Modulation of the cellular expression of circulating advanced glycation end-product receptors in type 2 diabetic nephropathy.
ABSTRACT Advanced glycation end-products (AGEs) and their receptors are prominent contributors to diabetic kidney disease.
Flow cytometry was used to measure the predictive capacity for kidney impairment of the AGE receptors RAGE, AGE-R1, and AGE-R3 on peripheral blood mononuclear cells (PBMCs) in experimental models of type 2 diabetes (T2DM) fed varied AGE containing diets and in obese type 2 diabetic and control human subjects.
Diets high in AGE content fed to diabetic mice decreased cell surface RAGE on PBMCs and in type 2 diabetic patients with renal impairment (RI). All diabetic mice had elevated Albumin excretion rates (AERs), and high AGE fed dbdb mice had declining Glomerular filtration rate (GFR). Cell surface AGE-R1 expression was also decreased by high AGE diets and with diabetes in dbdb mice and in humans with RI. PBMC expression of AGE R3 was decreased in diabetic dbdb mice or with a low AGE diet.
The most predictive PBMC profile for renal disease associated with T2DM was an increase in the cell surface expression of AGE-R1, in the context of a decrease in membranous RAGE expression in humans, which warrants further investigation as a biomarker for progressive DN in larger patient cohorts.
[show abstract] [hide abstract]
ABSTRACT: Age-associated increases in collagen cross-linking and accumulation of advanced glycosylation products are both accelerated by diabetes, suggesting that glucose-derived cross-link formation may contribute to the development of chronic diabetic complications as well as certain physical changes of aging. Aminoguanidine, a nucleophilic hydrazine compound, prevented both the formation of fluorescent advanced nonenzymatic glycosylation products and the formation of glucose-derived collagen cross-links in vitro. Aminoguanidine administration to rats was equally effective in preventing diabetes-induced formation of fluorescent advanced nonenzymatic glycosylation products and cross-linking of arterial wall connective tissue protein in vivo. The identification of aminoguanidine as an inhibitor of advanced nonenzymatic glycosylation product formation now makes possible precise experimental definition of the pathogenetic significance of this process and suggests a potential clinical role for aminoguanidine in the future treatment of chronic diabetic complications.Science 07/1986; 232(4758):1629-32. · 31.20 Impact Factor
Article: Interactions between advanced glycation end-products (AGE) and their receptors in the development and progression of diabetic nephropathy - are these receptors valid therapeutic targets.[show abstract] [hide abstract]
ABSTRACT: Diabetes, is a metabolic disorder characterised by chronic hyperglycaemia, hypertension, dyslipidaemia, microalbuminuria and inflammation. Moreover, there are a number of complications associated with this condition including retinopathy, neuropathy and nephropathy. Diabetic nephropathy, is the major cause of end-stage renal disease in Western societies affecting a substantial proportion (25-40%) of patients with diabetes. Advanced glycation end products (AGEs) have been identified as important modulators of the development and progression of diabetic nephropathy, through both receptor dependant and independent interactions. AGEs elicit their receptor mediated effects via their engagement with numerous receptors and binding proteins which are broadly thought to be either inflammatory (RAGE and AGE-R2) or clearance receptors (AGE-R1, AGE-R3, CD36, Scr-II, FEEL-1 and FEEL-2). Modulation of AGE receptor expression is an important potential therapeutic approach worth consideration as a treatment for diabetic nephropathy and likely applicable to other vascular complications.Current drug targets 02/2009; 10(1):42-50. · 3.93 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: The role of chronic hyperglycemia in the development of diabetic microvascular complications and in neuropathy has been clearly established by intervention studies. However, the biochemical or cellular links between elevated blood glucose levels, and the vascular lesions remain incompletely understood. This review focuses on the consequences of hyperglycemia on the formation of advanced glycation end-products (AGEs), and on the role of AGEs and of their specific receptors (RAGE) in the functional and anatomical alterations of the vascular wall. AGEs are formed during the Maillard reaction by the binding of aldoses on free NH(2) groups of proteins, which, after a cascade of molecular rearrangements, result in molecules of brown color and specific fluorescence. Experimental studies have indicated that the binding of AGEs to RAGE activates cells, particularly monocytes and endothelial cells. Activated endothelial cells produce cytokines, and express adhesion molecules and tissue factor. The role of AGEs in increased oxidative stress, and in the functional alterations in vascular tone control observed in diabetes, in part related to a reduction in nitric oxide, is also discussed. The microvascular retinal, glomerular and nerve lesions induced by experimental diabetes in animals are prevented by an inhibitor of AGEs formation, aminoguanidine. The administration in diabetic animals of recombinant RAGE, which hinders AGEs-RAGE interaction, prevents hyperpermeability and vascular lesions. These data suggest a central role of AGEs and RAGE in the development of chronic complications of diabetes.Diabetes & Metabolism 12/2001; 27(5 Pt 1):535-42. · 2.41 Impact Factor
Hindawi Publishing Corporation
Experimental Diabetes Research
Volume 2010, Article ID 974681, 9 pages
Modulation of theCellularExpressionof CirculatingAdvanced
GlycationEnd-Product ReceptorsinType2 DiabeticNephropathy
FeliciaY.T. Yap,1Amy L.Morley,1PhilipE.Morgan,3,4Michael J. Davies,3,4
Scott T. Baker,5,6George Jerums,5,6and Josephine M. Forbes1,2
1Glycation and Diabetes, Diabetes Division, Baker IDI Heart and Diabetes Institute, Melbourne 3004, Australia
2Departments of Immunology and Medicine, Monash University, Alfred Medical Research Education Precinct,
Melbourne 3004, Australia
3Heart Research Institute, Newtown, Sydney 2042, Australia
4Faculty of Medicine, University of Sydney, Sydney 2006, Australia
5Endocrine Centre, Austin Health, West Heidelberg, Victoria 3084, Australia
6Department of Medicine, The University of Melbourne, Melbourne 3010, Australia
Correspondence should be addressed to Karly C. Sourris, firstname.lastname@example.org
Received 27 July 2010; Revised 29 October 2010; Accepted 3 November 2010
Academic Editor: Shi Fang Yan
Copyright © 2010 Karly C. Sourris et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Background. Advanced glycation end-products (AGEs) and their receptors are prominent contributors to diabetic kidney disease.
and AGE-R3 on peripheral blood mononuclear cells (PBMCs) in experimental models of type 2 diabetes (T2DM) fed varied AGE
containing diets and in obese type 2 diabetic and control human subjects. Results. Diets high in AGE content fed to diabetic mice
decreased cell surface RAGE on PBMCs and in type 2 diabetic patients with renal impairment (RI). All diabetic mice had elevated
expression was also decreased by high AGE diets and with diabetes in dbdb mice and in humans with RI. PBMC expression of AGE
R3 was decreased in diabetic dbdb mice or with a low AGE diet. Conclusions. The most predictive PBMC profile for renal disease
associated with T2DM was an increase in the cell surface expression of AGE-R1, in the context of a decrease in membranous RAGE
expression in humans, which warrants further investigation as a biomarker for progressive DN in larger patient cohorts.
Advanced glycation end products (AGEs) are a heteroge-
neous and complex group of biochemical modifications,
which play an important role in the development of chronic
disease processes including the complications of diabetes
. AGEs can be produced endogenously as the result
of hyperglycaemia or other metabolic imbalances and are
also absorbed from foodstuffs, each of which contribute to
the body’s AGE burden. AGEs may elicit their effects via
receptors and binding proteins which are broadly thought to
be either inflammatory (e.g. RAGE) or “clearance” receptors
(such as AGE-R1, AGE-R3, CD36, Scr-II) [2, 3].
The receptor for advanced glycation end products,
RAGE, is a transmembrane receptor, expressed on the cell
surface of a number of circulating and organ-specific
cells including monocytes, macrophages, proximal tubular
cells, podocytes, and mesangial cells [4–6]. Gene splicing
results in three splice variants, including full-length cell
surface RAGE (contains the active binding site, extracellular,
and intracellular domains) and endogenous secretory es-
RAGE (lacks transmembrane and intracellular domain but
contains a secretory tail) [7, 8]. RAGE is also thought to
be proteolytically cleaved from the cell surface becoming
part of the circulating soluble RAGE pool thus considered
to be a decoy receptor [7, 8]. RAGE has been shown
2Experimental Diabetes Research
to be modulated by both AGEs [9, 10] and by diabetes
AGE-R1 is a type 1 integral membrane protein which
is also part of the oligosaccharide transferase complex
. AGE-R1 has a short extracellular domain and a long
cytoplasmic tail and is thought to be involved in both AGE
clearance  and apoptosis via the protein p66shc.
Cellular expression of AGE-R1 involves both circulating
and renal cells including mononuclear, endothelial ,
and mesangial cells . AGE-R1 has been shown to be
downregulated by both diets abundantly rich in AGEs 
and in diabetes [17, 18]. In addition, lymphoblasts from type
1 diabetic patients with progressive diabetic nephropathy
have also exhibited a decrease in AGE-R1 .
In contrast, AGE-R3 (also known as Galectin-3) is a
high affinity AGE-binding protein which is thought to be
primarily based in the cytoplasm and nucleus where it
binds to AGEs as well regulating cell cycle, replication, and
apoptosis . AGE-R3 has been shown to be elevated by
AGEs in cultured endothelial cells and within renal tissues
in the diabetic milieu . In addition, AGE-R3 has also
been shown to be expressed on circulating cells including
macrophages, eosinophils, and mast cells .
Modulation of AGE-receptor expression has been shown
both clinically and in experimental models [7, 8]. In the
present study, we employed flow cytometry as a novel
approach to measure AGE-receptor expression in PBMCs in
type 2 diabetes using both experimental models fed low or
high AGE diets and humans with renal impairment, in order
to assess the relevance of circulating cellular AGE receptor
expression profiles as biomarkers for diabetic nephropathy.
2.1. Experimental Mouse Model of Diabetes. Male Lepr(+/+)
C57BL/KsJ (dbdb) mice were originally purchased from
Jackson Laboratories and randomised at 8–10 weeks of age,
to consume diets low in AGE (LA; AIN-93G unbaked) or
high in AGE content (HA, AIN-93G baked for 1 hour at
160◦C), quantitated by HPLC as previously described .
This mouse strain develops diabetic nephropathy in the
context of severe metabolic syndrome (hypertension, hyper-
lipidaemia, obesity, insulin abnormalities) similar to that
seen in type 2 diabetes in humans. Male Lepr(+/−)C57BL/KsJ
(dbh) littermates were followed concurrently (n = 10/group)
and served as the appropriate control. All groups were
followed for 10 weeks.
At the completion of the study whole blood was isolated
by cardiac puncture. All procedures were in accordance
with the guidelines set out by the Alfred Medical Research
and Education Precinct Animal Ethics Committee and the
National Health and Medical Research Council of Australia.
2.2. Isolation of Peripheral Blood Mononuclear Cells (PBMCs).
Whole blood (∼1mL), was collected in heparinised tubes
(20IU Sodium Heparin). Blood was centrifuged at 500×g,
0.9% (w/v) saline. The whole blood suspension was then
diluted to a final volume of 6mL, underlaid with 3mLs of
Ficoll-Paque (Amersham, Upsalla Sweden), and centrifuged
at 800×g for 25mins to separate the peripheral blood
mononuclear cells. PBMCs were carefully removed from the
interface and transferred to a clean tube and washed twice in
Finally, PBMCs from three mice from the same group were
in PBSF (PBS, 2.5% (v/v) FBS, 0.1% (v/v) NaN3).
To isolate human PBMCs, baseline blood samples were
collected from participants who had provided written
informed consent prior to participation in the Diabesity
Health. Twenty-four-hour albumin excretion rates were
determined by radioimmunoassay, isotopic GFR using99Tc-
DTPA, and HbA1Cby HPLC as previously described in .
The protocol was approved by the Human Ethics Committee
of Austin Health and complied with the Declaration of
Helsinki, 2004. Whole blood (8mLs) from control (obese,
nondiabetic; n = 5), diabetic (obese, diabetic, n = 15) and
diabeticsubjectswithrenalimpairment(RI)(n = 7;Table 2)
was collected in BD CPT (362761, BD Australia) tubes and
processed as per manufacturer’s instructions.
2.3. Flow Cytometric Staining for Advanced Glycation End-
Product Receptors on PBMCs. PBMCs (1 × 106cells/tube),
isolated from experimental models and diabetic patients as
described above, were stained for cell surface receptors with
10μL goat-anti-RAGE (N-16; Santa Cruz, USA), AGE-R1
10μL (mouse) or 5μL (human) OST-48 H-300, Santa Cruz,
USA) for 30mins at room temperature. Cells were then
washed with excess PBSF and centrifuged (500g, 5min, RT).
Supernatant was removed, and the cell pellets were resus-
pended in 100μL PBSF. antigoat (F (ab’2) Chemicon, USA;
1:250) and Antirabbit (AP322F, Chemicon, USA; 1:500)
FITC- conjugated secondary antibodies were added to the
appropriate tubes and incubated at room temperature, for a
further 30mins in the dark. After washing, the supernatant
was removed and cell pellet resuspended in FACS fixative
(2% w/v paraformaldehyde in PBSF). Cells were fixed for
20mins 4◦C and washed with excess PBSF. Other tubes
of PBMCs were also concurrently stained with an isotype
control FITC-conjugated Rat IgG2bk(556923, BD Pharmin-
gen, USA, mouse PBMC) or FITC-conjugated Mouse
IgG1(349526, BD Pharmingen, USA) (human PBMC) and
CD45 FITC (FITC-conjugated rat antimouse CD45, 553079;
FITC-conjugated Mouse antihuman CD45, 555482) for
the determination of background binding and cell type,
For intracellular markers, following fixation PBMCs
were washed and cell pellet resuspended in 100μL 0.3%
w/v Saponin (diluted in PBSF, S4521, Sigma-Aldrich) to
permeabilise the cells. Cells were stained for intracellular
expression of AGE-R1 (OST48 (H-300) Santa Cruz, USA)
and AGE-R3 (5μL for mouse and 10μL for human, AF1197,
R&D Systems, USA), 30mins 4◦C. Cells were washed
once again and resuspended in saponin and stained with
Experimental Diabetes Research3
their appropriate secondary antibody, antigoat (F (ab’2)
Chemicon, USA; 1:250), and antirabbit (AP322F, Chemi-
con, USA; 1:500), at 4◦C for 30min, in the dark. At
completion of the incubation, cells were washed once
again with excess PBSF and cell pellet resuspended in
FACs fixative. For both intra- and extracellular receptors, a
BD Biosciences, USA. Cells were gated according to their
forward and side-scatter properties. Isotype and CD45
FITC controls were also employed for compensation of
the instrument. Data were analysed using WIN MDI v 2.9
cells were identified using histogram analysis and fluores-
cence relative to the secondary antibody alone control.
2.4. Measurement of Nε-Carboxymethyllysine (CML) in
Plasma, Urine, and Rodent Chow. CML was measured in
mouse plasma (1:8000) and urine (1:2) and in human
plasma (1:25600) at their respective dilutions, using an in-
house ELISA that has been previously described in .
In addition, Nε-carboxymethyllysine (CML) levels were
measured in low and high AGE diets using HPLC and
standardised to the amino acid serine (Ser) in that same
sample as previously described in .
2.5. Measurement of Thiamine in Rodent Diet. Thiamine
content was quantitated in 100gms of low and high AGE
diets by Pathwest Laboratories, Nedlands, WA, Australia.
2.6. Statistical Analysis. Data are expressed as means±SD,
unless otherwise stated. Analyses of data were performed
by ANOVA followed by post hoc analysis using Tukey’s test
or nonparametric t-tests. Data for albuminuria were not
normally distributed and therefore analysed after logarith-
mic transformation. P < .05 was considered statistically
3.1. Experimental Model of Metabolic Syndrome and Type 2
Diabetes (the dbdb Mouse). Male Lepr(+/+)C57BL/KsJ (dbdb)
mice were used as a model representative of type 2 diabetes.
Analysis of the low and high AGE diets administered to these
mice revealed that the CML content was 90 or 370nmoles
CML per mole of serine, respectively. In addition, Thiamine
content was found to be 2.61 and 3.51μg/g in the low
and high AGE diets. Renal and metabolic parameters from
this mouse model are shown in Table 1. Dbdb mice had
elevated blood glucose, renal hypertrophy and increased
urinary albumin excretion when compared with dbh mice
irrespective of diet. In addition, a decline in creatinine
clearance and increased plasma cholesterol were seen in high
AGE fed dbdb mice when compared with dbdb mice fed a
low AGE diet (Table 1). Serum CML concentrations were
not altered among any groups. In contrast however, urinary
CML concentrations were significantly elevated in diabetic
dbdb mice (dbh LA, 0.0025 ± 0.0021 versus dbdb LA,
5.654 ± 5.116μmol/mol/lysine/24hrs; P = .0476; dbh HA,
0.0155 ± 0.0120 versus dbdb HA, 3.517 ± 3.588μmol/mol
lysine/24hr, P = .0364).
In heterozygous dbh mice, a diet high in AGE did
not alter cell-surface expression of RAGE on PBMCs
(Figure 1(a)). By contrast, there was a significant loss of
cell surface RAGE expression on PBMCs from high AGE
fed dbdb mice as compared with low AGE fed dbdb mice
(Figure 1(a)). High AGE diets significantly declined the
PBMC cell surface expression of AGE-R1 in both dbdb and
dbh mice (Figure 1(b)), which was not altered by diabetes.
Intracellular levels of AGE-R1 were not altered in the dbh
mice by a high AGE diet (Figure 1(c)). However, dbdb
mice fed a low AGE diet had significantly lower intracellular
expression of AGER1 in PBMCs as compared to both high
AGE fed dbdb mice and low AGE fed dbh mice (Figure 1(c)).
HighAGE dietary intake increasedthe expression of AGE-R3
in dbh and to a lesser extent in dbdb mice. Overall, diabetic
dbdb mice exhibited significantly lower levels of AGE-R3
relative to dbh counterparts.
3.2. AGE-Receptors in PBMCs from Type 2 Diabetic Subjects.
We next investigated AGE-receptor expression on PBMCs
from control, diabetic, and diabetic subjects with renal
impairment, all of whom were obese. Renal and metabolic
parameters for these subjects are shown in Table 2. Type 2
diabetic individuals had a significant increase in HbA1cand
albuminuria tended to increase in concert with renal impair-
ment although this did not reach statistical significance
= .07). Diabetic patients with either a decline in
isotopic GFR to a level <60mL/min/1.73m2or an albumin
excretion rate >200μg/min were included as having early
renal impairment (Table 2). Diabetic individuals with renal
impairment also had lower diastolic blood pressure.
Consistent with the experimental models, cell surface
expression of RAGE and AGE-R1, in addition to intracellular
levels of AGE-R1 and AGE-R3, was readily measured by flow
cytometry in human PBMCs (Figure 2). Diabetes induced
a significant increase in cell surface RAGE expression on
PBMCs, which was significantly reduced in diabetic patients
with renal impairment (Figure 3(a)). By contrast, extracel-
lular AGE-R1 expression was not affected by diabetes per
se; however, PBMCs from diabetic subjects with diabetes
and renal impairment had a significant increase in this
receptor (Figure 3(b)). Diabetes increased the intracellular
PBMC expression of AGE-R1 (Figure 3(c)), while AGE-R3
expression was elevated in type 2 diabetic patients with renal
impairment, consistent with cell surface AGE-R1 expression.
In the present study we have identified that the most
predictive PBMC profile for progressive renal disease in type
2 diabetes in humans was an increase in the cell surface
expression of AGE-R1 in the context of a decrease in cell
surface RAGE. However, in contrast to a number of previous
studies [10, 25], we have not identified increases in circulat-
ing AGE modified protein concentrations in association with
early renal disease, in the diabetic mouse model used, nor
4Experimental Diabetes Research
Table 1: Renal and metabolic parameters in an experimental model of metabolic syndrome and type 2 diabetes the dbdb mouse followed
from weeks 10 to 20 of age. Dbh: non diabetic control mice, dbdb: diabetic mice.
24.2 ± 6.3§
Kidney to body
dbh Low AGE
dbh High AGE
dbdb Low AGE
dbdb High AGE
2608.0 ± 791.1$
2334.3 ± 520.2$
§P < .05 versus dbh Low AGE,#P < .05 versus dbdb Low AGE,$P < .05 versus corresponding dbh group.
Rage positive cells (%)
Cell surface expression
LA HALA HA
AGE-R1 positive cells (%)
Cell surface expression
LAHA LA HA
AGE-R1 positive cells (%)
LA HA LAHA
AGE-R3 positive cells (%)
Figure 1: Flow cytometric analysis for the cell surface expression of (a) RAGE, (b) AGE-R1 and intracellular levels of (c) AGE-R1 and (d)
AGE-R3 on PBMCs in dbh and dbdb mice at week 20 of age. Empty bars: low AGE diet (LA) and filled bars: high AGE (HA) groups.∗P < .05
versus corresponding low AGE group,§P < .05 versus dbh group within the same diet.
in type 2 diabetic individuals. In addition, there appears to
be no association among the expression of AGE receptors
studied on PBMCs and circulating CML concentrations in
4.1. Modulation of Circulating Levels of AGEs. Whilst CML
is one of the most well-characterised AGEs to date, there
are numerous others which have also been well charac-
terised including N-carboxyethyllysine (CEL), pentosidine,
(MGO). Given that HPLC analysis of the rodent chow
failed to detect measurable levels of other AGEs apart from
CML, we chose to focus on that modification. In addition,
the lack of differences in thiamine levels between baked
Experimental Diabetes Research5
Table 2: Metabolic and renal parameters in control and type 2 diabetic subjects. BMI: body mass index, SBP: systolic blood pressure, and
DBP: diastolic blood pressure.
Diabetes + RI
Circulating CML (μmol/mol lysine)
∗P < .05 versus control,†P < .05 versus diabetes,§P = .07 (ns) versus diabetes.
Cell surface markers
Cell surface markers
Figure 2: Representative histograms of flow cytometric analysis of AGE receptors in PBMCs isolated from type 2 diabetic patients. Cell
surface expression of (a) RAGE and (b) AGE-R1. Intracellular (c) AGE-R1 and (d) AGE-R3 within PBMCs. Receptor positive cells (M1 filled
histogram) were identified as those which fluoresced above their relative secondary antibody (empty histogram).
6Experimental Diabetes Research
ControlDMDM + RI
Positive cells (%)
Cell surface markers
ControlDM DM + RI
Positive cells (%)
Cell surface markers
ControlDM DM + RI
Positive cells (%)
Control DMDM + RI
Positive cells (%)
AGE-R1 positive cells (%)
P = .0056
Figure 3: Flow cytometric analysis for the cell surface expression of (a) RAGE and (b) AGER-1 and intracellular levels of (c) AGE-R1and (d)
AGE-R3 on PBMCs of human control, diabetic (DM) and diabetic individuals with renal impairment (DM + RI). (e) Positive correlation
between cell surface expression of AGE-R1 and Albumin Excretion Rate (AER), P = .0056. Empty bars: control, grey bars: diabetes, and
black bars are diabetes with renal impairment (iGFR < 90) groups. nd: not detected.∗P < .05 versus control,†P < .05 versus DM,ξP < .0001
versus control (n = 5–10/group).
Experimental Diabetes Research7
and unbaked chow suggests that the dietary effects demon-
strated in the present study are primarily via differences
in CML levels rather than via depletion of vitamin B. The
ELISA used in the present study for CML only recognises
protein-bound CML (no CML modified peptides or free
CML as are often recognised in many of the assays used pre-
viously), which likely explains the discrepancies between the
present study and others. The plasma to urinary CML ratio
is also important when considering the overall body AGE
burden in humans and in diabetic rodent models. Indeed,
type 2 diabetic dbdb mice have vastly elevated urinary excre-
tion of CML modified proteins in the absence of increases
in serum concentrations of CML. Therefore the kidneys of
diabetic mice in the present study are likely to be exposed
to more CML modified proteins (AGEs) than those kidneys
from nondiabetic mice. Since there were also no differences
in serum concentrations from obese type 2 diabetic subjects
with early renal impairment, it is prudent to suggest that
these individuals may also have an elevated urinary excretion
of CML, although this was not able to be measured in the
present study. Also, rodent models are often homogeneous
within groups, especially for markers of renal function
such as creatinine clearance due to their genetic back-
ground. However, there was significant heterogeneity across
the type 2 diabetic patients with renal impairment with
respect to iGFR, with some patients showing iGFR > 130,
often characteristic of obesity and about half having an
iGFR < 60ml/min/1.73m2
renal impairment. The greatest differences seen within this
group were in the spread of albuminuria which may have
been due to the early nature of the renal impairment or due
to the fact that in some studies in humans, urinary albumin
excretion does not correlate with progressive renal disease
[26, 27]. The concentration of CML-modified proteins in
type 2 diabetes is likely to be altered by a number of
parameters independent of glucose such as insulin levels,
food intake , and hyperlipidaemia , and therefore
CML modified proteins may not be directly responsible for
all of the changes observed in the AGE receptors in type 2
,indicative of more significant
4.2. Modulation of AGE Receptors. Importantly, in the
present study, changes seen in cellular expression of AGE
receptors were closely associated with renal impairment in
AGEs which demonstrate modulation of cellular RAGE
expression. Administration of diets low in AGE content has
been associated with reductions in RAGE expression which
are paralleled by improvements in renal function and insulin
sensitivity in mice [30, 31]. In contrast, in the present study
we found that administration of a diet low in AGE to
type 2 diabetic mice was associated with increases in RAGE
expression on PBMCs along with significant improvements
in renal function. The discrepancy seen with these findings
may be the result of the methodologies employed. In
our present study we have measured cell-surface RAGE
expression on circulating cells by flow cytometry, whilst
other studies have employed immunoblotting, which would
measure all isoforms of RAGE including soluble RAGE,
in addition to both cell surface and cytoplasmic RAGE
cellular content. Indeed our findings suggest that the use of
RAGE antagonists, which are currently under development
for diabetic complications (Phase 2B clinical trials), may
warrant further consideration for those patients with early
renal impairment, given that there may be some temporal
differences in RAGE expression over the course of the
development and progression of diabetic renal disease.
A decline in AGE-R1 expression on PBMCs has been
reported in various chronic diseases [19, 30]. In addition,
dietary AGEs have been shown to modulate renal AGE-R1
expression in mice, although these studies have also mea-
sured AGE-R1 expression by western immunoblot analysis
[9, 30], which does not differentiate between cell surface
or intracellular expression of these receptors. In particular,
this is important for AGE-R1 which has specific intracellular
function as part of the oligosaccharyltransferase complex
involved in N-glycosylation . In our present study, high-
AGE diets induced a decrease in cell surface AGE-R1 in dbdb
and dbh mice, our model of type 2 diabetes, independent
to glucose. In addition, the type 2 diabetic human subjects
studied with renal impairment clearly showed an increase
in AGE-R1 expression on PBMCs, and this correlated with
albumin excretion rate. Indeed, we have demonstrated,
for the first time, that the cell-surface and intracellular
expression patterns of AGE-R1 in circulating PBMCs differ
and in fact may be opposite to what has been previously
reported regarding diabetes-induced early renal impairment
in humans. This may be due to the fact that the patient
cohort studied had very early renal disease as compared to
the type 2 diabetic mouse model which was used in the
We have also demonstrated an increase in AGE-R3
expression in PBMCs in diabetic humans with renal impair-
ment, consistent with previous reports [33, 34]. Indeed,
previous studies in diabetic AGE-R3 knockout mice have
shown severe renal dysfunction . The interrelationship
among AGE clearance receptors in diabetic nephropathy
remains to be fully elucidated, although it is interesting
to note that, in the present study, AGE-R1 and AGE-R3
were each increased with renal impairment in diabetes in
humans, in addition to high AGE feeding in our mouse
models. This is likely representative of a higher AGE load
which requires clearance and detoxification as a protective
mechanism. Importantly, the decrease in AGE-R3 seen in
the high AGE fed dbdb group may be the result of the
advanced renal injury. By contrast the failure to demonstrate
this decrease in the patients with renal impairment could be
due to the absence of this advanced renal injury in this group
suggesting that there may be an initial increase in AGE-R3
expression as a protective mechanism to clear the increased
In conclusion, our findings have clearly demonstrated
differences between cell-surface and intracellular patterns of
diabetes. The most predictive PBMC profile for renal disease
in diabetes in humans was an increase in the cell surface
expression of the AGE-R1, in the context of a decrease in
8 Experimental Diabetes Research
RAGE, which warrants further investigation as a biomarker
for progressive DN in large patient cohorts since these mark-
ers were also associated with creatinine clearance in type
2 diabetes. Further investigation is warranted to determine
whether this noninvasive approach may be employed to
rationalise biomarkers for progressive DN in large patient
cohorts in type 2 diabetic patients.
Conflict of Interests
The authors have nothing to disclose.
K. Sourris carried out the animal studies, mouse and
human sample analyses, data analyses and drafted the paper.
B. Harcourt performed sample analyses on animal samples.
S. Penfold performed the flow cytometry of human and
animal samples and the statistical analysis. F. Yap assisted
with the animal and human studies and data analysis. P.
Morgan designed the CML HPLC experiments, and per-
and coordination of the CML HPLC experiments. S. Baker
carried out the human recruitment and patient collections.
G. Jerums conceived the human study, coordinated the
samples collection, and performed the statistical analysis of
the human data. J. Forbes conceived the study, participated
in its design and coordination, and helped to draft the paper.
All authors read and approved the final paper.
The authors would like to thank Kylie Gilbert for technical
assistance. They would also like to thank Harish Ramachan-
the rodent diets. This work was supported Juvenile Diabetes
Research Foundation International (JDRF), the National
Institutes of Health (USA), and the National Health and
Medical Research Council of Australia (NHMRC). They
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