Nephrology Dialysis Transplantation 27 (Supplement 2): ii77–ii85, 2012
doi:10.1093/ndt/gfs213
CELL SIGNALLING /
PATHOPHYSIOLOGY
FP014
ROLE OF INDOLE-DERIVED UREMIC SOLUTES ON TISSUE
FACTOR PRODUCTION VIA ARYL HYDROCARBON
RECEPTOR PATHWAY
Claire Cerini1, Bertrand Gondouin2, Laetitia Dou1, Ariane Duval-Sabatier2,
Philippe Brunet2, Francoise Dignat- George2 and Stephane Burtey2
1
Inserm U 1076, Marseille, France, 2Inserm U 1076, Université Aix Marseille,
Marseille, France
Introduction and Aims: Chronic kidney disease (CKD) markedly increases
cardiovascular risk. Tissue Factor (TF), whose levels are elevated in CKD patients,
may participate in the development of cardiovascular diseases.We hypothesized that
indolic uremic solutes, indoxyl sulfate (IS) and indole-3-acetic acid (IAA), are
involved in increased TF production.
Methods: IAA, IS and plasma tissue factor levels were measured in 72 hemodialysis
(HD) patients, 50 undialyzed CKD patients (CKD) and 37 control subjects
(controls).
We then studied in vitro the effect of IAA and IS at maximal uremic concentrations
(9 μg/mL and 250 μg/mL respectively) on TF production in Human Umbilical Vein
Endothelial Cells (HUVEC) and Peripheral Blood Mononuclear Cells (PBMC) . We
also measured TF-dependent procoagulant activity in HUVEC incubated with IS and
IAA. Finally, we studied the involvement of aryl hydrocarbon receptor (AhR) in this
TF production by using pharmacological antagonists and siRNA experiments.
Results: Soluble TF (sTF) levels were respectively 142+/- 48 pg/mL in HD, 77 +/- 66
pg/mL in CKD and 36+/-14 pg/mL in controls, with significant differences between
all groups. In CKD patients, sTF levels were negatively correlated with renal function
estimated by MDRD formula (r= - 0,333, p < 0,05). IS and IAA levels were positively
correlated with sTF (r= - 0,333, p < 0,05 and r= 0,32, p < 0,01).
In vitro, IS and IAA increased TF protein levels and TF membrane expression in
HUVEC and PBMC. IAA and IS also increased TF-dependent procoagulant activity
in HUVEC. This increased TF antigen production is preceded by increased mRNA
levels suggesting an elevated transcriptional activity. siRNA directed against AhR
abolished the increase in TF protein, procoagulant activity and mRNA levels induced
by IS and IAA. TF expression and activity were also increased by dioxin, a
well-known AHR agonist.
Conclusions: In conclusion, the indolic uremic solutes increase TF production in
endothelial cells and PBMC via AHR activation, evoking a “dioxin-like” effect. These
newly described mechanisms of uremic solute toxicity might help understand the
high cardiovascular risk of CKD patients.
FP015
IL-13 HAS AN INHIBITORY EFFECT ON TYPE I COLLAGEN
EXPRESSION BY SUPPRESSION OF SMAD3 ACTIVITIES IN
TGF-BETA1-STIMULATED RENAL TUBULOEPITHELIAL
CELLS
Kazuhiro Okano1, Kazuhiro Okano1, Tomihito Iwasaki2, Hikohiro Jinnai2,
Asako Hibi2, Naoko Miwa2, Naoki Kimata2, Kosaku Nitta3 and Takashi Akiba2
1
Department of Blood Purification and Department of Medicine, Kidney Center,
Tokyo Women’s Medical University, Tokyo, Japan, 2Department of Blood
Purification, Kidney Center, Tokyo Women’s Medical University, Tokyo, Japan,
3
Department of Medicine, Kidney Center, Tokyo Women’s Medical University,
Tokyo, Japan
Introduction and Aims: Interleukin (IL)-13 is an important mediator for tissue
fibrosis caused by T helper cell type 2 (Th2) inflammation. IL-13 overexpression
augments podocyte injury in rats (J Am Soc Nephrol 2007; 18: 1476). On the other
hand, IL-13 overexpression reduces renal tubulointerstitial damage in rat
ischemia-reperfusion injury (Kidney Int 2008; 73: 1364). These suggest that effect of
IL-13 may be dependent on pathological mechanisms of various renal diseases.
Transforming growth factor-beta1 (Tb1) is known as the most important mediators
for extracellular matrix (ECM) production in kidney. We hypothesized that IL-13
may have a role in renal fibrogenesis caused by Tb1. To test the hypothesis, we
examined the effects of IL-13 on type 1 collagen (COL1) production in
Tb1-stimulated renal cells.
Methods: Human renal tubular epithelial cells (HKC cells) were treated with Tb1
(4ng/mL) and/or IL-13 (10ng/mL). COL1 expression was examined by
immunofluorescence cytochemistry and western blot. Reporter activities were
measured using reporter constructs containing a2(I) collagen promoter (COL1A2),
Smad-binding element (SBE) promoter. Specific inhibitors against Smad3, several
mitogen-activated protein kinase (MAPK), or Janus kinase/signal transducers and
activators of transcription (JAK/STAT) pathways were used to examine contribution
of the signaling pathways in COL1 transcription.
Results: Stimulation with IL-13 alone augmented COL1A2 promoter activity and
COL1 expression. However, concurrent stimulation with IL-13 and Tb1 showed
reduction of COL1A2 promoter activities and COL1 expression compared to those
activated by Tb1 alone. Stimulation with both IL-13 and Tb1 also decreased SBE
promoter activities compared to those activated by Tb1 alone. Blockade of p38
MAPK pathways increased the COL1A2 reporter activities in the cells treated with or
without Tb1 and/or IL-13. Possible mechanism of the inhibitory effect by IL-13 is
that IL-13 affects Tb1/Smad pathway through p38 MAPK pathways, resulting in
decrease of COL1 transcription in Tb1-stimulated renal tubuloepithelial cells.
Conclusions: IL-13 is a bifunctional regulator for COL1 production in renal
tubuloepithelial cells stimulated by Tb1. The p38 MAPK pathway may be an
important mediator for cross-talk between Tb1 and IL-13 in COL1 production by
renal epithelial cells.
FP016
TELMISARTAN POTENTIATES ANTIANGIOGENIC EFFECTS
OF SUNITINIB IN A XENOGRAFT MODEL OF RENAL CELL
CARCINOMA
Thibault Dolley-Hitze1, Grégory Verhoest1, Florence Jouan1,
Yannick Arlot-Bonnemains1, Audrey Lavenu2, Marc-Antoine Belaud-Rotureau1,
Nathalie Rioux-Leclercq1 and Cécile Vigneau1
1
Cnrs Umr6061 / Ifr 140, Faculté de Médecine, Université Rennes 1,
2
Département de Pharmacologie Expérimentale Et Clinique, Université Rennes 1
Introduction and Aims: The prognosis of Renal Clear Cell Carcinoma (RCCC)
dramatically improved since antiangiogenic drugs, such as sunitinib, have been used.
These drugs mainly inhibit Vascular Endothelium Growth Factor Receptors
(VEGFR). In addition, it has been shown that angiotensin-2 type-1 receptor
antagonists, such as telmisartan, are not only potent antihypertensive drugs but can
also inhibit the production of Vascular Endothelium Growth Factor (VEGF) by
different tumour cells. We hypothesised that adding telmisartan to sunitinib could
potentiate antiangiogenic effects of sunitinib and we tested this additive effect in a
xenograft model of RCCC.
Methods: Human commercial RCCC cell lines (786-0) were injected in the flank of nude
mice. After tumour development (5 weeks — 100-200mm3), sunitib and/or telmisartan or
vehicle alone for 4 weeks were orally administered to mice and then animals were
sacrificed. Blood and tumours were collected for analysis of angiogenic characteristics,
growth, necrosis and apoptosis by ImmunoHistoChemistry (IHC), Western Blot (WB)
and ELISA methods. Statistical analyses were performed with Kruskal Wallis tests.
Results: Ten animals in each group were treated (n=40). Telmisartan added to sunitinib
significantly increases necrosis (HE staining p=0,038) and tends to reduce tumour size
(p=0,067) compared with sunitinib alone. In the association group, central microvascular
density is also decreased as well as circulating VEGF (ELISA p=0,003). VEGF produced by
tumours also seems to be decreased (WB). Association of both drugs doesn’t significantly
modify tumour cells proliferation (IHC Ki67 staining) or apoptosis (IHC activated
caspase-3 staining).
Conclusions: When telmisartan is added to sunitinib in xenograft model of RCCC in
nude mice, tumour necrosis is increased. It could be explained by decreased
neo-angiogenesis secondary to a best blockage of VEGF pathway.
FP017
ALTERED ANTIGEN HANDLING AND INVOLVEMENT OF THE
CX3CR1-FKN AXIS IN PROMOTING HEMATURIA IN IGA
NEPHROPATHY PATIENTS
Sharon Natasha Cox1, Fabio Sallustio2, Grazia Serino3, Antonia Loverre3,
Francesco Pesce1, Margherita Gigante4, Gianluigi Zaza5, Patrizia Stifanelli6,
Nicola Ancona7 and Francesco Paolo Schena8
1
Dept. of Emergency and Organ Transplantation, University of Bari, Bari, Italy,
2
Department of Emergency and Organ Transplantation, University of Bari, Bari,
Italy, 3University of Bari, Bari, Italy, 4Department of Biomedical Sciences,
University of Foggia, Foggia, Italy, 5Nephrology and Dialysis Unit, Department
of Medicine, University of Verona, Verona, Italy, 6Issia, Cnr, Bari, Italy, 7Issia, Cnr,
Bari, Italy., 8Nephrology Dialysis and Transplantation Unit, Department of
Emergency and Organ Transplantation (D.E.T.O.), University of Bari, Italy
Introduction and Aims: The hallmark of IgA nephropathy (IgAN) is gross
hematuria (GH) coinciding with or immediately following an upper respiratory or
© The Author 2012. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: [email protected]
Abstracts
gastrointestinal tract infection; thus a dysregulation of innate immunity has been
extensively postulated in IgAN. Oral immunization with a variety of common
pathogens and alimentary components is able to reproduce IgAN in experimental
mice models. Furthermore, some of these exogenous antigens can be detected in the
renal tissue of IgAN patients. Since GH can represent a disease triggering event, we
decided to study this important clinical time point in order to unravel the link
between mucosal encountered antigens and the occurrence of glomerular hematuria.
Methods: The first step of the study was to perform an exploratory/
hypothesis-generating analysis using microarray technology in order to identify
genes/pathways modulated in the acute phase of the disease. For this aim we
analyzed the genomic profile of PBMCs from 3 IgAN patients at two different
clinical time points, the first sample during the GH episode and the second during
the remission phase of the disease characterized by persistent microscopic hematuria
(MH). Identified genes and pathways were then validated with classical biomolecular
tools on a large cohort of 79 biopsy-proven IgAN patients, 5 membranous
nephropathy (MN), 3 membranoproliferative glomerulonephritis (MPGN) patients
and 10 Healthy blood donors (HBD).
Results: The modulated genes during GH showed a clear involvement of the
interferon signalling (e.g. STAT1), antigen presenting pathway (HLA-E, TAPBP) and
the immuno-proteasome (e.g. PSMB8, PSMB9, PSMB10). The gene characterizing
cytotoxic effector lymphocytes (CX3CR1), implicated in vascular endothelial damage,
was found up-regulated at both mRNA and protein level. Furthermore, a significant
increase of CX3CR1 surface expression was found in cell subsets with well known
cytotoxic effector functions. In vitro antigenic stimulation of PBMCs on an
independent set of IgAN patients demonstrated that patients up- regulate specifically
CX3CR1 and STAT1 in an enhanced and dose dependant manner, while an expected
down-regulation occurred in HBD and in other glomerulonephritis control groups.
This enhanced activation not only occurred in patients characterized by recurrent
GH or by permanent microscopic hematuria (MH) but also in IgAN patients
without hematuria. These results confirm that the deregulation of the interferon
signaling pathway through STAT1 and the enhanced CX3CR1 expression during
endotoxin challenge is specific to IgAN patients, however, the enhanced receptor
expression is not sufficient to cause GH. Then we analyzed glomerular fractalkine
(FKN) expression, since this ligand, is involved in the vascular gateway for CX3CR1+
cells towards the inflamed tissues and induces vascular injury. A significantly higher
glomerular and urinary FKN level was found in IgAN patients with recurrent GH
episodes compared to permanent MH patients and MPGN and MN patients,
suggesting a predisposition for cytotoxic cell extravasation only in recurrent GH
patients.
Conclusions: Taken together, our findings demonstrate, for the first time, a defect in
antigen handling in PBMCs of IgAN patients with a specific up-regulation of
CX3CR1 and STAT-1. Furthermore, the constitutive up regulation of
glomerular FKN, suggests an involvement of the CX3CR1-FKN axis in the
exacerbation of GH.
FP018
ARACHIDONIC ACID STIMULATES CA2+ ENTRY INTO
MOUSE TAL CELLS THROUGH AN APICAL TRPM7-LIKE
CA2+-PERMEABLE NON-SELECTIVE CATION CHANNEL.
Paulais Marc1 and Teulon Jacques1
Umrs872 - Erl7226
1
Introduction and Aims: We recently described a TRPM7-like, Ca2+-permeable,
non-selective cation channel in the apical membrane of mouse cortical thick
ascending limb (TAL) of Henle’s loop (Guinamard et al, BBA — Biomembranes,
in press), thought to be involved in Ca2+-dependent signaling pathways modulating
NaCl reabsorption. Since it is also known that arachidonic acid (AA), an
important regulator of potassium channels activity and of ion transport by the TAL,
raises intracellular calcium concentration ([Ca2+]i) in TAL (Firsov et al, Pflügers
Arch, 429:636-646, 1995), the aim of this study was to determine the potential
role of the apical cation channel in the AA-induced [Ca2+]i increase in
TAL tubule.
Methods: Mouse TAL tubules microdissected from collagenase-treated kidneys were
loaded with the Ca2+-sensitive fluorescent probe, Fura2, and intracellular Ca2+
concentration ([Ca2+]i) was monitored by fluorescence ratio imaging (Argus-50;
Hamamatsu).
Results: AA (3 - 30 mM) caused a dose-dependent, slow increase in [Ca2+]i of TAL
tubules, which was blocked by the presence of 10 mM Gd3+, an inhibitor of the
apical non-selective cation channel. The effect of AA on [Ca2+]i was reproduced by
linoleic acid (10 mM), a 18-C cis- polyunsatured fatty acid, but not by 10 mM ETA,
a 20-C cis-polyunsaturated fatty acid or oleic acid, a 18-C cis-monounsaturated fatty
acid, indicating the absence of a non-specific partitioning of AA into membrane fluid
lipid domain. In addition, 10 mM AA-CoA, an analog of AA with a CoA group
containing a negatively charged moiety preventing membrane insertion, caused a
very marginal increase in [Ca2+]i of cTAL tubules, clearly showing that AA rather
acts at an intracellular location.
The AA-induced [Ca2+]i increase mainly resulted from a Ca2+ entry into the cell
interior. First, external Ca2+ removal abolished the [Ca2+]i response to AA. Second,
AA led to a dramatic increase in the initial rate of Fura2 fluorescence quenching
observed upon addition of Mn2+, a Ca2+ surrogate for Ca2+-permeable channels, and
a rapid and high affinity Fura2 fluorescence quencher, indicating a AA-stimulated
Mn2+ influx, and therefore a Ca2+ influx, into TAL tubules.
ii | Abstracts
Nephrology Dialysis Transplantation
Finally, the AA-induced Ca2+ entry, as revealed by Mn2+-induced Fura2 quenching,
was blocked by 10 mM Gd3+ or 100 mM amiloride, another blocker of the apical
non-selective cation channel.
Conclusions: These results show that the AA-induced [Ca2+]i increase in the TAL is
mainly due to a Ca2+ entry, which occurs very likely through an activation of the
apical, TRPM7-like, Ca2+- permeable non-selective cation channel, and point towards
its major role in the modulation of NaCl reabsorption by Ca2+-dependent signaling
pathways.
FP019
EXTENDED FUNCTIONAL LIFESPAN OF THE
ERYTHROPOIETIN RECEPTOR (EPOR) FOLLOWING
STIMULATION WITH PEGINESATIDE
Jennifer M. Green1, Richard B. Mortensen2, Rakesh Verma3, Karen Leu2, Peter
J. Schatz2 and Don M. Wojchowski3
1
Affymax, Inc., Palo Alto, USA, 2Affymax, Inc., 3Maine Medical Center Research
Institute
Introduction and Aims: Peginesatide (formerly known as Hematide™) is a
PEGylated, investigational, peptide-based erythropoiesis stimulating agent (ESA) that
is designed to specifically stimulate the EPOR and is currently being developed for
the treatment of anemia due to chronic kidney disease in dialysis patients.
Peginesatide has a unique structure that consists of a synthetic peptide dimer (with
no sequence similarity to erythropoietin) conjugated to a 40 kDa PEG moiety. This
study was conducted to investigate the regulation of EPOR trafficking and
down-modulation after peginesatide vs. recombinant human erythropoietin
(rHuEPO, epoetin alfa) dosing.
Methods: We have developed a novel panel of specific rabbit monoclonal anti-EPOR
antibodies, and have used these tools in Western blotting, flow cytometry, and
electrochemiluminescence studies of EPOR cell surface expression, ubiquitination,
and degradation.
Results: In exponentially growing erythropoietin-dependent UT-7/EPO cells
maintained in rHuEPO, cell surface EPORs were detected at low levels, and EPOR
turnover/degradation appeared to predominate. In contrast, cell surface EPOR were
increased significantly (~ 5.9 fold) in cells maintained in functionally equivalent
levels of peginesatide, suggesting that EPOR turnover was lessened. After culture of
cells for 18 hours in the absence of EPOR stimulation, subsequent treatment with
rHuEPO led to the rapid tyrosine-phosphorylation of the EPOR, subsequent receptor
internalization, and the rapid appearance of the major 42 kDa and 30 kDa EPOR
degradation fragments observable within 15 minutes of treatment. In contrast, EPOR
phosphorylation occurred at reduced magnitude following peginesatide treatment,
but was sustained and correlated with an observed 30 minute delay in EPOR
fragmentation. Since the ubiquitin/proteasome system plays a major role in EPOR
internalization and down- modulation, we investigated EPOR ubiquitination after
peginesatide vs. rHuEPO treatment. Each ESA induced EPOR ubiquitination, but
peginesatide did so to a significantly lower extent (~ 50% less).
Conclusions: Cell surface EPOR levels are increased, ubiquitination levels are lower,
and receptor degradation is delayed after peginesatide vs. rHuEPO treatment. It is
proposed that peginesatide may not induce the ubiquitination and internalization of
the EPOR as efficiently as other ESAs, thereby allowing for a longer functional
lifespan of the EPOR. These studies provide new mechanistic insights into the
molecular basis for the extended erythropoietic activity of peginesatide.
FP020
SIGNAL TRANSDUCTION PATHWAY AND DIRECT ACTION ON
VASCULAR ENDOTHELIAL CELLS BY
ERYTHROPOIESIS-STIMULATING AGENT
Chieko Ihoriya1, Minoru Satoh1, Tamaki Sasaki1 and Naoki Kashihara1
Kawasaki Medical School, Kurashiki, Japan
1
Introduction and Aims: Previous studies have shown that high dose erythropoietin
(EPO) exerts vascular and tissue protective activities through non-hematopoietic
actions. However, it is still controversial whether therapeutic dosage of EPO is
beneficial for the cardiovascular protections. There are thought to be 2 receptors for
EPO. One is the homodimeric EPO receptor (EPO-R) that is responsible for
erythropoiesis. The second is the heterodimeric receptor that consists of the EPO-R
and the beta common receptor (bcR). We investigated that whether therapeutic
dosage of EPO administration could accentuate oxidative stress and modulate
endothelial function.
Methods: In vivo experiments, normal male Sprague-Dawley rats were treated with
either EPO (20 IU/kg/week, subcutaneously) three times a week or darbepoietin
(D-EPO; 0.1 μg/kg/week, subcutaneously) once a week for 4 weeks.
Endothelial-dependent vasodilatory response, NADPH oxidase activity, and gene
expression of ICAM-1 and MCP-1 were assessed. To further explore whether EPO is
involved in superoxide production, we stimulated human umbilical vein endothelial
cells with EPO and assessed NADPH oxidase activity. Next, we assessed EPO
induced signal transduction with or without small interfering RNA knockdown of
EPO-R and bcR.
Results: The used dosages of EPO and D-EPO had no effect on hemoglobin level. In
normal SD rats, the acetylcholine-dependent vasodilatory response decreased
Volume 27 | Supplement 2 | May 2012
Abstracts
Nephrology Dialysis Transplantation
significantly in both EPO and D-EPO treatment groups. NADPH oxidase activity
increased significantly in both groups. Aortic gene expression of ICAM-1 and
MCP-1 similarly increased in both groups. There was no difference between EPO
and D-EPO. We confirmed EPO-mediated superoxide production and extracellular
signal-regulated kinase (ERK) activation in vitro. Disruption of EPO-R and bcR
inhibited the ERK activation by EPO.
Conclusions: Administration of EPO and D-EPO increased oxidative stress and
impaired endothelial function in normal rats and in human endothelial cells.
FP021
SIRT1 OVEREXPRESSION MITIGATES CISPLATIN-INDUCED
ACETYLATES OF NF-KB P65 SUBUNIT AND CYTOTOXICITY
Yu Jin Jung1, Kyung Pyo Kang2, Ae Sin Lee1, Jung Eun Lee1, Sik Lee1, Sung
Kwang Park1, Won Kim1 and Kyung Pyo Kang1
1
Department of Internal Medicine, Research Institute of Clinical Medicine,
Chonbuk National University Medical School, Jeonju, 2Chonbuk National
University Medical School, Jeonju, Korea
Introduction and Aims: As the increased acetylation of RelA/p65 is linked to
nuclear factor-kB (NF-kB) activation, the regulation of p65 acetylation can be a
potential target for the treatment of inflammatory injury. Cisplatin-induced
nephrotoxicity is important issue in chemotherapy in cancer patients. Especially,
transcriptional activity of the RelA p65 subunit of NF-kB can be modulated by
cisplatin. Acetylation of p65 may control NF-kB transcriptional response. Sirt1, the
nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase, has been
implicated in a variety of cellular processes such as inflammatory injury and the
control of multidrug resistance in cancer. However, there is no report about the effect
of Sirt1 overexpression on cisplatin-induced acetylation of p65 subunit of NF-kB and
cell injury in HK2 cells. Thus, it can be suggested that deacetylation of NF-kB
transcriptional activity can be a possible target to attenuate cisplatin-induced cell
injury.
Methods: To investigate the effect of Sirt1 in cisplatin-induced acetylation of p65
subunit of NF-kB and cell injury, HK-2 cells were exposed with Sirt1 overexpression,
LacZ adenovirus or DN (dominant negative) virus after treatment with cisplatin.
Results: While the protein expression of Sirt1 was found to be decreased by cisplatin
treatment compared with control buffer treatment, acetylation of NF-kB p65 subunit
was significantly increased after treatment with cisplatin. Overexpression of Sirt1
increased protein expression and activity of Sirt1 in HK-2. Upon overexpression,
Sirt1 ameliorated the increased acetylation of p65 of NF-kB during cisplatin
treatment and cisplatin-induced cytotoxicity. Resveratrol also decreased acetylation of
NF-kB p65 subunit. Cisplatin treatment significantly decreased the cell viability in
HK-2 cells. Further. Sirt1 overexpression mitigates cisplatin- induced cytotoxicity.
Conclusions: Our findings suggests that the regulation of Sirt1 as a possible target to
attenuate cisplatin- induced proximal tubule cell damage.
FP022
CYSTEINE RESIDUES REGULATE TRANSIENT RECEPTOR
POTENTIAL CANONICAL TYPE 6 CHANNEL PROTEIN
EXPRESSION
Thilo Florian1, Martin Tepel2, Liu Ying3, Krueger Katharina1, Förste Nora1,
Wittstock Antje1 and Scholze Alexandra4
1
Charite Berlin Germany, 2Odense University Hospital, 3Sdu IMM Odense
Denmark, 4Sdu Ki Odense Denmark
Introduction and Aims: The regulation of calcium influx through transient receptor
potential canonical type 6 (TRPC6) channel is mandatory for the activity of human
monocytes. In the present study we investigated whether cysteine residues of
homocysteine (HC) or acetylcysteine (ACC) may affect TRPC6 expression in human
monocytes.
Methods: TRPC6 mRNA and protein expression in human monocytes were
investigated using quantitative real-time RT-PCR and quantitative in-cell western
assay. Protein expression of extracellular-signal-regulated kinase (ERK) and
phosphorylated ERK ( pERK) was measured after administration of ACC in the
absence and presence of cation-channel blockers, 2- aminoethoxydiphenyl borate or
gadolinium. Cytosolic calcium and intracellular stored calcium were measured using
fluorescent dyes.
Results: Patients with chronic kidney disease had significantly elevated HC levels
compared to control subjects (23.8±3.6 μmol/L vs. 11.3±1.0 μmol/L; p<0.01). TRPC6
mRNA expression was significantly higher in monocytes from 17 patients with
chronic kidney disease compared to 19 control subjects (normalized ratio, 100±39 vs.
22±7 arbitrary units; p<0.01). In monocytes from both, chronic kidney disease
patients and control subjects, the administration of HC (100 μmol/L) or ACC (500
μg/mL) significantly increased TRPC6 channel protein expression compared to
control conditions. ACC significantly increased pERK but not ERK protein
expression.
Conclusions: Experiments using homocysteine and acetylcysteine indicate that
cysteine residues increase TRPC6 channel protein expression in humans.
Volume 27 | Supplement 2 | May 2012
FP023
RAPAMYCIN PROMOTES PODOCYTE MIGRATION THROUGH
THE UPREGULATION OF UROKINASE RECEPTOR
Yung-Tsung Chiu1 and Ming-Ju Wu2
Taichung Veterans General Hospital, Taichung, Taiwan, 2Taichung Veterans
General Hospital
1
Introduction and Aims: Podocyte damage occurs commonly in rapamycin induced
proteinuria. Induction of urokinase receptor (uPAR) signaling in podocytes leads to
foot process effacement and urinary protein. In this study, we assess the expression of
uPAR in primary cultured podocytes with rapamycin treatment. The effect of
comination treatment with tacrolimus and rapamycin on the podocyte migration was
also evaluated.
Methods: The primary culture of rat podocytes is used in this study. The migratory
activity of podocyte is determined by the percentage of the reduction of gap
distance on the wound scratch assay. The expression of uPAR in podocytes
is detected by real-time PCR, immunofluorescence staining, and western blot
analysis.
Results: The treatment of 10 ng/mL and 100 ng/mL rapamycin significantly increase
the migratory activity of podocyte from 14.8% to 45%. Rapamycin does not increase
the apoptosis of podocytes or affect the podocyte cell viability and morphology. The
expression of uPAR in the podocyte is significantly increased by 1.8 ~1.9 fold after
rapamycin treatment. The upregulation of uPAR in podocytes is confirmed by
immunofluorescence staining, Real-time PCR and western blot analysis. Rapamycin
treatment also causes the activation of FAK and ILK. Compared to rapamycin
treatment only, combined treatment with tacrolimus and rapamycin significant
attenuates the podocyte migratory activity induced by rapamycin in a dose
dependent manner. Besides, the activation of FAK and ILK were also diminished by
the addition of tacrolimus.
Conclusions: Rapamycin could promote podocyte migration through the
upregulation of urokinase receptor. The co-treatment of tacrolimus effectively reduce
the podocyte migratory activity.
FP024
MICRORNA-146A DOWNREGULATES TOLL-LIKE RECEPTOR
SIGNALING PATHWAY MEDIATING PUROMYCIN
AMINONUCLEOSIDE INDUCED PODOCYTE INJURY
Zhi-Hong Liu1, Yaojun Liang1, Chun-Xia Zheng1, Zhao-Hong Chen1 and
Cai-Hong Zeng1
1
Research Institute of Nephrology, Jinling Hospital, Nanjing University School of
Medicine, Nanjing, China
Introduction and Aims: Through microRNA microarray expression profiling of
puromycin aminonucleoside podocyte injury model, we found microRNA-146a is
one of increased expression microRNAs. Using ISH technique, we found that
microRNA146a specific expression in glomerular podocytes of FSGS patients and
PAN rats. Analysis of Targetscan online software and reference reports, we found
that microRNA-146a specific targeting mRNA of IRAK1 and TRAF6 which are the
key molecules of Toll-like receptor signaling pathway. The aim of this work is to
study the role of microRNA146a in podocytes injury.
Methods: Using BLOCK-iT™ Pol II miR RNAi vector packaging microRNA-146a
down-expression vectors. And using Lipofectamine™ LTX and Plus agent transfer
plasmid to human glomerular podocytes, the number of viable cells using MTT
assay, apoptosis by flow cytometry, using phalloidin staining cytoskeletal damage,
using RealtimeRT-PCR detection of TLR1, TLR2, TLR4, TLR7, TLR9, IRAK1,
TRAF6, CD2AP, a-actinin4 mRNA and microRNA-146a expression, using western
blotting detection of IRAK1, TRAF6, TLR4, TLR9, CD2AP, a-actinin4 protein. And
detected by immunohistochemistry in patients with FSGS and renal puromycin rat
kidney TLR4, TLR9, IRAK1, TRAF6 protein expression.
Results: RealtimeRT-PCR detection shows that microRNA-146a down-expression
vector successfully been constructed, and selected stably transfected cell lines. By
MTT and flow cytometry showed that down-expression of miR-146a can significantly
improve the survival rate decreased podocyte apoptosis, phalloidin staining showed
that down-expression of miR-146a can reduce the damage of the cytoskeleton.
RealtimeRT-PCR and western blotting analysis showed that interference with the
down-expression of miR-146a can increas IRAK1, TRAF6, and a-actinin4 maintain
CD2AP expression. RealtimeRT-PCR analysis also show that TLR4 and TLR9 mRNA
in puromycin injury group is increased, other TLRs expression are no differences in
the two groups. WB shows TLR9 protein expression in the injury group is increased
with time extension of the puromycin intervention. Immunohistochemistry showes
renal tissue of puromycin rat, IRAK1, TRAF6, CD2AP, a-actinin4 in the glomeruli
are specifically expressed in podocytes, and the control group were significantly
different expression.
Conclusions: From the puromycin aminonucleosides podocyte injury models, the
expression of TLRs- IRAK1-TRAF6 signaling pathway which may play a key role in
puromycin aminonucleosides induced podocyte cytoskeletal protein damage is
decreased. Inhibit miR-146a function can increase Toll-like receptor signaling
protects podocyte injury.
doi:10.1093/ndt/gfs213 | ii
Abstracts
FP025
NANOTUBULAR CONNECTIONS BETWEEN HUMAN
PERITONEAL MESOTHELIAL CELLS
Julia Ranzinger1, Amin Rustom2, Lars Kihm3, Danijela Heide3, Peter Scheurich4,
Martin Zeier3 and Vedat Schwenger3
1
Nephrology, University of Heidelberg, Germany, 2Max-Planck Institute for
Intelligent Systems, Stuttgart, Germany, 3Nephrology, University of Heidelberg,
Heidelberg, Germany, 4University of Stuttgart, Stuttgart, Germany
Introduction and Aims: Success and efficacy of peritoneal dialysis (PD) strongly
depends on the integrity of the peritoneal membrane. Recently discovered membrane
channels, so called tunneling nanotubes (TNTs), mediate complex intercellular
communication processes between cells of different cellular systems and attain
growing physiological and pathological relevance. In this context, we recently
reported the existence of TNTs between human primary peritoneal mesothelial cells
(HPMCs). We showed that the number of TNTs is increased after incubation of the
cells with TNF-a, not with dialysis solution, demonstrating a stimulating effect of
TNF-a on the TNT-formation. Moreover, we could show that treatment of the cells
with simvastatin induces TNT-formation, whereas the immunosuppressant drug
rapamycin leads to a decrease of TNTs. It is known from other studies that the
protein kinases Akt, PI3K and mTOR are involved in the formation of TNTs,
although precise mechanisms remain elusive. The aim of our present study is to
investigate the underlying molecular mechanisms and the involvement of these
protein kinases during TNF-a and simvastatin induced TNT-formation between
HPMCs. Furthermore, we aim to study the role of cellular calcium channels in TNTformation processes.
Methods: HPMCs were isolated from i) omentum of healthy donors and ii) dialysis
fluid from patients undergoing PD. Calcium channels were blocked by incubating the
cells with the drug amlodipine. To investigate the effects of TNF-a, dialysis solution
and simvastatin on MAP- kinase signalling, cells were treated in a dose and time
dependent manner. High resolution fluorescence microscopy was applied to detect
TNTs and to assess the cellular topography of the target proteins. Western Blot
analyses were applied to detect and investigate protein expression levels. Gene
expression analyses were performed using real time PCR.
Results: We here show that stimulation of HPMCs with dialysis solution led to
increased levels of activated protein kinase Akt as compared to the control
situation and stimulation of cells with TNF-a. Furthermore, blocking of calcium
channels of HPMCs resulted in a significant decrease of TNT-numbers between
HPMCs.
Conclusions: The existence of TNTs connecting HPMCs in vitro points to a
distinctive communication among these cells at least under cell-culture conditions.
We show that dialysis solution leads to a strong decrease in the number of TNTs
between the cells and affects MAP-kinase signalling by activation of protein kinase
Akt. Moreover, our results provide evidence for a strong involvement of calcium
channels in the formation of TNTs between HPMCs. These findings may have major
importance for medical treatments.
Nephrology Dialysis Transplantation
FP027
Fang Zhong1, Fang Zhong1, Xia Liu1, Qiao Zhou1, Xu Hao1, Ying Lu1,
Shanmai Guo1, Weiming Wang1, Donghai Lin1 and Nan Chen2
1
Department of Nephrology, Ruijin Hospital, Shanghai Jiao Tong University
School of Medicine, 2Ruijin Hospital, Shanghai Jiaotong University
Introduction and Aims: In this study, we evaluated the renal protective effects of the
Chinese herb Cordyceps sinensis in rats with 5/6 nephrectomy.
Methods: Male SD mice were subjected to 5/6 nephrectomy. The rats were divided
into 3 groups: (a) untreated nephrectomized group (OP group, n=16), (b) oral
administration of Cordyceps sinensis-treated (4 mg/kg/d) nephrectomized group (CS
group, n=16) and (c) sham-operated group (SO group, n=16). Rats were sacrificed at
4 and 8 weeks after 5/6 nephrectomy, and the kidneys, serum and urine were
collected for 1H nuclear magnetic resonance (1HNMR) spectral analysis.
Multivariate statistical techniques and statistical metabolic correlation comparison
analysis were performed to identify metabolic changes in aqueous kidney extracts
between these groups.
Results: The levels of creatinine, BUN and proteinuria in the 5/6 Nx group were
progressively elevated, which were also accompanied by typical pathological changes.
Oral administration of C. sinensis was shown to ameliorate glomerulosclerosis and
renal interstitial fibrosis in this study, which indicated that C. sinensis has the same
renoprotective effects in the 5/6 Nx model as it was reported to ameliorate of
cyclosporin nephrotoxicity.Significant differences between these groups were
discovered in the metabolic profiles of the biofluids and kidney extracts. Pathways
including the citrate cycle, branched-chain amino acid metabolism and the
metabolites that regulate permeate pressure were disturbed in the OP group
compared to the SO group; in addition, these pathways were reversed by Cordyceps
sinensis treatment. Biochemistry and electron microscopic images verified that
Cordyceps sinensis has curative effects on chronic renal failure. These results were
confirmed by metabonomics results. The most important findings of this study are
(i) as a tonic, C. sinensis can affect the energy metabolism in kidney, particularly in
terms of the tricarboxylic acid cycle. (ii) C. sinensis can help balance the disturbed
osmotic pressure of the extracellular NaCl. (iii) C. sinensis might promote
branched-chain amino acids degraded in the kidney. (iv) C. sinensis further
decreased the concentration of triglycerides, lipoproteins and total cholesterol in
kidney. There might contribute to the effect of C. sinensis on the amelioration of
tubular and glomerular function.
Conclusions: Cordyceps sinensis has potential curative effects on CKD, and our
metabonomics results provided new insight into the mechanism of treatment of this
traditional Chinese medicine.
FP028
FP026
ROLE OF DNA METHYLATION IN RENAL APOPTOSIS
Liu1,
Jian Liu1, Fang Zhong1, Lili Xu1, Qiao Zhou1, Xu Hao1, Weiming Wang1
Jian
and Nan Chen2
1
Department of Nephrology, Ruijin Hospital, Shanghai Jiao Tong University
School of Medicine, 2Ruijin Hospital, Shanghai Jiaotong University
Introduction and Aims: DNA methylation, one kind of epigenetic modifications,
can regulate the generation of proteins by ways beyond alteration of gene sequence
and has been a popular topic in many diseases except kidney which still need more
detailed researches. The aim of the present study was to determine whether DNA
methylation in the progression of the kidney diseases is associated with apoptotic cell
deathin which FADD and APAF1.
Methods: Sprague-Dawley rats (180mg) were injected with adriamycin (ADR)
through penis vein twicely in doses of 5mg/kg and 2.5mg/kg to induce renal diseases
and the same weight Sprague-Dawley rats were treated with the same volume of
normal saline through the same way as negative controls. Tissue samples (8 weeks)
were evaluated for histology, TUNEL assay and the DNA methylation in the
promoter of genes(FADD and APAF1) was detected by Methylation-Sensitive
High-Resolution Melting-Curve Analysis(HRM, Roche) after DNA extracted from
the cortex of kidney (8 weeks) and bisudie-treated. Realtime-PCR was uses to
determine the expression of RNA.
Results: HE and PAS results showed increased mesangial cell proliferation, swelling
of tubules and inflammatory cell infiltration compared with the control group.
Besides, our Masson results indicated more fibrosis in glomeruli and interstitial areas
in model group. The TUNEL assay pointed out that more apoptosis in tubular
epithelial cells in model group. The promoter of FADD was hypermethylated in
ADR group whereas the level of methylation in promoter of APAF1 was the same as
the control group. At the same time, the realtime-PCR outcomes shows FADD
mRNA was up-regulated in ADR group which was opposite with hypothesis.
Conclusions: In the model of ADR rats, DNA methylation is not the major
factor affecting FADD and APAF1 expression. But further experiment is still
needed to prove the relationship between apoptosis in renal disease and DNA
methylation.
ii | Abstracts
1H NMR SPECTROSCOPY ANALYSIS OF METABOLITES IN
THE KIDNEYS PROVIDES NEW INSIGHT INTO
PATHOPHYSIOLOGICAL MECHANISMS: APPLICATIONS FOR
TREATMENT WITH CORDYCEPS SINENSIS
PHOSPHOPROTEOMIC ANALYSIS OF THE P2X7
SIGNALLING CASCADE IN MACROPHAGES: A PUTATIVE
ROLE IN GLOMERULONEPHRITIS
Annalisa Vilasi1, Simona Deplano2, Simona Deplano3, Pedro Cutillas4,
Robert Unwin1 and Frederick W.K. Tam3
1
Centre for Nephrology, University College London Medical School, Royal Free
Hospital, London, United Kingdom, 2Imperial College, 3Renal Medicine, Imperial
College Kidney and Transplant Institute, Hammersmith Hospital, London, United
Kingdom, 4Centre for Cell Signalling, Barts Cancer Institute, Bart's and the
London School of Medicine, Queen Mary University of London, London, UK.
Introduction and Aims: Introduction and aims: Recently there is increasing interest
in the role of P2X7 receptor (P2X7R) in the pathogenesis of glomerulonephritis
(GN). P2X7R knock-out mice in the experimental model of glomerulonephritis (GN)
demonstrated that P2X7R deficiency was renoprotective compared with wild-type
(WT) controls. Macrophages are the major effector cells in human and experimental
GN, in which P2X7R has a central role in IL-1β secretion and its upregulation
during chronic inflammation, suggesting a pro-inflammatory role for P2X7R in
immune mediated renal injury. Despite numerous studies that have documented
activation of several signalling pathways in response to ATP stimulation in different
cell lines, many questions still remain on the specific contribution of P2X7R in these
processes. Recently, quantitative proteomics of post-translational modification, has
emerged as a powerful tool to study cell signalling providing a quantitative analysis
of the phosphorylated peptides/proteins and on the level of phosphorylation.
Therefore, we used a phosphoproteomic approach to investigate the cell signalling
cascade following ATP stimulation in primary bone marrow derived macrophages
(BMDMs) from WT and P2X7R deficient mice.
Methods: BMDMs from WT and from two P2X7 deficient mouse lines
(GlaxosmithKline and Pfizer) were stimulated with or without 5 mM of ATP at
different time-points: 5-15-30 minutes. Cell pellets were then processed and analysed
by nanoflow-liquid chromatography tandem mass spectrometry (nano-LC—MS/MS).
PESCAL software was used to quantify the intensities of the phosphopeptides present
across all the samples.
Results: Thirty five distinct signalling pathways were activated in macrophages from
WT and P2X7 deficient mice. ATP stimulation led to increased phosphorylation of
Volume 27 | Supplement 2 | May 2012
Abstracts
Nephrology Dialysis Transplantation
15 different proteins within the ribosomal pathway in only macrophages from WT
mice and not in BMDMs from the two P2X7 deficient mice strains. MAPK and
mTOR pathways were also activated involving 20 and 9 proteins respectively.
Similarly, several proteins involved in the regulation of vesicle transport and actin
cytoskeleton organization were phosphorylated in only WT BMDMs.
Conclusions: To our knowledge this is the first report that P2X7R may play a
fundamental role in the activation of the ribosomal pathway and in the regulation of
vesicle trafficking. Furthermore, P2X7R also activates other important signalling
pathways, including MAPK and mTOR, where the inhibition of PI3K has been
shown, in a recent work, to block GN and extends lifespan in a murine model of
systemic lupus. These results may provide novel insights into the underlying
mechanism and cellular pathways affected in the progression of GN from
inflammation to fibrosis.
FP029
TWEAK UP-REGULATES ENDOTHELIN-1 SYSTEM IN HUMAN
ENDOTHELIAL CELLS
Diana Medrano-Andres1, Vanesa Lopez-Martinez1, Patricia Martinez-Miguel1,
Jose Luis Cano2, Ignacio Arribas1, Manuel Rodiguez-Puyol2 and
Susana Lopez-Ongil1
1
Fundacion Para la Investigacion Biomedica Del Hospital Principe de Asturias,
Alcala de Henares, Madrid, Spain., 2Universidad de Alcala, Alcala de Henares,
Madrid, Spain.
Introduction and Aims: Atherosclerosis is currently described as an inflammatory
disease. Vascular lesions are caused by inflammatory and fibroproliferative responses
to injury of the endothelium and vascular smooth muscle cells (VSMCs). Interaction
between members of the tumour necrosis factor (TNF) super family and their
receptors elicits diverse biological actions that participate in atherosclerosis
development. One of these members is the TNF-like weak inducer of apoptosis
(TWEAK), which has different biological functions, including induction of
inflammation, angiogenesis, activation of cell growth, and stimulation of apoptosis.
The receptor of this protein is TWEAKR/Fn14, the smallest reported member of the
TNF super family. TWEAK and Fn14 are expressed in different cell types, including
endothelial and VSMCs. Chronic kidney disease (CKD) is frequently associated with
cardiovascular disease (CVD), especially atherosclerosis, which constitutes one of the
major causes of morbidity and mortality in CKD. Atherosclerosis influence the
progression of renal disease, implying that deterioration of renal function is likely
provoked by atherogenic factors, such as hyperlipidemia. Both TWEAK and its
receptor, Fn14, are present at relatively low levels in healthy adult kidney and highly
induced after injury. Their possible effects on the endothelium are, however,
unknown. As patients with chronic kidney disease (CKD) present endothelial
dysfunction and endothelial cells present Tweak receptors, the following study was
contemplated in order to evaluate the intrinsic effects of TWEAK on the regulation
of vasoactive endothelial factors such as endothelin-converting enzyme-1 (ECE-1) in
endothelial cells.
Methods: Human endothelial cells (EA) were incubated with different doses and
times of TWEAK, to evaluate ECE-1 regulation. Protein levels were analysed by
Western blot, gene levels by Northern blot, the promoter’s activity by transfection
assays and ET-1 production and ECE-1 activity by ET-1 ELISA.
Results: TWEAK induced a dose and time-dependent increase in protein and gene
levels of ECE-1. Results were related to transcriptional changes, as ECE-1 promoter
activity was also increased. Transfections with serial deletions of ECE-1 promoter,
suggest a potential role for AP-1, which was confirmed by EMSA analysis. When
AP-1 activation was inhibited by the specific Erk1/2 inhibitor, PD98059, or a JNK
inhibitor, SP-600125, both were able to block TWEAK stimulation of ECE-1
promoter, suggesting that the effects of TWEAK on ECE-1 could be via AP-1. ET-1
production and ECE-1 activity increased significantly with TWEAK compared to
control cells.
Conclusions: Present results suggest the importance of TWEAK in the regulation of
ET-1 system in endothelial cells, which could be related with its effects on
inflammation, atherosclerosis and progression of renal disease. However, more
studies are needed in animals to study physiological consequences and confirm the
results found in cells.
FP030
ENDOTHELIAL DYSFUNCTION PROMOTES THE TRANSITION
FROM COMPENSATORY RENAL HYPERTROPHY TO KIDNEY
INJURY AFTER UNILATERAL NEPHRECTOMY IN MICE
Hiroyuki Kadoya1, Hajime Nagasu1, Minoru Satoh1, Tamaki Sasaki1 and
Naoki Kashihara1
1
Kawasaki Medical School
Introduction and Aims: Several studies demonstrated that both the endothelium
and endothelium-derived nitric oxide (NO) play critical roles in the compensatory
renal growth by modulating the hemodynamic changes. We and others have reported
that increased renal synthesis of NO is the initial step after unilateral nephrectomy
Volume 27 | Supplement 2 | May 2012
(UNx), which subsequently triggered the hemodynamic changes necessary for the
hypertrophy of the remnant kidney.Endothelial NOS (eNOS) is a major source of
NO. Studies in various animal models demonstrated that impaired availability of NO,
caused by either inhibition or genetic defect of eNOS, accelerates the progression of
kidney disease. Reduced NO bioavailability, characteristic of endothelial dysfunction,
is also widely recognized in hypertensive patients. The hypothesis tested in the
present study was that eNOS and downstream signal-transduction pathway play
crucial roles in compensatory renal hypertrophy after UNx, and that impairment of
NO availability caused by eNOS dysfunction promotes kidney injury rather than
compensatory renal hypertrophy.
Methods: Wild type (WT) and eNOS knockout mice (eNOSKO) were used in this
study. Endothelial- specific eNOS transgenic mice (EC-eNOSTG), generated by
targeting bovine eNOS overexpression to the vascular endothelium under the control
of the murine preproendothelin- 1 promoter were also used in this study. All mice
had the same background of C57BL/6J. Eight-week-old male mice weighing 23-28 g
were used in the study. WT and EC-eNOSTG mice were each randomly divided into
two groups (n=10-12 per group): right nephrectomized mice (WT-UNx,
EC-eNOS-TG-UNx) and sham-operated mice (WT-sham, EC-eNOS-TG- sham).
The eNOSKO mice were randomly divided into five groups (n=10-12 per group): Nx
group (eNOSKO-UNx), sham-operated mice (eNOSKO-sham), Nx group
administrated sGC stimulator (Bay 41-2272) (eNOSKO-UNx+Bay), sham-operated
mice administered Bay (eNOSKO-sham+Bay), and eNOSKO-UNx+Bay mice
administrated mammalian target of rapamycin (mTOR) inhibitor, rapamaycin. Bay
41-2272 (10 mg/kg/day) and rapamycin (1 mg/kg/day) were administrated
postoperatively by intraperitoneal injection once daily for two weeks.
Results: The present study highlighted the importance of the eNOS-sGC pathway in
compensatory renal hypertrophy after UNx. Compensatory renal hypertrophy was
blunted in eNOS-deficient mice and, in marked contrast, was enhanced in
EC-eNOSTG mice. In eNOS-deficient mice, UNx impaired renal function and
induced albuminuria as a result of mal-adaptation. Bay41- 2272, a sGC stimulator,
restored renal function and reduced urinary albumin excretion through the activation
of sGC in eNOS-deficient mice, whereas all these effects were suppressed following
administration of the mTOR inhibitor, rapamycin. These findings indicate that
impairment of NO availability associated with endothelial dysfunction promotes the
switching from compensatory renal hypertrophy to kidney damage.
Conclusions: Our results highlighted the importance of the eNOS-NO-PKG in
compensatory renal hypertrophy after unilateral nephrectomy. NO induced both
renal hemodynamic changes as well as directly stimulated sGC activation in tubular
cells.
FP031
CITRATE REDUCES CALCIUM PRECIPITATION FORMATION
AND PARTICLE INDUCED INFLAMMATION
Emma Lindeberg1 and Gunilla Grundström1
Gambro Research, Lund, Sweden
1
Introduction and Aims: During dialysis calcium precipitates are formed in the fluid
path and some of these particles are likely to reach the patient. Circulating particles
are known to cause inflammation in diseases such as gout and a major part of
atherosclerotic plaques consist of calcium phosphates deposits. Most dialysis patients
suffer from both increased inflammation and atherosclerosis and the aim of this
study was to investigate whether calcium precipitation may contribute to these
conditions. We also wanted to investigate the potential benefits of replacing
remaining acetate in dialysis fluids with citrate. Today citrate is mainly used as
anticoagulant but if used as an active ingredient in dialysis fluids citrate, being a
calcium chelator and antioxidant, has the potential to reduce both particle formation
and particle induced inflammation.
Methods: Calcium precipitates were produced by mixing NaHCO3 (35 mM) and
CaCl2 (1-5 mM) in Phosphate Buffered Saline. The inflammatory response to the
precipitates was evaluated as well as the cytotoxic effect. To evaluate cell toxicity a
murine fibroblast cell line (L929) was exposed to defined amounts of calcium
precipitates for 72 h with or without citrate (1 mM) present. Inhibition of Cell
Growth (ICG) was calculated as a measure of toxicity. The inflammatory response
was investigated by exposing Human Peripheral Blood Mononuclear Cells (PBMCs)
from healthy volunteers to defined amounts of particles for 24 h with or without
citrate (1 mM) present. Inflammation was measured as increased expression of IL-1β
and IL-6 analyzed by ELISA.
Results: The presence of calcium precipitates significantly induced both cell toxicity
and inflammation. Citrate significantly reduced particle formation which did not
occur until CaCl2 concentrations above 3mM with citrate compared to 2 mM CaCl2
without. The magnitude of particle induced cell toxicity was reduced by 30 % by
citrate and higher levels of calcium was tolerated (50 % ICG at 4,1 mM CaCl2 with
citrate vs. 2,4 mM without). Citrate reduced the inflammatory response to calcium
precipitates. CaCl2 EC50 values for both IL-1β (3,4 mM vs. 2,4 mM) and IL-6 (3,3
mM vs. 2,5 mM) were increased with citrate.
Conclusions: In this study citrate was shown to significantly reduce particle
formation and particle induced cell toxicity and inflammation. The results indicate
that citrate in the dialysis fluid may reduce treatment induced inflammation and
atherosclerosis.
doi:10.1093/ndt/gfs213 | ii
Abstracts
FP032
SUPEROXIDE DISMUTASE 1 (SOD1) IN CHRONIC KIDNEY
DISEASE
Scholze Alexandra1, Martin Tepel2, Krueger Katharina3 and Maier Alexandra3
Sdu Ki Odense Denmark, 2Odense University Hospital, 3Charite Berlin Germany
1
Introduction and Aims: Chronic kidney disease is associated with an increased
production of reactive oxygen species. Little is known about the regulation of
antioxidant enzyme protein amount and gene expression in chronic kidney disease
(CKD). The aim of our investigation was to characterize the antioxidant enzyme
SOD1 in CKD.
Methods: SOD1 enzyme characterization was performed in mononuclear leukocytes from
CKD patients (n=150), hemodialysis patients (n=100) and age-matched healthy controls
(n=35). SOD1 gene expression was investigated by quantitative real-time polymerase
chain reaction, protein amount was quantified by in-cell Western assay. SOD1 protein
species were separated using two-dimensional gel electrophoresis. The enzyme function
was assessed by immunfluorescence measurements. Survival analysis for hemodialysis
patients was performed by the Kaplan-Meier method and Mantel-Cox test.
Results: SOD1 gene transcription was increased in CKD and significantly higher in
hemodialysis patients compared to controls. SOD1 protein amount was reduced in CKD
and significantly lower in hemodialysis patients compared to controls. Specific SOD1
enzyme activity was significantly reduced in CKD, most pronounced in hemodialysis.
Two-dimensional gel electrophoresis revealed 6 different SOD1 protein species. These
protein species did not show pronounced differences in molecular weight or isoelectric
point between the groups. Analysis of survival data in hemodialysis patients (mortality of
all causes, mean follow up 12 months) showed a significant survival advantage in those
patients with higher specific enzyme activity.
Conclusions: We show an increase in SOD1 gene expression and a reduction in SOD1
protein amount and specific enzyme activity. Those findings suggest a higher portion of
dysfunctional SOD1 protein in CKD and a compensatory increase of gene expression. In
hemodialysis patients we found an according survival advantage with higher specific
SOD1 enzyme activity.
FP033
SIMVASTATIN INHIBITS ANGIOPOIETIN-2 RELEASE AND
PRODUCTION — A NOVEL PLEIOTROPIC EFFECT?
Chandra C Ghosh1, Sascha David2, Aditit Mukherjee1, Steven G John3,
Christopher W Mcintyre4, Hermann Haller5 and Samir M Parikh1
1
Beth Israel Deaconess Medical Center and Harvard Medical School, 2Medical
School Hannover, Department of Nephrology, 3Royal Derby Hospital, Renal
Medicine, 4Royal Derbe Hospital , Renal Medicine, 5Nephrology
Introduction and Aims: Given the repeated demonstrations that statins can reduce
inflammation, clinical trials are currently under way to evaluate its role in the
management of chronic kidney disease (CKD) patients. Yet, the mechanisms for
statins’ anti-inflammatory effect are incompletely understood. Angiopoietin-2
(Angpt-2) is pre-stored in Weibel-Palade bodies, released under stressed conditions,
and sensitizes endothelial cells to diverse inflammatory stimuli. We therefore
hypothesized that statins may interfere with this pathway.
Methods: We measured circulating Angpt-2 levels in sera from 128 CKD 4-5 patients
(48 on statins) by a commercially available enzyme linked immunosorbent assay (ELISA)
(R&D Systems). Confluent dermal and lung human microvascular ECs (d/l HMVEC),
and human umbilical vein ECs, (HUVEC) were treated with Simvastatin (10-100 μM)
or control vehicle. Angpt-2 was measured in supernatant and cell lysates by ELISA.
Immunofluorescence (IF) was performed using antibodies against human Angpt-2 and
vWF. Total RNA was isolated using the RNeasy Kit (Qiagen) and amplified with a
commercial TaqMan qPCR assay (Applied Biosystems).
Results: CKD 4-5 patients on statins had lower levels of circulating Angpt-2 than CKD
patients without statin treatment. A high amount of Angpt-2 accumulated in the media
in all three EC types, an effect that was not reversible by addition of the Tie2 agonist
Angpt-1. Simvastatin dose-dependently reduced Angpt-2 accumulation at all time points
studied (64% reduction at a dose of 100 μM after 24 h). Intracellular Angpt-2 storage
was also reduced by simvastatin (66% reduction at a dose of 100 μM after 24 h).
Immunofluorescence cytochemistry demonstrated that vWF - a major component of
WPBs - were unaffected by simvastatin whereas Angpt-2 transcript abundance was
reduced 10-fold by the drug.
Conclusions: CKD patients on maintenance statin therapy have lower circulating
Angpt-2 levels. In vitro Simvastatin specifically inhibits the release of pre-stored Angpt-2
as well as the de novo synthesis. Further studies should clarify if this observation might
represent one of the mysterious pleiotropic effects of statins?
FP034
INTEGRIN-LINKED KINASE (ILK) MODULATES CELLULAR
SENESCENCE REGULATING THE EXPRESSION OF
ANTI-AGING GENES
Nuria Troyano1, Maria Del Nogal1, Gemma Olmos2, Ines Mora2, Sergio DE
Frutos3, Manuel Rodriguez-Puyol2 and Maria Piedad Ruiz1
1
Physiology Department, Alcala University, Alcala de Henares, Madrid, España,
2
Universidad de Alcala. Facultad de Medicina. Alcala de Henares. Madrid. Spain,
3
Alcala University. Alcala DE Henares. Spain
ii | Abstracts
Nephrology Dialysis Transplantation
Introduction and Aims: Cellular senescence is a cell cycle arrest related with
alteration of the cell structure and functions. Senescence state can be promoted
by the replicative life of the cells or by stress stimuli. Fibrosis is characteristic feature
in renal aging and Integrin linked kinase (ILK) is a protein involved in the
relationship between cells and surrounding extracellular matrix. It has been
previously described that ILK was increased in old animals. The aim of the
present work was to explore the role of ILK in the cellular senescence induced by
different stressful stimuli in renal cells and the mechanism involved in this process
with the special attention to the regulation of the antioxidant enzymes and Klotho
protein.
Methods: Experiments were performed in vitro and in vivo. In vitro experiments
were made in cultured mouse proximal tubular cells (MCT) and human mesangial
cells (HMC) which were treated with glycated albumin (GA) (100μg/ml) or Glucose
oxidase (GOx) for different periods of time. P53, P16 and ILK protein expression
and activity were measured by inmunoblotting. Cellular senescence was analyzed
measuring the senescence associated b-galactosidase activity by fluorescence.
Down-regulation of ILK was performed by transfection with specific siRNA to
silence ILK. To explore the mechanism involved in senescence process we analyzed
catalase expression by immunoblot and Klotho expression by quantitative RT-PCR.
In vivo experiments were performed in a conditional knock out for ILK obtained by
the CRE- LOX system.
Results: The treatment with GA promoted time-dependent cellular senescence in
MCT and HMC and increases the expression of p53 protein. In the same way, GOx
induce senescence 24 to 72 hours after addition to cells increasing the expression
levels of p53 and p16 proteins. Both stimuli increased the expression and activity of
ILK in a time dependent way. Inhibition of ILK expression with specific siRNA
prevented the increase in p53 and cellular senescence induced by GA or GOx. To
investigate the mechanism involved, the expression of antioxidant enzymes and
Klotho protein was analyzed after GA or GOx addition. Stimuli promoted a fall in
the expression of catalase in HMC and MCT and a down-regulation in the gene
expression of Klotho in MCT. MCT over-expressing Klotho protein did not undergo
to senescence after GA or GOx addition. The role of ILK in renal senescence was
examined in vivo by using mice deficient in ILK. These mice showed an increase
gene expression of Klotho and lower levels of p53 and p16 expression in renal cortex
than wild type mice.
Conclusions: Stressful stimuli such as GA or GOx promoted cellular senescence in
renal cells through the increase in ILK expression and activity. ILK could be
down-regulating the expression of catalase and Klotho proteins and then modulating
the expression of senescence genes such as p53 and p16.
FP035
DIMERIZATION OF THE CALCIUM-SENSING RECEPTOR AND
RESPONSE TO CINACALCET HCL
Hansjörg Rothe1, Hansjörg Rothe2, Warren Shapiro3 and Markus Ketteler4
Klinikum Coburg, Coburg, Germany, 2Klinikum Coburg, Germany, 3Brookdale
University Hospital & Medical Center, 4Klinikum Coburg, Coburg Germany
1
Introduction and Aims: A paradox initial increase in parathormone levels in the
first two hours following administration of cinacalcet HCl was noted in a minority of
patients in the initial dose- response studies. In the meantime, dimerization of
G-protein-coupled receptors including the calcium-sensing receptor (CaSR) has been
described. This leads to the question, whether dimerization might cause receptor/
receptor interactions influencing the dose-response-curves of allosteric activators
such as cinacalcet HCl, with genetic polymorphisms altering these interactions.
Especially heterozygosity in CaSR gene polymorphisms could conceivably influence
such receptor/receptor interactions, since in heterozygotes about 50% of CaSR dimers
are chimeric, while in homozygotes all dimers consist of two identical receptor
molecules.
Methods: Polymorphism status for Ala986Ser, Arg990gGly and Glu1011Gln was
determined in 20 patients on maintenance haemodialysis with secondary
hyperparathyroidism (iPTH levels above 300pg/ml). Baseline corrected serum
calcium and phosphate levels and iPTH levels at baseline and at 1h, 2h, 4h, 6h, 24h
and 48h post one dose of 60mg cinacalcet HCl were measured, as well as cinacalcet
plasma levels at 4h post dose.
Results: In all patients a reduction in iPTH was found after 24h with minimal iPTH
levels after 4h. 12 patients were homozygous for the arginine, 3 for the glycine allele
in position 990, the remaining 5 patients were heterozygous. No polymorphism was
found in position 986, only 1 pt. was heterozygous for Gln1011Glu. After 1h,
heterozygotes showed an increase in iPTH of 66.3 % from baseline (26.29-306.4),
homozygotes showed a decrease of 35.89 % ( 35.21- 92.98) ( p=0.011 ) After 2h,
heterozygotes showed an increase in iPTH of 30.3 % from baseline (-123.8-384.4),
homozygotes showed a decrease of 64.91 % ( 12.63-57.54) ( p=0.046) After 4h, the
difference had levelled off. No significant differences between arginine and glycine
homozygotes were detected at these timepoints. There were also no differences in
plasma cinacalcet levels or baseline corrected calcium or phosphate levels.
Conclusions: Our data show that the previously described paradox initial increase
in parathormone levels in the first two hours following administration of cinacalcet
HCl is associated with Arg990gGly heterozygosity. Since heterozygote individuals
differ from homozygotes in that 50% of their receptor dimers are chimeric, this
paradox effect is probably due to altered receptor/receptor interactions in the CaSR
dimers.
Volume 27 | Supplement 2 | May 2012
Abstracts
Nephrology Dialysis Transplantation
FP035
FP036
THE CALCIUM RECEPTOR CASR AND PARATHYROID
HORMONE DETERMINE THE PARACELLULAR PATHWAY
PERMEABILITY TO CALCIUM IN THE THICK ASCENDING
LIMB.
Ramakrishnan1,
Loupy1
Alexandre
and Pascal
Suresh Krishna
Cordeliers Research Centre, 2Cordelier Research Centre
signaling pathway (ERK phosphorylation).
Methods: Confluent immortalized human MC were incubated during 24 hr,
according to the experimental groups as follows: Control group with standard
glucose concentration (NG; 10mM glucose); HG (30 mM) stimulated group; HG
groups in the presence of 100 nM Losartan (HG+Los) or 100nM Candesartan (HG
+Cand). The mRNA expression levels of PRR, fibronectin and β-actin were
determined through the qPCR technique. PRR, fibronectin, ERK, p-ERK,
angiotensinogen and prorenin protein expression levels were estimated by Western
blot technique. Extracellular levels of AII (culture medium) and the intracellular
content (cell lysate) were quantified by HPLC.
Results: HG increased intra- and extracellular AII levels by 50% and 70%
respectively. The PRR expression increased (50%), p-ERK/ERK (76%), prorenin
(50%) and fibronectin (87%). The upregulation of prorenin, PRR and pERK/ERK
protein expressions induced by HG were completely reversed by Losartan but not by
Candesartan, although both drugs were efficient to reverse the effects of HG on AII
levels.
Conclusion: AII exert a modulatory action on prorenin and PRR genes. Besides the
well described beneficial effects of the cell surface AT1 receptors inhibition (Los and
Can) the additional blockade of the intracellular AT1 receptors (Los) may be
potentially more effective to reduce RAS activity by a mechanism involving a
suppression of PRR gene expression.
FP038
Houillier2
1
Introduction and Aims: Renal tubular calcium absorption is a major determinant of
urinary calcium excretion and, together with net bone calcium release, of blood
calcium concentration. In the kidney, calcium is reabsorbed in the proximal tubule,
the cortical thick ascending limb (cTAL) of the loop of Henle and the distal/
connecting tubule. Renal tubular calcium absorption is negatively altered by the
extracellular calcium concentration, an effect that likely involves the calcium-sensing
receptor (CaSR); conversely, it is positively altered by parathyroid hormone (PTH).
Both factors act in the cTAL where calcium is reabsorbed paracellularly thanks to a
lumen-positive transepithelial voltage and a measurable permeability of the
paracellular pathway to calcium. The objective was to determine the mechanism(s)
by which CaSR and PTH both control calcium absorption in the cTAL.
Methods: The rate of transepithelial calcium, sodium and chloride absorption and
paracellular passive permeability for calcium by the cortical thick ascending limb of
the loop of Henle (cTAL) was measured in invitro microperfused cTAL from male
Sprague-Dawley rats weighting 70-95 g. Calcium, sodium and chloride
concentrations were measured by microfluorometry, microcoulometry, and
ion-exchange chromatography, respectively. Transepithelial voltage was measured by
a differential electrometer.
Results: An allosteric CaSR inhibitor (NPS2143, 1μM in bath) reversibly increased
calcium absorption (4.9±0.43 to 7.2±0.54 pmol/mm/min) without altering the
transepithelial voltage or sodium and chloride absorptions. In addition, NPS2143
reversibly increased the paracellular pathway permeability to calcium (2.2±0.2 to 3.4
±0.5 10-6 cm.s-1). Parathyroid hormone (3.10-10 M in bath) reversibly increased
calcium absorption (4.2±0.3 to 6.1±0.8 pmol/mm/min) without altering the
transepithelial voltage. In addition, when added in the presence of PTH, NPS2143
still reversibly increased calcium absorption (by 0.40±0.01 pmol/mm/min) but its
effect was smaller in the absence of PTH (2.25±0.29 pmol/mm/min).
Conclusions: Both CaSR and PTH directly alter calcium absorption in the cTAL.
Both affect the paracellular pathway permeability to calcium. CaSR and PTH share,
in part, common pathways for their action on the paracellular permeability.
FP037
INTRACELLULAR BLOCKADE OF AT1 RECEPTORS
DOWNREGULATES THE (PRO)RENIN RECEPTOR (PRR)
GENE EXPRESSION IN MESANGIAL CELLS.
Luciana Guilhermino Pereira1, Mirian Boim1, Danielle Aragão1 and
Dulce Casarini1
1
Federal University of São Paulo, São Paulo, Brazil
Introduction: Losartan and Candesartan are two examples of AT1 receptor blockers,
but their biochemical properties make them different in their AT1 blocking capacity.
Candesartan appears to bind tightly to AT1 and remains placed at the cell surface
whereas Losartan, besides binding to cell nmembrane AT1, is sufficiently
hydrophobic to freely penetrate cell membranes. This difference is crucial due to the
presence of cytoplasmic and nuclear AII receptors (AT1 like), thus, Losartan is
probably more efficient to blockade intracellular AII actions. High glucose (HG)
concentration increases intracellular AII content in many cell types including the
mesangial cells (MC). The expression of the (Pro)Renin Receptor (PRR) is also
stimulated by HG contributing to increase AII formation and we have previously
shown that Losartan completely prevented this increase, thus we hypothesized that
intracellular AII may have a role in the regulation of PRR gene activity. In order to
obtain better evidences of this property of intracellular AII, we stimulated MC with
HG and compared the effect of Losartan and Candesartan on PRR expression and its
Volume 27 | Supplement 2 | May 2012
THE ROLE OF INTERLEUKIN-10 IN THE FIBROSIS AND
INFLAMMATION OF UNILATERAL URETERAL OBSTRUCTION
IN MICE
Yuanmeng Jin1, Yuanmeng Jin1 and Nan Chen2
Department of Nephrology, Ruijin Hospital, Shanghai Jiao Tong University,
School of Medicine, 2Ruijin Hospital, Shanghai Jiaotong University
1
Introduction and Aims: Interleukin-10 functions as a general immunosuppressive
cytokine, which down-regulates chronic inflammatory responses through many
mechanisms. Consistent with its role as a suppressive cytokine, recent studies
suggested that IL-10 could inhibit fibrosis in numerous models. Here, we further
investigated the effect of IL-10 in renal tubulo-interstitial fibrosis through unilateral
ureteral obstruction (UUO) model in 8-week old IL-10 gene knockout (-/-) male
mice.
Methods: We performed sham or unilateral ureteral obstruction surgery on
IL-10-/-mice and wild type (WT) male mice and then sacrificed mice on 7 days after
surgery. Collagen deposition and concentration were determined by Picrosirius red
staining and Hydroxyproline assay. Gene and protein expression of fibrotic molecules
and pro-inflammatory pathways activity were quantified by real-time PCR and
Western blot analysis.
Results: The collagen synthesis and deposition in IL-10-/- mice is more serious than
wide type mice after 7 days of UUO operation. Hydroxyproline assay showed that
the collagen content in kidney is increased by 6.24±0.89 folds in IL-10-/- mice after
UUO, compared to 3.3 folds increase in wide type mice with same treatment. The
mRNA levels of collagen I in IL-10-/- mice is up-regulated by 15.43±1.45 folds than
sham-operated mice, compared to 6.03±0.33 folds up-regulation in wide type mice
after UUO. The positive sirius-red staining area was enlarged by 20.18±3.12 folds in
IL-10-/- mice than sham-operated mice, compared to 13.54±1.51 folds increase in
wild type mice after UUO. The fibrotic genes expression, including fibronectin,
MMP-2, a-SMA, FSP-1 and vimentin, was more highly up-regulated in UUO
IL-10-/- mice. IL-10 deficiency also promoted more serious activation of
inflammatory pathways including TGF-β pathway, JAK/STAT3 pathway, NF-kB
pathway which have been verified to play important roles in renal fibrosis progress.
Conclusions: These data constitutes new evidence that IL-10 is implicated in
collagen deposition, fibrotic genes expression and imflammatory pathways activation
in response to ureteral obstruction in mice. Regulation of IL-10 expression levels may
be an useful treatment target for renal tubulo-interstitial fibrosis.
FP039
ANGIOTENSIN II-INDUCED MITOCHONDRIAL NOX4 IS A
MAJOR ENDOGENOUS SOURCE OF OXIDATIVE STRESS IN
KIDNEY PROXIMAL TUBUALR CELLS
Ju-Young Moon1, Yang-Gyun Kim1, Sang-Ho Lee1, Tae-Won Lee2,
Chun-Gyoo Ihm2, Eun-Young Kim1, Hong-Ju Lee2, Jeong-Guk Wi2 and
Kyung-Hwan Jeong2
1
Kyung Hee University Hospital at Gangdong, College of Medicine, Kyung Hee
University, 2Kyung Hee University
Introduction and Aims: Angiotensin II (Ang II)-induced activation of NAD(P)H
oxidase (Nox) leads to increased production of reactive oxygen species (ROS), an
important intracellular second messenger in renal disease. Recent novel findings
suggest that Ang II stimulation induces mitochondrial potential and further amplifies
ROS generation by mitochondria. We examined the hypothesis that ROS injury
mediated by Ang II-induced mitochondrial Nox4 plays a pivotal role in
mitochondrial dysfunction in normal rat kidney epithelial (NRK-52E) cells. In
addition, we assessed whether Ang-(1-7) could counteract Ang II-induced
ROS-mediated cellular injury.
doi:10.1093/ndt/gfs213 | ii
Abstracts
Nephrology Dialysis Transplantation
down SCAP. HK2 cells were also transfected with empty plasmid (NC) or plasmid
with SCAP cDNA. Twenty four hours after transfection, the cells were treated with
serum free, lipopolysaccharide (LPS) 1μg/ml, LDL 200μg/ml and LPS plus LDL for
24 h. The purpose of LPS treatment is to induce inflammatory cytokines production.
Intracellular cholesterol content was assessed by Oil Red O (ORO) staining and
quantitative assay. The mRNA and protein expression of LDLr, HMGCoAR, SCAP
and proinflammatory cytokines tumor necrotic factor-a (TNF-a) and interleukine-6
(IL-6) were examined by real-time quantitative RT-PCR and Western blotting.
Results: Knocking down SCAP significantly decreased while over-expressing SCAP
increased LDLr and HMGCoAR in gene and protein levels. Consequently,
intracellular cholesterol content was significantly reduced after knocking down SCAP
and remarkably increased after over- expressing SCAP as evidenced by ORO staining
and quantitative assay. Interestingly, proinflammatory cytokines TNF-a (3 fold in
gene levels and 2 fold in protein levels vs. control) and IL-6 (2.6 fold in gene and 1.6
fold in protein levels vs. control) significantly increased after over-expressing SCAP.
On the other hand, TNF-a and IL-6 gene expression were decreased up to 80% and
70%, respectively, after knocking down SCAP. As expected, LPS increased TNF-a (2.8
fold) and IL-6 (3 fold) gene expression respectively, while knocking down SCAP
remarkably attenuated the up-regulation of TNF-a and IL-6 by LPS and also reduced
tubular foam cell formation.
Conclusions: SCAP is not merely a cholesterol sensor and regulator but is also
involved in regulation of inflammation response and cytokines production. SCAP
may serve as a new target for anti- inflammation therapy in renal tubular injury.
FP039
FP041
THE UREMIC TOXIN INDOLE-3-ACETIC ACID INDUCES
ENDOTHELIAL COX-2 EXPRESSION BY INCREASING
OXIDATIVE STRESS
Laetitia Dou1, Bertrand Gondouin2, Claire Cerini2, Stephane Poitevin2,
Philippe Brunet2, Francoise Dignat-George3 and Burtey Stephane3
1
Inserm U 1076, Marseille, France, 2Inserm U1076 Université Aix-Marseille,
Marseille, France, 3Inserm U 1076 Université Aix-Marseille, Marseille, France
FP039
Methods: Cultured NRK-52E cells were stimulated with 10-6 M Ang II for 24 h with
or without Ang-(1-7) or apocynin.
Results: Ang II simulated mitochondrial Nox4 and resulted in abrupt production of
mitochondrial superoxide (O2-) and hydrogen peroxide (H2O2). Ang II also induced
depolarization of the mitochondrial membrane potential and cytosolic secretion of
cytochrome C and AIF. Ang-(1-7) attenuated the Ang II-induced mitochondrial
Nox4 activation and apoptosis, and its effect was comparable to that of the NAD(P)
H oxidase inhibitor apocynin.
Conclusions: These findings suggest that Ang II-induced activation of mitochondrial
Nox4 plays a pivotal role in cell survival and the ACE2-Ang-(1-7)-Mas receptor axis
should be investigated further as a novel target of Ang II-mediated ROS injury.
FP040
STEROL REGULATORY ELEMENT BINDING PROTEIN
(SREBP) CLEAVAGE-ACTIVATING PROTEIN (SCAP)
MEDIATES THE INFLAMMATORY STRESS IN RENAL
PROXIMAL TUBULE CELLS
Xiong Zhong Ruan1, Lung-Chih LI1, Zac Varghese2, Jin-Bor Chen3,
Chien-Te Lee4 and John Moorhead1
1
Centre for Nephrology, Ucl Medical School, Royal Free Campus, 2Centre for
Nephrology, Ucl Medical School, Roayal Free Campus, 3Division of Nephrology,
Department of Internal Medicine, 4Division of Nephrology, Department of Internal
Medicine, Kaohsiung Chang-Gung Memorial Hospital
Introduction and Aims: Sterol regulatory element binding protein (SREBP)
cleavage-activating protein (SCAP) is an endoplasmic reticulum protein that regulates
LDL receptor (LDLr) and 3-hydroxy-3- methylglutaryl coenzyme A reductase
(HMGCoAR) transcription and maintains the intracellular cholesterol homeostasis.
We have previously shown that dysregulation of SCAP/ SREBP2, together with
inflammatory stress contribute to renal glomerulosclerosis and tubulointerstitial
fibrosis by increasing renal lipid rich foam cell formation. However, the cross-talk
between SCAP and inflammatory response in renal tubular cells remains unclear.
The aim of this study is to investigate if cholesterol sensor SCAP regulates
inflammatory stress and causes renal tubular injury.
Methods: Human renal proximal tubule cell lines (HK2 cells) were transfected with
negative conrol (NC) or small interfering RNA (siRNA) against SCAP to knock
ii | Abstracts
Introduction and Aims: Patients with chronic kidney diseases (CKD) have a high
risk of cardiovascular diseases, associated with inflammation, endothelium
dysfunction, and oxidative stress. Our hypothesis is that uremic toxins retained in
these patients are involved in these processes. Indoleacetic acid (IAA) is an indolic
protein-bound uremic toxin. Here, we hypothesized that IAA causes endothelial
inflammation and oxidative stress in vitro by inducing reactive oxygen species (ROS)
production and subsequent cyclooxygenase -2 (COX-2) expression.
Methods: Oxidative stress induced by IAA in cultured human umbilical vein
endothelial cells (HUVEC) was assessed by measuring the production of reactive
oxygen species (ROS) by cytofluorimetry. COX-2 expression was measured by
comparative RT-PCR. We also investigated whether the signaling pathways p38
MAPK was involved in COX-2 expression.
Results: IAA significantly increased ROS production in HUVEC. We then showed
that IAA induced endothelial p38 phosphorylation, which was inhibited by the
antioxidant N-acetyl cysteine (NAC), demonstrating that ROS induce p38
phosphorylation. IAA increased COX2 mRNA, which was inhibited by NAC and by
the MAPK p38 inhibitor SB203580, supporting that COX-2 transcription was
mediated by ROS and subsequent p38 activation. Finally, we demonstrated that the
COX-2 inhibitor NS-398 decreased ROS production, supporting the participation of
COX-2 in ROS production.
Conclusions: The uremic solute indole-3 acetic acid increases ROS production; ROS
then mediate p38 MAPK phosphorylation and stimulation of COX-2 expression,
which in turn produces ROS. Our data suggest a pathway by which IAA may
contribute to sustained oxidative stress and endothelial inflammation in CKD
patients.
FP042
ROLE OF CAVEOLIN-3 IN ALTERED PI3K/AKT SIGNALING IN
SKELETAL MUSCLE OF PATIENTS WITH CKD
Alice Bonanni1, Daniela Verzola1, Davide Maggi2, Giuliano Brunori3,
Antonella Sofia1, Irene Mannucci1, Sara Maffioli4, Barbara Salani4,
Elena D'amato1, Stefano Saffioti1, Alessandro Laudon5, Renzo Cordera4 and
Giacomo Garibotto6
1
Department of Internal Medicine, Nephrology Section, Genoa University, Genoa,
Italy, 2Department of Internal Medicin, Endocrinology Section, Genoa University,
Genoa, Italy, 3Nephrology Section Ospedale Santa Chiara, Trento, Italy,
4
Department of Internal Medicine, Endocrinology Section, Genoa University,
Genoa, Italy, 5Nephrology Section, Ospedale Santa Chiara, Trento, Italy, 6Genoa
University
Introduction and Aims: Recently, a series of changes in muscle post-receptor
signaling of the PI3K/Akt pathway, which lead to decreased phosphorylation of the
downstream effector phospho-Akt ( p-Akt), has been identified in rodent models of
CKD as a major cause for accelerated proteolysis and wasting. However, the initial
signal for defective insulin/IGF-1 action is still unknown.Caveolin family proteins are
the principal components of caveolae, the uncoated invaginations of plasma
membranes. Within this family Caveolin-3 (Cav-3) is specifically expressed in muscle
Volume 27 | Supplement 2 | May 2012
Nephrology Dialysis Transplantation
where it modulates insulin uptake and signalling. In this work, we used both an
ex-vivo (muscle biopsies) and an in vitro approach (myotubes cultures) to investigate
if CKD modifies Cav-3 expression and insulin signaling in skeletal muscle.
Methods: Muscle biopsies (rectus abdominis) were obtained from 21 CKD (Stage V)
patients who underwent the placement of a PD catheter (13M/8F, age 67±11 yrs,
eGFR 8±2 ml/min, HCO3- 25±1 mmol/l,) and from 9 healthy subjects (6M/3F, age
67±4 yrs). Cav-3, IGF-I, p- AKT and Myostatin were examined by
Immunohistochemistry and Western Blot.
Results: Cav-3 expression was significantly decreased (-35%, p<0.01) in muscle
biopsies of CKD patients vs. controls. The p-AKT expression (an index of anabolic
hormone signalling) was reduced by 36% ( p<0.001). Myostatin (a negative regulator
of muscle regeneration) mRNA and protein expressions were enhanced by ~60%
( p<0.05). As a next step, we evaluated whether serum factors may directly account
for the reduced Cav-3 expression. The expression of Cav-3 was evaluated in C2C12
mouse myotubes stimulated with 10% serum of pooled hemodialysis patients (uremic
serum) and normal subjects (normal serum). Myotubes were incubated for 5,18, 24,
30 h and Cav-3 expression was studied by western blot. Compared with controls,
uremic serum inhibited caveolin-3 (-30%, p=0.001), as well as p-Akt (-60%, p<0.05).
Conclusions: The present study identifies a potential mechanism for the altered
intracellular muscle anabolic signal observed in rodent models of uremia and in
muscle of patients with CKD. Cav-3, a cell surface system signal, is markedly
impaired in skeletal muscle of CKD patients and is associated with impaired
intracellular pAkt signaling. The uremic milieu per se is able to reproduce these
findings in myotubes, suggesting that circulating inhibitors of the caveolae system
play a major role to downregulate the PI3K/Akt pathway in uremic patients.
FP043
POTENTIAL RENOPROTECTIVE EFFECT OF CALCITRIOL IN
TUBULOINTERSTITIAL FIBROSIS INDUCED BY A
PROTEINURIC NEPHROPATHY.MODEL.
Edgar Maquigussa1, Mirian Boim1, Carine Arnoni1 and Luciana Guilhermino Pereira1
Federal University of São Paulo, São Paulo, Brazil
1
Volume 27 | Supplement 2 | May 2012
Abstracts
Introduction and Aims: Proteinuria plays a key role in the tubulointerstitial changes
that ultimately lead to renal insufficiency. Increased protein filtration may have direct
toxic effects on tubular epithelial cells, that in turn, undergo to the
epithelial-mesenchymal transition (EMT) acquiring a myofibloblast phenotype. The
activation of the Renin angiotensin system (RAS) - TGF-b axis and NF-?B
production take place in this process, leading to inflammatory infiltration, tubular
atrophy and interstitial fibrosis. Vitamin D (VD) is thought to exert a potential
renoprotective effect in many renal diseases by the inhibiting renin gene expression
and reducing NF-?B production. This study investigated the benefits of Calcitriol, a
synthetic vitamin D analogue treatment on a proteinuric nephropathy model induced
by puromycin.
Methods: Adult male Wistar rats were submitted to uninephrectomy followed by
intraperitoneal administration of puromycin (100mg/kg) or vehicle. After 8 weeks
animals were separated in 2 groups, vehicle (V, n=5) or Calcitriol (VD, 0.5μg/kg,
n=5) treated, for over 4 weeks. After treatment proteinuria levels were determined in
24 hr urine samples and then animals were sacrificed. Remnant kidney was
excised and the expression levels extracellular matrix components (collagen an
fibronectin) and infiltration of macrophages (ED-1) were determined by
immunohistochemistry, The expression levels of EMT markers (a-SMA, FSP1), renin,
TGF-b and its signaling pathway (Smad3 and the inflammatory cytokines IL6
and IL10 were estimated by real time PCR, Western blot and
immunohistochemistry.
Results: Vehicle-treated proteinuric rats developed progressive albuminuria and
tubulointerstitial fibrosis after 12 weeks. Increased collagen deposition and
fibrosis were significantly ameliorated by VD treatment. In addition, VD was
effective to prevent the increase of EMT markers (aSMA, FSP1), and to decrease
macrophages infiltration (ED-1). These effects were paralleled by a reduction in
renin mRNA, and TGF-β /Smad3 protein expressions. VD also prevented the
upregulation of the inflammatory cytokine (IL-6) and increased the
antiinflamatory cytokine (IL-10).
Conclusions: These data demonstrate that the VD analogue Calcitriol may have
an excellent therapeutic potential in this proteinuric nephropathy model by
inhibiting the RAS-TGF-b axis and reducing EMT.
doi:10.1093/ndt/gfs213 | ii