinin (DALBK) reverses the progression of adriamycin C
(ADM)-induced nephropathy after 28 days of study. The administration of DALBK on days 4–6 after ADM injection diminished the levels
of albuminuria (a) and also prevented increases in the mRNA (b) and protein levels (c) of podocin. The mRNA level of the fibrotic marker
transforming growth factor-b (TGF-b; d) were at basal levels in the ADM þ DALBK group, and the level of tumor necrosis factor-a (TNF-a; e)
showed the same pattern; n ¼ 5 animals per group. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HPRT, hypoxanthine
phosphoribosyltransferase 1; NPHS-2, nephrosis 2, idiopathic, steroid-resistant (podocin). *Po0.05 vs control, #Po0.05 vs ADM. Bars ¼ mean
and s.e.m.

1.80
*# 0.95
1.0
Urine albumin/creatinine ratio

NPHS-2/HPRT 2–ΔΔCt

1.44 0.76
BK +

0.8
DA DM

NPHS-2/GAPDH
l
tro

M
A
on

AD
C

1.08 0.57 0.6

* * GAPDH
0.72 0.38 NPHS-2
0.4

0.36 0.19
*#
0.2

0.00 0.00 0.0

BK +

BK +
BK +
l

M
l

l
DA DM

DA DM
DA DM
tro

tro

tro
AD

AD

AD
on

on

on
A

A
A
C

4 85
2.25
*
–ΔΔCt

–ΔΔCt

3 1.80 68

*
TGF-α pg/ml
TGF-β/HPRT 2

TGF-α/HPRT 2

1.35 51
2
0.90 34
1
0.45 17

0 0.00 0
BK +

BK +
BK +

M
l

M
DA DM

DA DM
DA DM

ro
tro

tro
AD
AD

AD
t
on
on

on
A

A
A

C
C

Figure 7 | Bradykinin 1 receptor (B1R) agonist des-Arg9-bradykinin (DABK) treatment aggravates the symptoms of adriamycin
(ADM)-induced nephropathy. DABK administration on days 1–3 after ADM injection induced more albuminuria (a) than ADM treatment
alone. The administration of DABK diminishes the mRNA levels of podocin (b). A significant downregulation of podocin, as analyzed by
western blot, is seen in the ADM þ DABK group (c). No differences were found when we analyzed transforming growth factor-b (TGFb)
mRNA (d). There was no difference between the groups in mRNA and serum protein levels of tumor necrosis factor-a (TNF-a; e, f). GAPDH,
glyceraldehyde 3-phosphate dehydrogenase; HPRT, hypoxanthine phosphoribosyltransferase 1; NPHS-2, nephrosis 2, idiopathic, steroid-
resistant (podocin). *Po0.05 vs control, #Po0.05 vs ADM. Bars ¼ mean and s.e.m.

6 Kidney International
RL Pereira et al.: Bradykinin B1 receptor in FSGS original article

number of proliferating cell nuclear antigen-positive cells

40
(Figure 9d).
* Effect of B1RBK modulators on interleukin-1b expression

Macrophage infiltration (%)

32
1.34 We observed that delayed treatment with B1RBK antagonist,
24 DALBK, decreased interleukin-1b expression, compared
with adriamycin-induced nephropathy group at day 7.
16 In contrast, on day 4, there was no difference in inter-
leukin-1b level among all groups (Supplementary Figure S11
6.23 8
online).
0
Control ADM ADM+DABK DISCUSSION
Because B1RBK is involved in many inflammatory and
fibrotic disorders,8,12,13,17,22–26 we hypothesized that its
SSCA

7.78
14.0 * blockade could have a protective role in adriamycin-induced
d
FSGS. Several studies have shown that the blockade and
deletion of this receptor are associated with less inflamma-
Macrophage infiltration (%)

11.2

tion and fibrosis,23,24,27,28 but no data were reported in FSGS.

8.4
The upregulation of B1RBK has previously been associated
11.4
e with glomerular diseases.29
5.6
To assess the role of B1RBK in FSGS, we observed that this
#
2.8
receptor is quickly upregulated during the disease, from days 1
to 28, as observed in other studies,13 which in turn helped us to
13.8 0.0 determine a strategy to modulate this receptor. Initially, we
f Control ADM ADM+DALBK
evaluate whether the early blockade of B1RBK influence the
development of FSGS. Because FSGS is a podocyte-related
disease,6,17,29,30 we analyzed the mRNA and protein expression
of podocyte markers, such as nephrin, podocin, and a-actinin-
0.55 4.31,32 Indeed, animals treated with the antagonist were
F4/80 protected, with decreased levels of proteinuria and podocyte
Figure 8 | The bradykinin 1 receptor (B1R) antagonist damage. Conversely, when we used an agonist for B1RBK, we
Des-Arg9-[Leu8]-bradykinin (DALBK) diminished the level of observed the opposite result, corroborating the idea that the
macrophages in renal tissue, and the B1 receptor agonist
des-Arg9-bradykinin (DABK) augmented the macrophage bradykinin acting through B1RBK is deleterious to FSGS
level. (a) Negative control; (b) saline-treated animals; (c) kidney progression, as observed in other fibrotic pathologies.8,10–13,23,33
sample collected 4 days after adriamycin injection; (d) kidney In a delayed treatment with DALBK, we observed a
samples from mice treated with adriamycin and on days 1–3 with progressive augmentation in renal fibrosis, which was
DABK; (e) kidney samples collected after 7 days of adriamycin
treatment; (f) kidney samples collected from animals injected with associated with an increase in mRNA and protein expression
adriamycin and with DALBK on days 4–6 after adriamycin of fibrosis-related proteins, such as TGF-b, PAI-1, and
injection; (g, h) index of macrophage infiltration. *Po0.05 vs vimentin, and with a downregulation of podocyte-specific
control, #Po0.05 vs ADM. Bars ¼ mean and s.e.m. SSCA, side or
orthogonal scatter: measures cell complexity or granularity.
proteins. We investigated the long-term outcomes of early
and delayed B1RBK blockade. We quantified renal mRNA
and/or protein levels of TGF-b, PAI-1, and vimentin.13,34–36
on the other hand, adriamycin induced TIMP-1 (tissue TGF-b has been associated with many fibrotic diseases,13,34
inhibitor of matrix metalloprotease-1) expression, a MMP-9 and together with upregulation of TNF-a, is implicated in
inhibitor, whereas DALBK diminished it (Supplementary the progression of proteinuria in FSGS, especially because of
Figure S10B and D online). DABK treatment did not alter the its role in the downregulation of nephrin.37 Interestingly, the
levels of these proteins (Supplementary Figure S10A and treatment with DALBK prevented the upregulation of these
B online). molecules. TGF-b is also associated with the downregulation
of MMP-9 and, consequently, augmentation of extracellular
Effect of B1RBK modulators on cell proliferation matrix proteins, contributing to fibrosis.18 Here, TIMP-1, an
Cell proliferation is closely related to tubular and glomerular inhibitor of MMP-9, was upregulated in adriamycin-treated
injury.21 We observed strong nuclear staining for proliferating animals, whereas DALBK diminished its expression.
cell nuclear antigen in the tubular and glomerular area of the Shin et al.38 showed that adriamycin accumulates in the
kidneys in adriamycin-induced nephropathy (Figure 9). Ani- glomerulus, and its delayed clearance causes a continuous
mals treated with DALBK presented less prominent staining noxious insult associated with reactive oxygen species
(Figure 9c and f). In contrast, DABK treatment augmented the release that consequently leads to podocyte damage.39–41

Kidney International 7
original article RL Pereira et al.: Bradykinin B1 receptor in FSGS

PCNA deposition

Figure 9 | Early treatment with bradykinin 1 receptor (B1R) antagonist des-Arg9-[Leu8]-bradykinin (DALBK) diminished the
proliferating cell nuclear antigen (PCNA) deposition induced by adriamycin (ADM), whereas the B1 receptor agonist des-Arg9-
bradykinin (DABK) did not. Both were analyzed on days 4 and 7 after its injection. (a) Control; (b) ADM at day 4; (c) ADM þ DALBK (DALBK
treatment at days þ 1, þ 2, and þ 3) at day 4; (d) ADM þ DABK at day 4 (DABK treatment at days þ 1, þ 2, and þ 3); (e) ADM at day 7; and
(f) ADM þ DALBK (DALBK treatment at days þ 4, þ 5, and þ 6) at day 7. All photographs were taken with an original magnification of  40.

Corroborating this finding, we observed upregulation of adriamycin (doxorubicin hydrochloride; Pfizer, New York, NY),14,17
heme oxygenase-1 in adriamycin-treated mice; however, this whereas an equal volume of saline was given to control mice.
marker was not upregulated by DALBK in our work. Another Modulation of B1RBK. B1RBK was modulated using three
factor associated with DALBK-induced protection was the different protocols. First, we treated animals with intraperitoneal
decreased glomerular and tubular cell proliferation, as injection of 10 mg/kg of the B1RBK antagonist DALBK (Sigma,
St Louis, MO)22 on days 1–3 after adriamycin injection. The animals
assayed by proliferating cell nuclear antigen staining.21
were killed on days 4 or 7. In the second protocol (delayed treatment),
A possible role of macrophages in FSGS was verified in animals received DALBK on days 4–6 after adriamycin injection13 and
other studies.42 Here, we observed that DALBK-treated were killed on days 7 and 28. In the third protocol, we treated animals
animals presented macrophage infiltration levels similar to on days 1–3 after adriamycin injection, and then the animals were
control, which could be associated with the downregulation killed on day 28. Finally, some animals were also treated with a B1RBK
of macrophage chemokines as seen by Klein et al.43 agonist (DABK; Sigma). Animals received an intravenous injection of
The redistribution of slit diaphragm proteins is a common 1.5 mg/kg of DABK on days 1–3 after adriamycin injection. Mice were
feature of glomerular diseases.32,44–46 In our model, we killed on day 4 for further analysis.13
observed that adriamycin-induced nephropathy caused a
similar redistribution of podocin and podoplanin, as Renal function analyses
previously observed.45 All these alterations observed under On days 1, 4, 7, 10, 14, 21, and 28 after adriamycin injection, urinary
adriamycin treatment were prevented by DALBK. and blood samples were collected. Serum creatinine and urea, the
Our study is the first to evaluate the role of B1RBK in urinary protein/creatinine ratio, and albuminuria were used to
FSGS. We observed that B1RBK has an important role in estimate renal and podocyte functions. At the time of killing, blood
and urine were collected. All samples were analyzed using Labtest
FSGS progression and that its blockage is important for the
Diagnosis (Belo Horizonte, State of Minas Gerais, Brazil) for creatinine
prevention and effective reversion of adriamycin-induced measurements and Sensiprot for protein measurements. To estimate
FSGS. Therefore, our findings should provide new and the urinary albumin concentration, 10 ml of urine (1 mg/ml), corrected
valuable perspective on FSGS management. for urinary creatinine level, was separated by 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and stained with Coomas-
MATERIALS AND METHODS sie. The density of the bands was analyzed using the software
Methods GeneSnap and Gene Tools (Syngene, Cambridge, UK).
Animals. Isogenic male BALB/c mice, age 8–12 weeks (23–28 g),
were obtained from the Animal Care Facility at the Federal Expression of slit diaphragm-related genes
University of São Paulo (UNIFESP). All animals were housed in Kidney samples were frozen in liquid nitrogen. Total RNA
individual standard cages and had free access to water and food. All was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA).
procedures were previously reviewed and approved by the internal First-strand cDNAs were synthesized using Moloney murine
ethical committee of the Institution. leukemia virus reverse transcriptase (Promega, Madison, WI).
Experimental model of FSGS induced by adriamycin. FSGS Real-time PCR was performed using the TaqMan primers and
was induced in mice by a single tail vein injection of 10 mg/kg probes for TIMP-1 (Mm 00441818), MMP-9 (Mm01240560),

8 Kidney International
RL Pereira et al.: Bradykinin B1 receptor in FSGS original article

Table 1 | Base pair sequence of primers (probes) used in real-time PCR assays
Gene Sense Antisense
HPRT 50 -CTCATGGACTGATTATGGACAGGAC-30 50 -GCAGGTCAGCAAAGAACTTATAGCC-30
B1R 50 -CCATAGCAGAAATCTACCTGGCTAAC-30 50 -GCCAGTTGAAACGGTTCC-30
B2R 50 -ATGTTCAACGTCACCACACAAGTC-30 50 -TGGATGGCATTGAGCCAAC-30
TGF-b 50 -AACTATTGCTTCAGCTTCACAGAGA 30 50 -AGTTGGATGGTAGCCCTTG-30
ACTN-4 50 -CGCTGAGAGCAATCACATCA-30 50 -AGTGCAATGGTCCCTCTTTGG-30
NPHS-2 50 -ATGCTCCCTTGTGCTCTGTTG-30 50 -TTTGCCTTTGCCATTTGACA-30
IL-1-b 50 -CCTAAAGTATGGGCTGCACTGTTT-30 50 -TAGAGATTGAGCTGTCTGCTCATTC-30
MCP-1 50 -AAGAGAATCACCAGCAGCAGGT-30 50 -TTCTGGACCCATTCCTTATTGG-30
Abbreviations: ACTN-4, actinin, a-4; B1R, bradykinin 1 receptor; B2R, bradykinin 2 receptor; HPRT, hypoxanthine phosphoribosyltransferase 1; IL-1-b, interleukin 1b; MCP-1,
monocyte chemotactic protein-1; NPHS-2, nephrosis 2, idiopathic, steroid-resistant (podocin); TGF-b, transforming growth factor-b.

NPHS-1 (Mm004497831_g1), vimentin (Mm 00801666-g1), TNF Bio-Plex suspension array system, and the data were analyzed using
(Mm0136932), and PAI-1 (Mm 009312) (Applied Biosystems, Bio-Plex Manager software version 4.0. Standard curves ranged from
Foster City, CA). For the analyses of B1RBK, B2RBK, TGF-b, 32,000 to 1.95 pg/ml.
NPHS-2, ACTN-4, interleukin-1b, and MCP-1 expression, real-time
PCR was performed using a SYBRGreen assay (Applied Biosystems; Renal histology analysis
Table 1). Kidney samples were fixed in 10% neutral formalin. Paraffin
The cycling conditions of both TaqMan and SYBRGreen primers sections (3 mm in thickness) were cut and stained with hematoxylin
were as follows: 10 min at 95 1C, followed by 45 cycles of 30 s at and eosin. The sections were analyzed in a trinocular optical
95 1C, 30 s at 60 1C, and 30 s at 72 1C. The relative quantification of microscope (Olympus Corporation, Tokyo, Japan). Photographs
mRNA levels was performed as described in detail in User Bulletin 2 were taken through the digital camera coupled with the microscope,
(PerkinElmer, Applied Biosystems, Branchburg, NJ, 1997). Briefly, and the images were captured with the software Pinnacle Studio
the target gene amount was normalized to the endogenous reference Plus (Pinnacle Systems, Bucks, UK). All sections were evaluated at
(hypoxanthine phosphoribosyltransferase 1 (HPRT); SYBRGreen)  40 magnification.
and then related to a calibrator (sample with the lowest expression, Glomerulosclerosis was evaluated as described by Mu et al.47
namely the controls) using the formula 2DDCt. Hence, all data The extent of glomerulosclerosis and glomerular collapse was
were expressed as an N-fold difference related to the expression of evaluated in each kidney by consecutive examination under light
matched controls. Analyses were performed with the Sequence microscopy. Tubulointerstitial injury was defined as tubular dilation
Detection Software 1.9 (Applied Biosystems). and/or atrophy or as interstitial fibrosis.48
Tubular injuries were examined in at least 20 areas using the
Immunohistochemistry following scoring system: 0 ¼ changes in o10% of the cortex,
The localization of a-smooth muscle actin (diluted 1:600; DAKO, 1 þ ¼ changes in up to 25% of the cortex; 2 þ ¼ changes in up to
Glostrup, Denmark) and proliferating cell nuclear antigen (diluted 50% of the cortex; and 3 þ ¼ changes in 450% of the cortex sections.
1:300; DAKO) was performed according to the manufacturer’s
instructions for the staining procedures. Flow cytometry analysis
The localization of nephrin (NPHS-2 antibody diluted to Animals were killed, and the kidneys were collected for flow
10 mg/ml; Abcam, Cambridge, UK) and podoplanin diluted in a cytometry analysis, following the standard manufacturer’s proceed-
1:100 concentration (Biolegends, San Diego, CA) was detected in ing and the compensation process was made according to the
frozen sections of the kidney. The sections were fixed and performed ‘fluorescence minus one’ method.49
according with the manufactures’ instructions. We analyzed the renal macrophage population by multicolor
flow cytometry. The monoclonal antibody used was F4/80 PerCP
Determination of TGFb-1 protein by enzyme-linked (BD Biosciences, Franklin Lakes, NJ). Samples were acquired on a
immunosorbent assay FACSCanto, using FACSDIVA software (BD Biosciences) and then
Total renal TGFb-1 protein was measured using a TGFb-1 Emax were analyzed with FLOWJO software (Tree Star, San Carlo, CA).
immunoassay system (Promega), according to the manufacturer’s Fluorescence voltages were determined using matched unstained
instructions. The results are presented as TGFb-1 pg/mg of total cells. Compensation was carried out using cells (BD Biosciences)
protein measured using the Bradford assay (Bio-Rad, Hercules, CA). single-stained with CD3 PerCP, CD4 FITC, CD8 APC-CY7, CD4
PE-CY7, CD3 PE, or CD3 APC. Samples were acquired up to at least
Western blotting for podocin 200,000 events in a live mononuclear gate.
Briefly, 100 mg of total protein from renal tissue was collected and
then diluted in sample buffer (Bio-Rad), containing 20 mg/ml of Electron microscopy analysis
2-b-mercaptoethanol (Sigma). NPHS-2 and GAPDH antibodies Samples for electron microscopy were processed according to
were purchased from Abcam, and western blot was done according standard methods as described by Ertmer et al.50 The glomerular
to the manufacturer’s instructions. and podocyte foot processes structures were analyzed.

TNF-a serum measurement Statistical analysis

A Bio-Plex mouse cytokine assay kit (Bio-Rad) was used to test All data are presented as the mean±s.e.m. Different results among
samples for the presence of TNF-a. The assay was read on the groups were compared using analysis of variance. Significance was

Kidney International 9
original article RL Pereira et al.: Bradykinin B1 receptor in FSGS

established as Po0.05. All statistical analyses were performed using 6. Donoviel DB, Freed DD, Vogel H et al. Proteinuria and perinatal lethality
in mice lacking NEPH1, a novel protein with homology to NEPHRIN.
GraphPad PRISM (GraphPad, La Jolla, CA).
Mol Cell Biol 2001; 21: 4829–4836.
7. Drenckhahn D, Franke RP. Ultrastructural organization of contractile and
DISCLOSURE cytoskeletal proteins in glomerular podocytes of chicken, rat, and man.
All the authors declared no competing interests. Lab Invest 1988; 59: 673–682.
8. Wang PH, Cenedeze MA, Campanholle G et al. Deletion of bradykinin B1
receptor reduces renal fibrosis. Int Immunopharmacol 2009; 9:
ACKNOWLEDGMENTS
653–657.
This work was supported by FAPESP (Fundac¸ão de Apoio à Pesquisa 9. Marin-Castano ME, Schanstra JP, Praddaude F et al. Differential induction
do Estado de São Paulo) grants 04/08226-9 and 07/07139-3, the of functional B1-bradykinin receptors along the rat nephron in endotoxin
FAPESP/Inserm (08/55125-4), the International Associated Laboratory induced inflammation. Kidney Int 1998; 54: 1888–1898.
(CNPq/Inserm), and INCT Complex Fluids. ACK is a recipient of grants 10. Schanstra JP, Marin-Castano ME, Praddaude F et al. Bradykinin B(1)
from FAPESP (2007/07120) and CNPq (501848/2009-6). We thank receptor-mediated changes in renal hemodynamics during endotoxin-
Bernardo Albe for preparing the histology slides and Meire Hiyane, induced inflammation. J Am Soc Nephrol 2000; 11: 1208–1215.
Claudia Cunha, Marina Burgos, and Angela Castoldi for technical 11. Wang PH, Cenedeze MA, Pesquero JB et al. Influence of bradykinin B1
and B2 receptors in the immune response triggered by renal
assistance.
ischemia-reperfusion injury. Int Immunopharmacol 2006; 6: 1960–1965.
12. Kakoki M, McGarrah RW, Kim HS et al. Bradykinin B1 and B2 receptors
both have protective roles in renal ischemia/reperfusion injury.
SUPPLEMENTARY MATERIAL Proc Natl Acad Sci USA 2007; 104: 7576–7581.
Figure S1. Characteristics of adriamycin-induced nephropathy. 13. Klein J, Gonzalez J, Duchene J et al. Delayed blockade of the kinin B1
Figure S2. Early treatment with the B1R antagonist des-Arg9-[Leu8]- receptor reduces renal inflammation and fibrosis in obstructive
bradykinin (DALBK) protects mice from adriamycin (ADM)-induced nephropathy. FASEB J 2009; 23: 134–142.
nephropathy. 14. Zheng Z, Pavlidis P, Chua S et al. An ancestral haplotype defines
Figure S3. B1R antagonist des-Arg9-[Leu8]-bradykinin (DALBK) susceptibility to doxorubicin nephropathy in the laboratory mouse.
treatment promotes a sustained protection of mice from the J Am Soc Nephrol 2006; 17: 1796–1800.
15. Zheng Z, Schmidt-Ott KM, Chua S et al. A Mendelian locus on
progression of ADM-induced nephropathy after 28 days. chromosome 16 determines susceptibility to doxorubicin nephropathy in
Figure S4. Serum urea kinetics on adriamycin nephropathy. the mouse. Proc Natl Acad Sci USA 2005; 102: 2502–2507.
Figure S5. Delayed treatment with the B1R antagonist des-Arg9- 16. Seguin T, Buleon M, Destrube M et al. Hemodynamic and renal
[Leu8]-bradykinin (DALBK) reverses the progression of adriamycin involvement of B1 and B2 kinin receptors during the acute phase of
(ADM)-induced nephropathy. endotoxin shock in mice. Int Immunopharmacol 2008; 8: 217–221.
Figure S6. The B1R antagonist des-Arg9-[Leu8]-bradykinin (DALBK) 17. Wang Y, Wang YP, Tay YC et al. Progressive adriamycin nephropathy in
reverses the upregulation of inflammatory and fibrotic markers and mice: sequence of histologic and immunohistochemical events.
Kidney Int 2000; 58: 1797–1804.
restores the levels of podocyte markers after 28 days of ADM-induced
18. Hattori M, Horita S, Yoshioka T et al. Mesangial phenotypic changes
nephropathy. associated with cellular lesions in primary focal segmental
Figure S7. Heme oxygenase kinetics in adriamycin-induced glomerulosclerosis. Am J Kidney Dis 1997; 5: 632–638.
nephropathy. 19. GLee VW, Wang Y, Qin X et al. Adriamycin nephropathy in severe
Figure S8. B1R agonist des-Arg9-bradykinin (DABK) treatment combined immunodeficient (SCID) mice. Nephrol Dial Transpl 2006; 21:
aggravates the symptoms of adriamycin-induced nephropathy. 3293–3298.
Figure S9. Treatment with B1 receptor modulators DALBK (at days 4, 20. Rerrole J-P, Hertig A, Nguyen G et al. Plasminogen activator inhibitor type
5 and 6 after adriamycin injection) and DABK (at days 1, 2 and 3 after 1 is a potential target in renal fibrogenesis. Kidney Int 2000; 58:
1841–1850.
adriamycin injection) alters the mRNA and renal tissue levels of
21. Geleilete TJM, Costa RS, Dantas M et al. a-Smooth muscle actin and
chemokines associated with macrophage infiltration. proliferating cell nuclear antigen expression in focal and segmental
Figure S10. Early treatment with B1 receptor modulators DALBK and glomerulosclerosis: functional and structural parameters of renal disease
DABK alters the mRNA levels of matrix-associated proteins, which progression. Bras J Med Biol Res 2001; 34: 985–991.
were (A-B) analyzed 4 days after adriamycin injection and (C-D) 7 days 22. Ni A, Yin H, Agata J et al. Overexpression of kinin B1 receptors induces
after adriamycin injection. hypertensive response to des-Arg9-bradykinin and susceptibility to
Figure S11. Treatment with B1 receptor modulators DALBK (at days inflammation. J Biol Chem 2003; 278: 219–225.
23. Westermann D, Lettau O, Sobirey M et al. Doxorubicin cardiomyopathy-
4, 5 and 6 after adriamycin injection) and DABK (at days 1, 2 and 3
induced inflammation and apoptosis are attenuated by gene deletion of
after adriamycin injection) alters the renal mRNA and protein levels of the kinin B1 receptor. Biol Chem 2008; 389: 713–718.
IL-1b (A-C) analyzed 4 days after adriamycin injection and (B-D) 7 days 24. Westermann D, Walther T, Savvatis K et al. Gene deletion of the kinin
after adriamycin injection. receptor B1 attenuates cardiac inflammation and fibrosis during the
Supplementary material is linked to the online version of the paper at development of experimental diabetic cardiomyopathy. Diabetes 2009;
http://www.nature.com/ki 58: 1373–1381.
25. Ahluwalia A, Perretti M. Involvement of bradykinin B1 receptors in the
polymorphonuclear leukocyte accumulation induced by IL-1 beta in vivo
REFERENCES in the mouse. J Immunol 1996; 156: 269–274.
1. LeBrun CJ, Diehl LF, Abbott KC et al. Life expectancy benefits of cancer 26. Ricupero DA, Romero JR, Rishikof DC et al. Des-Arg(10)-kallidin
screening in the end-stage renal disease population. Am J Kidney Dis engagement of the B1 receptor stimulates type I collagen synthesis via
2000; 35: 237–243. stabilization of connective tissue growth factor mRNA. J Biol Chem 2000;
2. Seikaly M, Ho PL, Emmett L et al. The 12th Annual Report of the 275: 12475–12480.
North American Pediatric Renal Transplant Cooperative Study: renal 27. Ferreira J, Campos MM, Araujo R et al. The use of kinin B1 and B2 receptor
transplantation from 1987 through 1998. Pediatr Transplant 2001; 5: knockout mice and selective antagonists to characterize the nociceptive
215–231. responses caused by kinins at the spinal level. Neuropharmacology 2002;
3. Franceschini N, Hogan SL, Falk RJ. Primum non nocere: should adults with 43: 1188–1197.
idiopathic FSGS receive steroids? Semin Nephrol 2003; 23: 229–233. 28. Lagneux C, Bader M, Pesquero JB et al. Detrimental implication of B1
4. Kerjaschki D. Caught flat-footed: podocyte damage and the molecular receptors in myocardial ischemia: evidence from pharmacological
bases of focal glomerulosclerosis. J Clin Invest 2001; 108: 1583–1587. blockade and gene knockout mice. Int Immunopharmacol 2002; 2:
5. Kretzler M, Teixeira VP, Unschuld PG et al. Integrin-linked kinase 815–822.
as a candidate downstream effector in proteinuria. FASEB J 2001; 15: 29. Christopher J, Jaffa AA. Diabetes modulates the expression of glomerular
1843–1845. kinin receptors. Int Immunopharmacol 2002; 2: 1771–1779.

10 Kidney International
RL Pereira et al.: Bradykinin B1 receptor in FSGS original article

30. Deegens JK, Dijkman HB, Borm GF et al. Podocyte foot process 40. Hino M, Nagase M, Kaname S et al. Expression and regulation of
effacement as a diagnostic tool in focal segmental glomerulosclerosis. adrenomedullin in renal glomerular podocytes. Biochem Biophys Res
Kidney Int 2008; 74: 1568–1576. Commun 2005; 330: 178–185.
31. Saleem MA, Ni L, Witherden I et al. Co-localization of nephrin, podocin, 41. Susztak K, Raff AC, Schiffer M et al. Glucose-induced reactive
and the actin cytoskeleton: evidence for a role in podocyte foot process oxygen species cause apoptosis of podocytes and podocyte
formation. Am J Pathol 2002; 161: 1459–1466. depletion at the onset of diabetic nephropathy. Diabetes 2006; 55:
32. Boute N, Gribouval O, Roselli S et al. NPHS2, encoding the glomerular 225–233.
proteinpodocin, is mutated in autosomal recessive steroid-resistant 42. Nikolic-Paterson DJ, Lan HY, Hill PA et al. Macrophages in renal injury.
nephrotic syndrome. Nat Genet 2000; 24: 349–354. Kidney Int Suppl 1994; 45: S79–S82.
33. Wang PH, Campanholle G, Cenedeze MA et al. Bradykinin [corrected] B1 43. Klein J, Gonzalez J, Decramer S et al. Blockade of the kinin B1
receptor antagonism is beneficial in renal ischemia-reperfusion injury. receptor ameloriates glomerulonephritis. J Am Soc Nephrol 2010; 21:
PLoS One 2008; 3: e3050. 1157–1164.
34. Ng YY, Chen YM, Tsai TJ et al. Pentoxifylline inhibits transforming 44. Kawachi H, Koike H, Kurihara H et al. Cloning of rat nephrin: expression in
growth factor-beta signaling and renal fibrosis in experimental developing glomeruli and in proteinuric states. Kidney Int 2000; 57:
crescentic glomerulonephritis in rats. Am J Nephrol 2009; 29: 43–53. 1949–1961.
35. Chang HR, Yang SF, Lian JD et al. Prediction of chronic allograft damage 45. Breiteneder-Geleff S, Matsui K, Soleiman A et al. Podoplanin, novel 43-kd
index of renal allografts using serum level of plasminogen activator membrane protein of glomerular epithelial cells, is down-regulated in
inhibitor-1. Clin Transplant 2009; 23: 206–212. puromycin nephrosis. Am J Pathol 1997; 151: 1141–1152.
36. Semedo P, Correa-Costa M, Antonio Cenedeze M et al. Mesenchymal 46. Coward RJ, Foster RR, Patton D et al. Nephrotic plasma alters slit
stem cells attenuate renal fibrosis through immune modulation and diaphragm-dependent signaling and translocates nephrin, Podocin, and
remodeling properties in a rat remnant kidney model. Stem Cells 2009; CD2 associated protein in cultured human podocytes. J Am Soc Nephrol
27: 3063–3073. 2005; 16: 629–637.
37. Lai KN, Leung JC, Chan LY et al. Podocyte injury induced by mesangial- 47. Mu W, Ouyang X, Agarwal A et al. IL-10 suppresses chemokines,
derived cytokines in IgA nephropathy. Nephrol Dial Transplant 2009; 24: inflammation, and fibrosis in a model of chronic renal disease.
62–72. J Am Soc Nephrol 2005; 16: 3651–3660.
38. Shin M, Matsunaga H, Fujiwara K. Differences in accumulation of 48. Zeisberg M, Kalluri R. Experimental strategies to reverse chronic renal
anthracyclines daunorubicin, doxorubicin and epirubicin in rat tissues disease. Blood Purif 2004; 22: 440–445.
revealed by immunocytochemistry. Histochem Cell Biol 2010; 133: 49. Roederer M. Spectral compensation for flow cytometry: visualization
677–682. artifacts, limitations, and caveats. Cytometry 2001; 45: 194–205.
39. Ricardo SD, Bertram JF, Ryan GB. Antioxidants protect podocyte foot 50. Ertmer C, Köhler G, Rehberg S et al. Renal effects of saline-based 10%
processes in puromycin aminonucleoside-treated rats. J Am Soc Nephrol pentastarch versus 6% tetrastarch infusion in ovine endotoxemic shock.
1994; 4: 1974–1986. Anesthesiology 2010; 112: 936–947.

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