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University of Groningen
Novel roles for syndecan-1 in renal transplantationAdepu, Saritha
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75
Am J Transplant 2014; 14: 2328-‐38
Saritha Adepu Kirankumar Katta
Uwe Tietge Arjan Kwakernaak
Wendy Dam Harry vanGoor Robijn Dullaart Gerjan Navis
Stephan Bakker Jacob van den Born
Chapter 4
Hepatic syndecan-‐1 changes associate with dyslipidemia after renal transplantation.
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ABSTRACT
Syndecan-‐1 is a transmembrane heparan sulfate proteoglycan present on hepatocytes
and involved in uptake of triglyceride-‐rich lipoproteins via its heparan sulfate (HS)
polysaccharide side chains. We hypothesized that altered syndecan-‐1 metabolism could
be involved in dyslipidemia related to renal transplantation. In a rat renal
transplantation model elevated plasma triglycerides were associated with fivefold
increased expression of hepatic syndecan-‐1 mRNA (p<0.01), but not protein. Expression
of syndecan-‐1 sheddases (ADAM17, MMP9) and heparanase was significantly
upregulated after renal transplantation (all p<0.05). Profiling of HS side chains revealed
loss of hepatic HS upon renal transplantation accompanied by significant decreased
functional capacity for VLDL binding (p=0.02). In a human renal transplantation cohort
(n=510), plasma levels of shed syndecan-‐1 were measured. Multivariate analysis
showed plasma syndecan-‐1 to be independently associated with triglycerides
(p<0.0001) and inversely with HDL cholesterol (p<0.0001). Last, we show a physical
association of syndecan-‐1 to HDL from renal transplant recipients (RTRs), but not to
HDL from healthy controls. Our data suggest that after renal transplantation loss of
hepatic HS together with lipoprotein-‐bound syndecan-‐1 hampers lipoprotein binding
and uptake by the liver contributing to dyslipidemia. Our data open perspectives
towards improvement of lipid profiles by targeted inhibition of syndecan-‐1 catabolism
in renal transplantation.
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INTRODUCTION
Increased plasma levels of triglyceride rich lipoproteins (TRLs) is common after renal
transplantation and conceivably contribute to the increased cardiovascular disease risk
consistently observed in these patients (1-‐3). A persistent increase in plasma
triglycerides can result into cardiovascular disease (4-‐6). TRLs and chylomicron
remnants from the circulation can enter the space of Disse in the liver where they are
cleared by endocytosis, facilitated by receptors present on hepatocytes (7). The
clearance of TRLs in liver is takes place by most well-‐known LDL receptor (LDLR)
pathway (8, 9), and also a recent player in the field via HS dependent pathway that is
syndecan-‐1 mediated uptake (10). The importance of syndecan-‐1 mediated uptake and
clearance of TRLs has been shown in syndecan-‐1 KO mice studies where the presence of
the LDLR could not compensate for lack of syndecan-‐1 in TRL clearance (11). Among all
HS proteoglycans present in liver, syndecan-‐1 is the primary HS proteoglycan mediating
hepatic clearance of TRLs (11-‐13). On the other hand, it has also been reported that
very high level overexpression and subsequently increased shedding of liver syndecan-‐
1 result in increased plasma levels of triglycerides and cholesterol in mice (14).
Syndecan-‐1 is a major type I transmembrane HS proteoglycan. It consists of a core
protein, which can be divided into a long ectodomain, a conserved transmembrane
domain and a short cytoplasmic domain. The ectodomain provides attachment sites for
HS distally and eventual chondroitin sulfate at the proximal end (15,16). It also contains
protease-‐sensitive cleavage sites for various matrix metalloproteinases (MMPs),
resulting in release and shedding of (parts of) the ectodomain of syndecan-‐1 (17-‐19).
Syndecan-‐1 is predominantly expressed on epithelial cells in a basolateral pattern (20),
on plasma cells (21) and abundantly on the sinusoidal basal surface of hepatic
parenchymal cells (22). Via its HS polysaccharide side chains syndecan-‐1 acts as a co-‐
receptor and is involved in diverse processes such as triglyceride clearance (11), cell-‐
cell adhesion, migration, wound healing, growth factor binding, inflammation, and
binding of chemokines (23-‐27). The core-‐protein of syndecan-‐1 is implicated in
adhesion, angiogenesis, raft-‐dependent endocytosis and response to IGF-‐1 (28-‐30).
Under pathological conditions the expression and shedding of hepatic syndecan-‐1 may
become abnormal. Serum syndecan-‐1 levels are increased in patients with
hepatocellular carcinoma (31). In patients with chronic hepatitis C serum levels of
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syndecan-‐1 serves as non-‐invasive marker to predict liver fibrosis (32).
Immunohistochemical analysis of syndecan-‐1 in liver specimens of patients with
chronic cholestatic liver disease and in primary hepatic stellate cells revealed increased
expression of syndecan-‐1 which might contribute to matrix deposition leading to
fibrosis (33). Adenovirus-‐mediated hepatic overexpression of syndecan-‐1 in mice is
associated with hyperlipidemia (14).
Recently, we showed in renal transplant recipients (RTR) an increased expression of
renal tubular epithelial syndecan-‐1, which as co-‐receptor for growth and survival
factors, was associated with epithelial repair and improved graft outcome (34). In RTR,
loss of renal function is often associated with dyslipidemia (1-‐3). A persistent increase
in plasma triglycerides can result in atherosclerosis and adds to the highly increased
cardiovascular risk in these patients (6,9,35). However, besides renal changes in
syndecan-‐1 in RTR, potential alterations in hepatic syndecan-‐1 metabolism have not
been addressed thus far. We therefore evaluated in a rat renal transplantation model
the expression of hepatic syndecan-‐1 and its sheddases, heparanase and SULF2, and we
profiled HS side chains in rat livers from control and renal allograft recipients. To reveal
whether these changes have clinical relevance we measured plasma levels of shed
syndecan-‐1, and associated these values with parameters of lipid metabolism in a
human renal transplantation cohort, and we also checked for physical association of
shed syndecan-‐1 with plasma lipoproteins. Our results suggest hepatic loss of HS and
syndecan-‐1 shedding to be implicated in dyslipidemia in RTR, opening the possibility
for novel therapeutic intervention strategies aimed at blocking syndecan-‐1 shedding in
transplantation patients.
MATERIALS AND METHODS
Rat renal transplantation model
In this study 5 to 10 weeks old inbred female Dark Agouti rats (donors) and 5 to 10
week old inbred male Wistar Furth rats (recipients) were used. Dark Agouti and Wistar
Furth rats were obtained from Harlan (Horst, The Netherlands) and Charles River
Laboratories Inc. (I’Arbresle, France; Wilmington, MA) respectively. All animals
received care in compliance with the Principles of Laboratory Animal Care (NIH
Publication No.86-‐23, revised 1985), the University of Groningen guidelines for animal
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husbandry and the Dutch Law on Experimental Animal care, and were approved by the
ethical committee on animal experiments of the University of Groningen. Female Dark
Agouti kidneys were orthotopically transplanted into male Wistar Furth recipients as
described previously (n=5) (36). Non-‐transplanted, sham-‐operated age and sex
matched Wistar Furth rats served as control group (n=5). Recipients received
cyclosporine A (Sandimmune, Novartis, Basel, Switzerland; 5mg/kg BW)
subcutaneously for the first 10 days after transplantation. The contralateral kidney was
removed 12 to 14 days after transplantation. Total follow up was 9 weeks. The
transplanted animals showed loss of renal function and developed proteinuria and
hypertension. Blood plasma obtained at baseline, 4 and 8 weeks after transplantation,
was analyzed for urea, creatinine and triglycerides on a multi test analyzer system
(Roche Modular; F.Hoffmann-‐La Roche Ltd, Basel, Switzerland). Kidney and liver tissues
were obtained 9 weeks after transplantation and cryopreserved for RNA isolation and
histology.
Quantitative reverse transcriptase (qRT-‐) PCR
Total RNA was isolated from liver tissues of RTR group and control rats using RNeasy
mini kit; 1µg of RNA was reverse transcribed to cDNA using QuantiTect Reverse
Transcriptase Kit (Qiagen, Germany) according to the manufacturer’s instructions. To
detect the expression of selected target genes QuantiTect Primer Assay (Qiagen) on
demand primers were used. Endogenous GAPDH was used as housekeeping gene along
with the following primers: syndecan-‐1, ADAM17, MMP9, Sulf2, heparanase-‐1 and -‐2,
albumin, apolipoproteins B, E and AV, and the LDL receptor. Real time polymerase chain
reaction (PCR) was performed by CFX384 touch real-‐time PCR (Bio-‐Rad CA,USA) with
SYBR Green I dye (Bioline, Dublin, Ireland) according to the manufacturer’s
instructions. Fluorescent data were converted to cycle threshold (CT) values. Relative
mRNA levels were calculated as 2-‐∆CT, in which ∆CT is CT gene of interest – CT
housekeeping gene.
Dot Blot Assay
Proteins were isolated from cryosections of livers of RTR and control rats with RIPA
buffer (Santa Cruz sc-‐24948, Dallas, Texas, USA). Tissue was lysed on ice, re-‐suspended
and undissolved material was spun down. Protein concentration was determined with
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the Pierce BCA protein Assay kit (Product # 23227 Thermo scientific, Waltham, MA,
USA). 1.25µg/µl of protein was blotted on an activated polyvinylidene difluoride
membrane with the aid of a BIO-‐DOT (BioRad, Hercules, CA, USA) device. After blotting
the membrane was dried, incubated with methanol for 1 min and washed three times
with demi water. Thereafter the membrane was blocked for endogenous peroxidase
activity with 3% H2O2 in water for 10 min, followed by overnight blocking with 5%
skimmed milk in TBS + 0.05% Tween-‐20. The membrane was incubated for 1 h with
anti-‐syndecan-‐1 N18 antibody (sc7100, Santa Cruz Biotechnology INC), followed by
rabbit anti goat HRP (DAKO Glostrup, Denmark) secondary antibody. Detection and
quantification were done with Western Lightning Ultra from PerkinElmer
NEL112001EA (Waltham, MA, USA). Data are expressed as fold increase compared to
the control group. As a positive control human recombinant syndecan-‐1 was spotted on
the membrane. Wells without any spotted sample, but incubated with anti-‐syndecan-‐1
and HRP-‐labeled secondary antibody served as negative controls.
Immunofluorescence
Four µm liver cryosections were fixed in acetone and blocked for endogenous
peroxidase activity with 0.03% H2O2 followed by blocking with 5% bovine serum
albumin in phosphate buffered saline (PBS), with normal goat serum or normal rabbit
serum. Sections were incubated for 1hr with goat polyclonal anti-‐syndecan-‐1 (N-‐18,
1:100 in PBS), rabbit polyclonal anti-‐LDLR (Pab8804; Abnova, Taipei city, Taiwan) and
mouse anti-‐HS mAbs 10E4, JM403, and 3G10 (37-‐39). Binding of primary antibodies
was detected by incubating with rabbit anti goat Ig-‐HRP (DAKO, 1:100 in PBS), goat
anti-‐mouse IgM-‐HRP (Southern Biotech, Birmingham, AL) or rabbit anti mouse Ig-‐HRP
(DAKO) 1:100 in PBS for 30 min. HRP activity was visualized using the
Tetramethylrhodamine System (PerkinElmer). DAPI was used to stain the nuclei. Liver
staining was quantified in ten randomly taken photomicrographs at 200X magnification
by using Mac Biophotonics ImageJ program (Rasband,W.S., ImageJ, U.S. National
Institute of Health, Bethesda, MD). Data is expressed as fold increase compared to the
control group.
Ligand binding assay
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To detect binding capacity of hepatic HS proteoglycans to bind the HS-‐binding proteins
FGF2 and L-‐selectin, a ligand binding assay on hepatic tissue sections was performed as
described previously (36). HS epitope requirements for binding of FGF2 and L-‐selectin
have been defined before (40,41). To confirm that the observed binding pattern (for
both FGF2 and L-‐selectin) was specifically mediated by HS proteoglycans, the sections
were pretreated with 0.05 U/ml heparitinase I (Flavobacterium heparinum, EC 4.2.2.8;
Seikagaku, Tokyo, Japan) for 1h at 37°C in a humidified chamber.
Labelled VLDL binding assay
VLDL fractions were isolated from control healthy human plasma by density gradient
ultra-‐centrifugation. 1milliliter of purified VLDL (1mg/ml) was added to 2 ml of
lipoprotein deficient plasma and was incubated with 100µl (3mg/ml) DiI (Sigma
Aldrich, St.Louis, MO, USA) for 8 hrs at 37ºC. VLDLs were re-‐isolated by density gradient
ultra-‐centrifugation by adjusting with KBr. The re-‐isolated VLDL-‐DiI fraction was
dialyzed against PBS overnight to remove KBr. DiI labelled VLDL were incubated on
4µm thick liver cryosections from RTR and control rats for 2hrs at room temperature
and after washing mounted with vectashield DAPI (DAKO). Quantification of VLDL
binding was done as described above.
Renal Transplant recipient cohort and healthy individuals
A number of 510 stable RTR with a functioning graft for >1 year were included in the
study. Data were collected between August 2001 and July 2003 at a median of 6 years
after transplantation. All patients available for analysis signed written informed
consent. Approval for this study has been obtained by the Institutional Review Board
(METC 2001/039). Detailed description of this study has been published before (42-‐
44). Plasma from 21 healthy sex-‐ and age-‐matched volunteers (age 48±7 years, 38%
females) was used as controls.
ELISA
Plasma syndecan-‐1 was determined in RTR and control samples using sCD 138
sandwich ELISA kit (Diaclone, Besancon, France) according to the manufacturer’s
instructions.
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Lipoprotein isolation
One milliliter of plasma from four RTR and two control individuals were subjected to
fast protein liquid chromatography size exclusion gel filtration using a Superose 6
column (GE Healthcare, Uppsala, Sweden) as described (45). Samples were
chromatographed at a flow rate of 0.5 ml/min, and fractions of 500 µl each were
collected. Syndecan-‐1 was determined in the respective lipoprotein fractions by ELISA
as detailed above.
Statistics
Between-‐group differences in the rat transplantation experiment were tested with the
Mann-‐Whitney U test. Statistical calculations in the human transplantation study were
done using version 20 of SPSS (Inc. Chicago, IL). Data are given as means ± SD or as
medians (IQ ranges). Log-‐transformed data are shown for nonparametrically
distributed data. Transplantation patients were divided into sex stratified tertiles based
on plasma syndecan-‐1 levels. Analyses were performed by analysis of variance and by
Kruskal-‐Wallis test where appropriate. Besides, univariate analysis was done to show
associations between syndecan-‐1 and other continuous variables. To determine
whether plasma syndecan-‐1 was independently associated with triglycerides and HDL
cholesterol linear regression analysis was performed with log plasma triglycerides as
the dependent variable in one analysis and HDL cholesterol as the dependent variable in
the other (model 1), with adjustments for recipient age, sex and body mass index
(model 2), followed by adjustments for renal function (model 3), donor age, donor type,
CMV seropositivity, type of dialysis, duration of dialysis (model 4), for diabetes, use of
statins and liver function parameters (model 5) and for plasma CRP (model 6). Two-‐
sided p-‐values < 0.05 were considered statistically significant.
RESULTS
Liver syndecan-‐1 mRNA, but not protein is increased in RTR group
In a rat model of renal transplantation, chronic transplant dysfunction developed over
time as indicated by gradually increasing plasma levels of creatinine and urea (Fig. 1A
and B). As a consequence of progressive renal failure, the allografted rats developed
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dyslipidemia, as indicated by increased plasma triglyceride values at week 8 after renal
transplantation (Fig. 1C).
Figure 1: Progressive renal dysfunction in experimental renal transplantation resulted in hypertriglyceridemia. Renal transplantation in rats led to progressive renal function loss evidenced by plasma creatinine (A) and plasma urea (B) levels over time. In the same timeframe rats developed hypertriglyceridemia (C). P-‐values are indicated in the graphs.
Renal transplantation did not change hepatic lipoprotein synthesis but receptors involved in TRL uptake are up-‐regulated. No differences were observed in apolipoprotein mRNA expression between livers of RTR and control rats evidenced for ApoE (2A), ApoB (2B) and ApoAV (2C). Hepatic lipoprotein lipase also did not differ between the groups (2D), however the receptors involved in TRL uptake, LDLR (2E) and syndecan-‐1 (2F) are significantly upregulated in livers of RTR rats compared to control rats. Data are expressed relative to GAPDH expression. P-‐values are indicated in the graphs.
Given the key role of the liver in TRL metabolism, nine weeks after renal
transplantation, by qRT-‐PCR we evaluated the mRNA expression of apolipoproteins
with a link to the metabolism of apoB-‐containing lipoproteins apoE, apoB and apoAV,
triglyceride hydrolysis (lipoprotein lipase) and TRL uptake receptors (LDLR and
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syndecan-‐1) in the liver. Results are given in Fig. 2 and show that renal transplantation
did not change hepatic mRNA expression of major apolipoproteins with a link to the
metabolism (Fig.2A-‐2C), nor lipoprotein lipase expression (Fig 2D). Despite the
increased plasma triglyceride levels, the mRNA expression of both TRL uptake
receptors LDLR and syndecan-‐1 were significantly increased in the livers of RTR rats
(Fig. 2E and F). Therefore, we next determined syndecan-‐1 localization and protein
expression in the livers of control Wistar Furth and RTR rats. Both in control and RTR
rats, syndecan-‐1 was exclusively expressed in the sinusoids of the liver (Fig. 3A and B
respectively). No syndecan-‐1 expression was seen in blood vessels, bile ducts and
Kupffer cells. Staining intensity of syndecan-‐1 was not significantly changed in RTR as
compared to control rats (Fig. 3C). In addition, also no differences in protein levels of
syndecan-‐1 were found in hepatic lysates of RTR and control rats using semi-‐
quantitative dot-‐blot analysis (Fig. 3D and E).
Liver syndecan-‐1 sheddases are increased in RTR group
As there is a difference in hepatic syndecan-‐1 mRNA expression between RTR and
control groups, which however was not reflected at the protein level, we next evaluated
hepatic mRNA expression profiles of the known syndecan-‐1 sheddases ADAM17 and
MMP9 (Fig. 4A and B). Both sheddases were significantly up regulated in the RTR
compared to the control group. As it has been described that shedding of syndecan-‐1 is
promoted by heparanase (46), we further evaluated the expression of heparanase-‐1 and
its homologue heparanase-‐2, which were indeed both significantly increased in RTR
rats (Fig. 4C and D). Taken together, these data indicate increased hepatic syndecan-‐1
shedding after experimental renal transplantation.
Hepatic HS side chains are largely lost in RTR
Since HS side chains of liver syndecan-‐1 are involved in binding and uptake of TRLs, we
profiled for HS polysaccharide structures to evaluate potential changes/modifications in
HS structure in livers of the RTR group. In a combined approach we used anti-‐HS
mAbs10E4, JM403, and 3G10 to visualize the respective HS epitopes as well as ligand
binding assays for the known HS-‐binding proteins FGF2 and L-‐selectin. Liver staining
with anti-‐HS mAbs 10E4, JM403 and 3G10 (Fig. 5A-‐C) showed a significant reduction in
staining intensity in livers of RTR group compared to controls. Consistent with these
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data also the binding capacity of hepatic HS for FGF2 was significantly reduced in the
RTR compared to the control group (Fig. 5D). Hepatocyte HS did not bind L-‐selectin
(data not shown) either in control or in RTR livers, despite L-‐selectin
Protein levels of hepatic syndecan-‐1 did not differ between RTR and control rats. Sinusoids in the livers of control (3A) and RTR rats (3B) showed comparable distribution and staining intensity for syndecan-‐1. Quantification of hepatic syndecan-‐1 protein by digital image analysis (3C) and by dot blot assay (3D and 3E) did not reveal differences between control and RTR rats. Sinusoids in the livers of control (3F) and RTR (3G) also showed expression of LDLR, with increased expression in RTR rats (3H) Scale bar in A and B is 50 µm. Data in C and E are expressed relative to controls, which was set to 1.
binding with basement membrane HS in the same liver sections. We also evaluated by
qRT-‐PCR the expression of SULF2, an exo-‐sulfatase that removes 6-‐O sulfates from HS
and was found to be increased in the liver under diabetic conditions (47,48). However,
no significant difference was found in hepatic SULF2 expression between control and
RTR rats (not shown), suggesting that loss of hepatic HS-‐staining is not related to
increased SULF2 activity.
Reduced binding of VLDL to hepatic HS in RTR
3B
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We next aimed to investigate if the apparent reduction of hepatic HS after renal
transplantation also translates into a difference in the binding capacity of hepatic HS for
lipoproteins. Incubation of liver sections with DiI-‐labelled VLDL showed a significantly
reduced HS binding in liver sections of the RTR group (p=0.02; Fig. 5E). All VLDL
binding was prevented by pretreatment of the sections with heparitinase (not shown).
Based on these findings we conclude that renal transplantation results in loss of hepatic
HS and a subsequently reduced lipoprotein binding capacity in RTR rats.
Increased plasma syndecan-‐1 in RTR associates with dyslipidemia
To translate the relevance of the results generated in the rodent model into a clinical
setting plasma syndecan-‐1 was measured in a cross-‐sectional cohort of 510 RTR and
associated to clinical parameters. In 43 of these patients, syndecan-‐1 concentrations
were below the detection level therefore set at the lower limit of 8ng/ml. Median
plasma concentration was 27 (16-‐42) ng/ml. This is significantly higher than median
Liver syndecan-‐1 sheddases are increased in RTR. The mRNA expression levels of syndecan-‐1 sheddases ADAM17 (A), MMP9 (B), Heparanase-‐1 (C) and Heparanase-‐2 (D) are upregulated in RTR livers compared to control livers. Data are expressed relative to GAPDH expression. P-‐values are indicated in the graphs.
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values in sex-‐ and age-‐matched healthy control individuals, namely 16 (10-‐36) ng/ml,
p<0.05, indicating increased syndecan-‐1 shedding in renal transplantation patients.
Table 1 shows sex stratified tertiles of syndecan-‐1, and the respective associations with
dyslipidemia and renal function. Plasma syndecan-‐1 was positively associated with
triglycerides, ApoB and serum creatinine, and inversely with HDL cholesterol. These
associations were also analyzed by univariate analyses showing significant associations
between continuous variables (Table 1, last column). Since the differences in
triglyceride, ApoB, and HDL values in the three syndecan-‐1 tertiles are relatively small,
we more extensively analyzed these associations by regression analysis. Table 2 shows
the multivariate regression analysis of syndecan-‐1 as independent variable with either
plasma triglycerides, or HDL cholesterol as dependent variable upon adjustment for
clinical parameters (age, sex and body mass index), renal function, transplant related
variables, diabetes, and liver function. Even after adjustment for all these co-‐variables,
the association between plasma syndecan-‐1 and triglycerides or HDL cholesterol
remained very robust (in both analyses: p<0.0001). The association of plasma
creatinine and ApoB with syndecan-‐1 levels was lost in multivariate analysis. These
data imply that in RTR plasma syndecan-‐1 is independently positively associated with
triglycerides and inversely with HDL cholesterol.
Physical association of plasma lipoproteins with shed syndecan-‐1
In order to see whether shed syndecan-‐1 in human plasma is physically associated with
lipoproteins, we selected four RTR with high plasma levels of syndecan-‐1 (median 237
ng/ml) and two control individuals with normal (<20 ng/ml) plasma syndecan-‐1 values.
Lipoproteins were isolated by FPLC and syndecan-‐1 was determined by ELISA in both
HDL and LDL fractions. Very low protein content of VLDL fractions did not allow
evaluation in ELISA. The Results showed that shed syndecan-‐1 was abundantly present
in HDL from RTR patients, whereas in both control HDL fractions no syndecan-‐1 was
detected (Table 3). In LDL fractions low amounts of measurable syndecan-‐1 was
present both in control and RTR samples, however without apparent differences.
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Hepatic HS side chains are largely lost in RTR. Hepatic HS side chain profiling using anti-‐HS monoclonal antibodies10E4 (5A), JM403 (5B) and 3G10 (5C) revealed reductions in staining intensity in livers of the RTR compared to the control group. The ligand binding capacity of hepatic HS for FGF2 (5D) and DiI labeled VLDL (E) was significantly reduced in livers of the RTR compared to the control group. For every mAb / HS-‐binding protein representative photomicrographs of control and RTR liver sections are shown, with the corresponding quantification, expressed relative to control values. P-‐values are indicated in the graphs.
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Table 1. Sex stratified tertiles of plasma syndecan-‐1
Tertile I Tertile II Tertile III P-‐value P-‐value
(Univariate
analysis)
N 169 172 169
Men (%) 56 55 56
Plasma syndecan-‐1 men (ng/ml) 14 (8-‐17) 28 (26-‐33) 55 (43-‐75)
Women (%) 44 45 44
Plasma syndecan-‐1 women (ng/ml) 11 (8-‐14) 25 (22-‐28) 51 (37-‐78)
Recipient demographics
Age (years) 53±12 52±13 50±12 0.09 0.06
Body composition
Hip circumference (cm) 99.8±7.6 98.7±9.6 100.1±9.1 0.32 0.91
Waist circumference (cm) 96.4±13.3 96.5±13.5 98.6±13.7 0.22 0.08
BMI 25.8±4.1 25.8±4.1 26.5±4.3 0.19 0.17
Blood pressure
Systolic pressure (mm Hg) 154±23 151±23 153±22 0.41 0.58
Diastolic pressure (mm Hg) 90 ±10 89±10 90±9 0.19 0.74
Mean arterial pressure (mm Hg) 142±16 139±16 141±15 0.23 0.74
Lipids
Triglycerides (mmol/L) 1.74 (1.24-‐2.19) 1.94 (1.49-‐2.48) 2.15 (1.47-‐2.96) <0.0001 <0.0001
LDL cholesterol (mmol/L) 3.56±0.79 3.52±0.98 3.50±0.97 0.81 0.69
HDL cholesterol (mmol/L) 1.19±0.31 1.06±0.34 1.03±0.30 <0.0001 <0.0001
ApoB (g/L) 1.05±0.20 1.08±0.22 1.12±0.25 0.005 0.002
Insulin Conc. (µU/ml) 11.1 (7.6-‐15.7) 11.7(8.2-‐16.3) 10.9(8.4-‐16.4) 0.457 0.267
Liver function
ASAT (U/L) 22 (19-‐27) 22 (18-‐27) 22 (19-‐26) 0.84 0.81
ALAT (U/L) 18 (14-‐22) 17 (13-‐25) 20(14-‐25) 0.21 0.29
Total bilirubin (µmol/L) 18 (15-‐21) 16 (14-‐21) 16 (13-‐21) 0.24 0.56
C-‐reactive protein (mg/L) 1.73 (0.82-‐4.25) 1.88 (0.75-‐5.64) 2.42 (1.03-‐5.15) 0.22 0.26
Renal function
Serum creatinine ( µmol/L) 120 (95-‐157) 127 (99-‐168) 134 (104-‐198) 0.04 0.001
Creatinine clearance (ml/min) 65±20 60±21 61±27 0.12 0.01
Proteinuria > 0.5g (n (%)) 43 (25) 47 (27) 54(31) 0.41 0.04
Transplantation type
Postmortem donor ( n (%)) 149 (88) 150 (87) 143 (84) 0.61 0.38
Living donor (n (%)) 20 (12) 22 (13) 26 (16) 0.61 0.38
*Normally distributed data are given as mean ± SD, skewed distributed data as median (interquartile range) and categorical distributed variables as number (percentage).
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Table 2. Multivariate regression analysis of log plasma syndecan-‐1 with log plasma triglycerides and HDL cholesterol in renal transplant recipients
Log plasma triglycerides HDL cholesterol
Standardized β 95% CI P-‐value Standardized β 95% CI P-‐value
Model 1 0.21 0.07; 0.51 <0.0001 -‐0.21 -‐ 0.11; -‐0.05 <0.0001
Model 2 0.29 0.06; 0.16 <0.0001 -‐0.18 -‐ 0.10; -‐0.04 <0.0001
Model 3 0.18 0.06; 0.15 <0.0001 -‐0.17 -‐ 0.10; -‐0.03 <0.0001
Model 4 0.18 0.05; 0.15 <0.0001 -‐0.18 -‐ 0.10; -‐0.03 <0.0001
Model 5 0.18 0.05; 0.15 <0.0001 -‐0.18 -‐ 0.10; -‐0.40 <0.0001
Model 6 0.18 0.05; 0.15 <0.0001 -‐0.18 -‐ 0.10; -‐0.03 <0.0001
Log plasma triglycerides and HDL cholesterol were entered as dependent variable in this analyses: Model 1 : Crude Model 2 : Model 1 + adjustments for recipient age, sex and BMI Model 3 : Model 2 + adjustments for creatinine clearance and proteinuria Model 4 : Model 3 + adjustments for donor age , donor type, CMV seropositivity, type of dialysis, duration of dialysis Model 5 : Model 4 + adjustments for diabetes, use of statins , liver function Model 6 : Model 5 + adjustments for log plasma CRP
Table 3: Presence of syndecan-‐1 in plasma and lipoprotein size exclusion fractions, isolated from two control individuals and four RTR patients
Syndecan-1 sandwich
ELISA (ng/mg protein)
Control individuals RTR patients
Plasma
HDL
LDL
<20 (<20-<20)
<8 (<8-<8)
23 (22-24)
273 (217-376)
160 (9-197)
31 (13-45) RTR, renal transplant recipient Results are given as median values (range) DISCUSSION
In this study we show a pathophysiologically relevant decrease in hepatic syndecan-‐1
function in a rat model of renal transplantation. Secondly, we found plasma syndecan-‐1
levels in RTR to be increased and to be associated with dyslipidemia. Despite rather
small differences in triglyceride and HDL values in the three syndecan-‐1 tertiles, the
regression analysis, even after adjustment for many potential confounders, showed
strong associations between plasma syndecan-‐1 and both lipid parameters, which
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indicates that this finding is robust and relevant for lipid metabolism. Since syndecan-‐1
is involved in TRL uptake in the liver, our data indicate that shedding of hepatic
syndecan-‐1 and loss of hepatic HS post renal transplantation hamper proper TRL
uptake by the liver. These mechanisms conceivably contribute to dyslipidemia in RTR
and might provide novel opportunities for treatment of dyslipidemia in RTR and
probably other (renal and/or transplantion) cohorts.
In recent years, the abundant syndecan-‐1 expression in hepatocytes and its crucial role
in TRL clearance was shown (11). Besides, loss of hepatic syndecan-‐1 due to increased
shedding, hampered hepatic TRL uptake, thus leading to hypertriglyceridemia (13).
Others showed that overexpression of hepatic syndecan-‐1 leads to increased plasma
triglyceride levels (14). This suggested to us, that in our study increased plasma
syndecan-‐1 might originate (at least partly) from the liver. In livers of RTR rats we
showed syndecan-‐1 mRNA to be upregulated ~5-‐fold, along with upregulation of
sheddases and heparanases, however no increase in syndecan-‐1 protein levels, despite
a clear loss of hepatic HS side chains. These findings are compatible with increased
hepatic syndecan-‐1 synthesis, followed by syndecan-‐1 HS deglycanation by heparanase
and subsequent shedding (46). As a consequence, TRL cannot be cleared properly by
syndecan-‐1 HS on hepatocytes, resulting in hypertriglyceridemia. This proposed
mechanism may explain the strong independent association of plasma syndecan-‐1 with
triglyceride levels in RTR patients. Since in general decreased HDL cholesterol follows
hypertriglyceridemia, this might explain the inverse correlation of plasma syndecan-‐1
with HDL-‐cholesterol. However, our data would suggest an additional mechanism that
may explain this syndecan-‐1 – HDL relationship. We showed a physical association of
syndecan-‐1 with HDL from RTR, but not from control individuals. We thus speculate
that shed syndecan-‐1 is largely deglycanated and via core-‐protein interaction bind with
HDL proteins such as α1-‐antitrypsin, which by proteomic analysis have been found in
HDL fractions (49) and binds specifically with syndecan-‐1 core protein, but not with HS-‐
glycanated syndecan-‐1 (50). Eventual functional consequences of HDL-‐syndecan-‐1
association are not known yet warrant further investigation.
Which mechanism(s) or pathways are involved in these changes in hepatic syndecan-‐1
metabolism? Increased plasma syndecan-‐1 have been described before in various liver
diseases such as liver fibrosis, chronic hepatitis C infection and liver cancer (31,32).
Moreover, hepatic syndecan-‐1 is increased under conditions of chronic cholestatic liver
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diseases (33). These findings suggest that upregulation of hepatic syndecan-‐1 turnover
is part of a stress response upon various noxi. The mechanism behind this is not clear
yet and needs further research. As described by Siedel (51) allograft transplantation in
itself can create a systemic pro-‐inflammatory condition resulting in shedding by
proteases such as ADAM17 (52) and rise of serum syndecan-‐1 levels. We earlier
documented a pro-‐inflammatory profile of our renal transplantation cohort (43).
Alternatively, urinary loss of albumin in renal transplant recipients might induce
increased albumin synthesis in the liver to compensate albuminuria. Indeed, by qRT-‐
PCR we found increased albumin synthesis in livers of RTR rats compared to control
rats (not shown). It might be possible that in response to urinary protein loss, increased
protein synthesis in the liver is not restricted to albumin alone, but includes many
others proteins including syndecan-‐1 and its sheddases too. However, quantification of
hepatic apolipoprotein synthesis (apoB, -‐E, and -‐AV mRNA expression) did not reveal
any difference between RTR recipient and control rats, arguing against a uniform
upregulation of all hepatic proteins upon renal transplantation. We cannot formally
exclude that increased syndecan-‐1 expression and shedding is a consequence of the use
of immunosuppressive medication, although comparison between patients with the
highest and the lowest plasma syndecan-‐1 values did not reveal any significant
difference in immunosuppressive regimen (not shown). Moreover, in the rat renal
transplantation model, rats only received cyclosporine A in the first ten days after
transplantation, whereas the tissues were harvested nine weeks after transplantation.
Generalized oxidative stress is also increased in RTR (53) and could also be involved in
the changes in hepatic syndecan-‐1 metabolism. Focus of current research is the
identification of the mechanism(s) involved in syndecan-‐1 changes upon renal
transplantation.
The data presented in this study is consistent with the concept that in RTR, liver
syndecan-‐1 synthesis, deglycanation and shedding is increased. Shed syndecan-‐1 ends
up in the plasma and associates with HDL. Hepatic loss of HS side chains hampers
binding and uptake of TRLs and might contribute to dyslipidemia. We speculate that
targeted intervention in syndecan catabolism, e.g. by specific inhibitors of ADAM17 (52)
could be a promising therapy to reduce hepatic dysfunction related to shedding of
syndecan-‐1.
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Acknowledgements
S. Adepu and K. Katta are financially supported by the Graduate School of Medical
Sciences of the University of Groningen. We thank Andre Zandvoort, Annemieke Smit –
van Oosten and Michel Weij for their excellent technical assistance.
Disclosure
The authors of this manuscript have no conflicts of interest to disclose as described by
the American Journal of Transplantation.
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