Upregulation of Angiotensin II Type 1 Receptor,
Inflammatory Mediators, and Enzymes of Arachidonate
Metabolism in Obese Zucker Rat Kidney
Reversal by Angiotensin II Type 1 Receptor Blockade
Zhong-Gao Xu, MD, PhD; Linda Lanting, BS; Nosratola D. Vaziri, MD; Zhen Li, MD;
Lili Sepassi, BA; Bernardo Rodriguez-Iturbe, MD; Rama Natarajan, PhD
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Background—Severe obesity can result in proteinuria and progressive glomerulosclerosis in humans and experimental
animals. The associated renal disease is ameliorated by weight reduction and/or blockade of the renin-angiotensin
system. Various growth factors, cytokines, and lipid mediators are implicated in the pathogenesis of renal disease. To
explore the possible involvement of these mediators in obesity-induced renal disease, we examined the expression of
key enzymes of arachidonate metabolism and inflammatory genes in untreated and losartan-treated obese Zucker rats,
a model of obesity, insulin resistance, and renal injury.
Methods and Results—Seven-week-old male obese Zucker rats were randomized to losartan-treated (100 mg/L drinking
H2O) and untreated groups, with lean Zucker rats as controls. After 4 months, RNA and protein were obtained from renal
cortical tissue for relative reverse transcription–polymerase chain reaction, Western blots, and immunohistochemistry.
Compared with the lean controls, obese Zucker rats showed significant glomerular matrix expansion and increased
mRNA expression of the extracellular matrix protein fibronectin, inflammatory mediators interleukin-6 and monocyte
chemoattractant protein-1, and 2 major enzymes of arachidonate metabolism, namely, 12/15-lipoxygenase and
cyclooxygenase-2. This was associated with significant increases in p38 and extracellular signal-regulated kinase (ERK)
1/2 mitogen-activated protein kinase activities and marked upregulation of angiotensin II type 1 receptor (AT1R) mRNA
and protein expression. These abnormalities and the associated glomerulopathy and proteinuria were prevented by
administration of the AT1R blocker losartan.
Conclusions—These findings indicate that obesity-induced glomerulopathy is associated with upregulation of key
inflammatory mediators. These events are associated with and perhaps in part due to upregulation of AT1R, as
evidenced by their reversal with AT1R blocker treatment. (Circulation. 2005;111:1962-1969.)
Key Words: obesity 䡲 angiotensin 䡲 inflammation 䡲 kidney
M
assive obesity in humans can result in focal segmental
glomerulosclerosis (FSGS), which presents with glomerular proteinuria.1,2 In addition, by promoting insulin
resistance and type 2 diabetes mellitus, obesity can lead to
diabetic nephropathy (DN), which has emerged as the major
cause of end-stage renal disease in the United States and
many other countries.3 The associated proteinuria in patients
with massive obesity can be ameliorated by weight reduction
and ACE inhibition.1 Obese Zucker rats (fa/fa rats) exhibit
hyperphagia, obesity, peripheral insulin resistance, hyperinsulinemia, and hyperlipidemia.4 –7 This is due to the autosomal recessive mutation of the gene that encodes the leptin
receptor.8 With aging, obese Zucker rats develop proteinuria
and FSGS, which eventually leads to advanced renal fail-
ure.5,7,9 The associated proteinuria and glomerulosclerosis in
obese Zucker rats can be ameliorated by food restriction,7
ACE inhibitors,10 lipid-lowering agents,11 and insulin
sensitizers.12
The prominent features of glomerular lesions early in the
course of renal disease in the obese Zucker rat consist of marked
increases in glomerular monocyte/macrophage counts and evidence of podocyte injury marked by a significant de novo
desmin expression.13,14 It is of note that micropuncture studies
have revealed no significant differences in glomerular capillary
pressure, single-nephron glomerular plasma flow, or glomerular
filtration rates among 9- to 13-week-old obese and lean Zucker
rats.15 These observations exclude glomerular capillary hypertension or hyperfiltration as a primary cause of glomeruloscle-
Received November 10, 2004; revision received December 22, 2004; accepted December 28, 2004.
From Gonda Diabetes Research Center (Z.-G.X., L.L., R.N.), Beckman Research Institute of the City of Hope, Duarte, Calif; Division of Nephrology
and Hypertension (N.D.V., Z.L., L.S.), University of California at Irvine, Irvine, Calif; and Servicio Nephrologia (B.R.-I.), Hospital Universitario,
Universidad del Zulia Maracaibo, Venezuela.
Correspondence to Rama Natarajan, PhD, Gonda Diabetes Research Center, Beckman Research Institute of the City of Hope, 1500 E Duarte Rd,
Duarte, CA 91010. E-mail [email protected]
© 2005 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/01.CIR.0000161831.07637.63
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rosis in young prediabetic Zucker rats. Taken together, the above
observations suggest that severe obesity and the associated
metabolic consequences in prediabetic obese Zucker rats may
contribute in part to the glomerulosclerosis process via an
inflammatory pathway that involves recruitment of monocytes/
macrophages. These events can lead to podocyte injury, proteinuria, mesangial expansion, glomerulosclerosis, and progressive
renal disease. Moreover, the favorable effects of renin-angiotensin system blockade in retarding FSGS in this model must be
mediated in part by such nonhemodynamic mechanisms as an
antiinflammatory action.
Chemokines (particularly monocyte chemoattractant
protein-1 [MCP-1]), cytokines, and products of the major
enzymes of arachidonate metabolism play an important role
in promoting cell proliferation, macrophage recruitment, and
inflammation.16 –20 In this regard, lipid mediators and oxidized lipids participate in the pathogenesis of various renal
diseases, including DN. In particular, products of the cyclooxygenase (COX) and lipoxygenase (LO) pathways of arachidonate metabolism exert numerous physiological and
pathological effects in the kidney. For instance, 12-LO
activation can lead to the formation of oxidized lipids such as
12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE].21 It is of
note that the leukocyte-type 12-LO and 15-LO are classified
as 12/15-LO because they have high structural homology and
can form both 12(S)-HETE and 15(S)-HETE from arachidonic acid.21 12/15-LO is present in the kidney,22,23 and
12/15-LO mRNA and protein are increased in parallel with
fibronectin in a type 1 diabetes mellitus model of experimental DN.23 Moreover, urinary excretion of 12(S)-HETE is
increased in diabetic patients.24 Factors relevant to the pathogenesis of DN, such as high glucose and angiotensin II (Ang
II), increase 12/15-LO activity and expression in rat mesangial cells.16,23 Furthermore, 12(S)-HETE directly stimulates
cellular hypertrophy and extracellular matrix (ECM) protein
(fibronectin) expression in rat mesangial cells. Likewise, it
mediates Ang II–induced mesangial cell growth and ECM
production.16 12(S)-HETE-induced effects are mediated, at
least in part, by p38 mitogen-activated protein kinase
(MAPK) and its target transcription factor, cAMP responseelement binding protein (CREB).16,25 Furthermore, cellular
growth, matrix production, oxidant stress, and activations of
MAPK and transcription factor CREB are attenuated in
12/15-LO knockout mice.26 Taken together, the observations
cited above illustrate the relevance of 12/15 LO pathway to
the pathogenesis of DN and Ang II–induced renal injury. In
addition, COX-2 can participate in the pathogenesis of DN
and inflammation mainly through hemodynamic and prooxidant effects.17,18,27–29
In contrast to diabetes, very little is known regarding the
role of the enzymes of the arachidonate pathway in the
pathogenesis of kidney disease associated with obesity and
insulin resistance. The present study was designed to examine
the expression of 12/15-LO and COX enzymes of arachidonate metabolism, inflammatory genes such as MCP-1 and
interleukin-6 (IL-6), and MAPK activities in the renal cortex
of prediabetic obese Zucker rats relative to control lean
Zucker rats. In addition, Ang II type 1 receptor (AT1R)
expression and response to the AT1 receptor blocker (ARB)
ARB Effect on Obese Zucker Rat Kidney
1963
losartan were tested. The latter was justified by the fact that
Ang II can activate the 12/15-LO and COX pathways, the
products of which, in turn, can activate MAPKs and inflammatory genes.
Methods
Materials
AT1R antibody was from Santa Cruz Biotechnology; fibronectin
(FN-Ec) antibody was from Chemicon; COX-2 antibody was from
Cayman Chemical Company; and antibodies for phosphospecific
and nonphospho-p38 and -ERK1/2 (extracellular signal-regulated
kinase) MAPKs were from Cell Signaling. We also used a 12/15-LO
peptide antibody as described previously.23 In addition, we used
horseradish peroxidase– conjugated secondary antibodies from Cell
Signaling; ␤-actin antibody from Sigma; Supersignal chemiluminescence reagent from Pierce; relative multiplex reverse transcription–
polymerase chain reaction (RT-PCR) kits and primers for Quantum
RNA 18S internal standards from Ambion Inc; and RNA-STAT60
reagent from Tel-Test. Losartan was obtained from Merck Co.
Animals and Experimental Design
All animal studies were conducted under a protocol approved by the
Animal Care and Use Committee of the University of California,
Irvine. Seven-week-old male lean Zucker rats (n⫽6) and male obese
Zucker rats (n⫽12) were used in the present study. Obese animals
were further randomized into 2 groups of 6 rats each. One group of
obese Zucker rats was administered losartan in the drinking water
(100 mg/L) for 4 months. Under general anesthesia, the rats were
killed by exsanguination with cardiac puncture. Renal cortical tissues
were removed and stored at ⫺70°C for further study. Additional
specimens were fixed in 10% formalin for histological evaluation.
Body weight, tail arterial pressure, 24-hour urine albumin, serum
glucose, cholesterol, and triglyceride concentrations were determined by standard methods.
Relative and Competitive RT-PCR
Total RNA was isolated with RNA-STAT60 reagent according to the
manufacturer’s instructions. cDNA was synthesized with 1 ␮g of
RNA using murine leukemia virus reverse transcriptase and random
hexamers. Primers for the 18S ribosomal RNA (489 or 324 bp) were
included in each reaction as an internal control. RNA was similarly
reverse transcribed. The primers used are summarized in Table 1.
Relative RT-PCRs were performed as described previously.16
For AT1R mRNA expression, we used a quantitative competitive
RT-PCR method. The AT1R competitor cDNA (212 bp) used as
internal standard was designed to contain the same base pair
sequence as the target cDNA that would allow efficient priming, but
it had a portion deleted so that the competitor PCR-generated
fragment could be easily distinguished electrophoretically by size.
Band densities were analyzed by laser densitometry (Bio-Rad
Laboratories). The RT-PCR products were separated by electrophoresis, and the values were log transformed against the competitor
concentration for each PCR tube. The quantity of cDNA in the test
sample was defined as the amount at which the competitor and
wild-type optical density bands were equal.23
Western Blot Analysis
Cortical tissue samples were lysed in SDS sample buffer (2% SDS,
10 mmol/L Tris-HCl, pH 6.8, 10% [vol/vol] glycerol). Lysates were
centrifuged at 12 000 rpm for 15 minutes at 4°C and the supernatant
stored at ⫺70°C. Fifty micrograms of protein per lane was separated
on 10% SDS-PAGE gels (Bio-Rad), transferred onto a nitrocellulose
membrane. and immunoblotted with antibodies to AT1R (1:1000),
12/15-LO (1:500), COX-2 (1:500), phospho-p38 (1:500), and phospho-ERK1/2 (1:500). The blots were stripped and then reprobed with
an antibody to ␤-actin (1:5000), total p38 MAPK (1:1000), or total
ERK1/2 (1:1000). Immunoblots were scanned with a GS-800 den-
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TABLE 1. Primer Sequences for Amplification of
Various Transcripts
Target
Sequence
12/15-LO
TABLE 2.
Product, bp
290
General Animal Data
Lean
Obese
Obese⫹ARB
Body weight, g
423⫾8*
688⫾11
632⫾15
Systolic blood pressure, mm Hg
145⫾10
131⫾8
101⫾8*
259⫾23
Sense
5⬘-GTCTACTCCACCACCTATTTTC-3⬘
Serum glucose, mg/dL
218⫾14
277⫾54
Antisense
5⬘-CTGTGCTCATTGCCTTGTC-3⬘
Urine albumin, mg/24 h
6⫾3
15⫾2*
10⫾2
Serum creatinine, mg/dL
0.3⫾0.04
0.3⫾0.1
0.14⫾0.04
Serum cholesterol, mg/dL
79⫾5*
164⫾12
116⫾12†
Serum triglyceride, mg/dL
86⫾17*
549⫾105
506⫾64
COX-2
298
Sense
5⬘-ACACTACATCCTGACCCAC-3⬘
Antisense
5⬘-TGAACTCTCTCCTCAGAAGAAAC-3⬘
COX-1
254
Sense
5⬘-CTGGCCGGATTGGTGGGGGTAG-3⬘
Antisense
5⬘-GTACTCTGGGGAACAGAT-3⬘
MCP-1
Sense
5⬘-CCTGCTGCTACTCATTCAC-3⬘
Antisense
5⬘-TCTCACTTGGTTCTGGTCC-3⬘
IL-6
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Sense
5⬘-GGATACCACCCACAACAGAC-3⬘
Antisense
5⬘-GAAACGGAACTCCAGAAGAC-3⬘
Fibronectin
191
Statistical Analyses
256
Data are expressed as mean⫾SEM from multiple experiments.
ANOVAs with Tukey post tests for multiple groups were used in
statistical evaluation of the data with PRISM software (Graph Pad).
Statistical significance was detected at the 0.05 level.
Results
446
Sense
5⬘-GCAAGCCTGAACCTGAAGAGACC-3⬘
Antisense
5⬘-CCTGGTGTCCTGATCATTGCATC-3⬘
Values are mean⫾SEM.
*P⬍0.05 vs other groups; †P⬍0.05 vs untreated obese group.
sitometer and protein bands quantified with Quantitation One software (Bio-Rad).
Histological Evaluation
Renal cortical slices for routine light microscopy were placed into
alcoholic Bouin’s solution processed in the standard fashion, and
sections were stained with periodic acid-Schiff (PAS). Renal cortical
slices for immunohistochemical staining were fixed in 10% neutral
buffered formalin and paraffin embedded by standard techniques,
and 5-␮m sections were used. Fibronectin staining was performed
with a commercial kit (Dako Corporation) according to the manufacturer’s instructions and as described previously.23 Glomeruli in
each section were examined with light microscopy at ⫻400
magnification.
General Animal Data
Untreated obese Zucker rats had significant increases in body
weight, plasma cholesterol, and triglyceride concentrations
and significant albuminuria compared with lean Zucker rats
(Table 2); however, nonfasting serum glucose and creatinine
concentrations and arterial pressure did not significantly
differ between these groups. Losartan (ARB) administration
significantly lowered blood pressure and normalized urinary
albumin excretion without altering body weight or serum
glucose concentration in treated obese Zucker rats (Table 2).
Histological Data
One of the most striking characteristics of FSGS and progression of renal disease is mesangial expansion, which results
from accumulation of ECM proteins. The glomeruli in obese
Zucker rats showed marked mesangial expansion characterized by increased PAS staining (Figure 1B) and heavy
fibronectin staining (Figure 1E) relative to lean Zucker rats
Figure 1. PAS staining and immunohistochemical staining for fibronectin protein in lean Zucker rat, obese Zucker rat,
and obese Zucker rat with ARB (losartan)
treatment (Obese⫹ARB) after 4 months.
Top, PAS staining (original magnification
⫻400). Mesangial regions have normal
width and morphology in lean Zucker rat
(A). Mesangial regions in untreated
obese Zucker rat show marked expansion with PAS-positive material without
hypercellularity (B). There is only mild
mesangial expansion in ARB-treated
group (C), which is less than untreated
group. Bottom, Fibronectin immunohistochemistry (original magnification ⫻400).
Fibronectin immunostaining was greater
in obese Zucker rat glomeruli (E) than in
lean Zucker rat glomeruli (D), and this
was reduced by ARB treatment (F).
Xu et al
ARB Effect on Obese Zucker Rat Kidney
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Figure 2. Fibronectin mRNA expression in lean Zucker rat,
obese Zucker rat, and obese Zucker rat with ARB treatment
(Obese⫹ARB) after 4 months. Cortical fibronectin mRNA/18S
ratio was significantly higher in obese than in control rats, and
this was significantly blocked by ARB treatment for 4 months in
obese Zucker rats. Top, PCR data. Bar graph data are
mean⫾SEM from 6 rats per group. *P⬍0.01 vs lean Zucker rats;
⫹P⬍0.05 vs obese Zucker rats (analyzed by ANOVA).
(Figures 1A and 1D). These abnormalities were prevented by
ARB administration (Figures 1C and 1F). The observed
changes in immunodetectable fibronectin were accompanied
by parallel changes in fibronectin mRNA in the 3 groups
(Figure 2), which points to transcriptional regulation of this
matrix protein. In addition to the glomerular changes cited
above, significant tubulointerstitial abnormalities were found
on histological examination of the kidneys in untreated obese
Zucker rats. These included tubular dilation, tubular epithelial
cell effacement, and peritubular cellular infiltrations that
affected 25% to 50% of tubulointerstitial space. The observed
tubulointerstitial abnormalities were mitigated by long-term
ARB administration.
Expression of AT1R
To determine the changes of AT1R mRNA, we performed
quantitative competitive RT-PCR with AT1R-specific primers. As internal standard, we used an AT1R deletion mutant
that is coamplified with the endogenous gene. The use of the
same primers for the mutant and for the endogenous gene
ensures comparable amplification efficiencies. Competitive
RT-PCR data showed a significant increase in AT1R mRNA
expression in obese Zucker rat cortex, and this increment was
reversed by ARB therapy (Figure 3A). Similarly, AT1R
protein expression was increased significantly in obese
Zucker rat kidney cortex and was normalized by ARB
treatment (Figure 3B).
p38 and ERK1/2 MAPK Activities
The MAPK pathway has been implicated in the pathogenesis
of renal disease. We therefore compared the activation of key
growth- and stress-related MAPKs in cortical tissues from
lean, obese, and obese-plus-ARB groups. The results showed
increased activation of both p38 and ERK1/2 MAPKs, as
Figure 3. AT1R expression in lean Zucker rat, obese Zucker rat,
and obese Zucker rat with ARB treatment (Obese⫹ARB) after 4
months. AT1 mRNA expression in cortex was determined by
quantitative competitive RT-PCR. Fixed amount of AT1R wildtype cDNA and fixed amount of AT1R competitor cDNA were
used (A). AT1R protein expression was detected by Western
blot (B). Data are mean⫾SEM from 6 rats in each group.
*P⬍0.01 vs lean ; ⫹P⬍0.01 vs obese Zucker (by ANOVA).
assessed by phosphorylated kinase levels, in obese Zucker rat
cortex compared with lean Zucker controls. Furthermore,
these changes were significantly attenuated by ARB treatment (Figures 4A and 4B).
12/15-LO and COX-2 Expression
Several studies indicate that enzymes of arachidonate metabolism, such as 12/15-LO and COX-2, play important roles in
the pathogenesis of renal disease via hemodynamic and
nonhemodynamic actions and inflammatory responses. We
observed that 12/15-LO and COX-2 protein and mRNA
expressions were increased in the cortex of obese Zucker rats
relative to the lean controls (Figures 5A and 5B, Figures 6B
and 6C). Furthermore, ARB treatment significantly attenuated the rise in 12/15-LO and COX-2 expressions in obese
Zucker rats. In contrast, COX-1 expression was unchanged
(Figure 6A). These results suggest that obesity-induced glomerulopathy is associated with altered arachidonic acid enzymes and their oxidized lipid products that may contribute to
renal damage. The results further suggest that renin-angiotensin system activation is involved in the upregulation of these
enzymes in animals with obesity and insulin resistance.
Expression of Inflammatory Genes MCP-1 and IL-6
Inflammatory factors such as MCP-1 and IL-6 have been
implicated in glomerular and tubulointerstitial injury. We
found significant upregulation of MCP-1 and IL-6 mRNAs in
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Figure 4. p38 and ERK1/2 MAPK activities in lean, obese, and
obese Zucker rat with ARB treatment (Obese⫹ARB) after 4
months. Cortical p38 MAPK phosphorylation (A) and ERK1/2
phosphorylation (B) were significantly increased in obese Zucker
rats, whereas administration of ARB to obese Zucker rats significantly inhibited these parameters. Data are mean⫾SEM from 6
rats per group. *P⬍0.01 vs lean Zucker; ⫹P⬍0.05 vs obese
Zucker rat (by ANOVA).
the kidney cortex of obese Zucker rats. Inflammation and
fibrosis are pathological processes that are regulated in part
by signaling through the MAPK pathway. Because MAPK
activation can increase expression of inflammatory genes, we
hypothesized that the ARB-induced reduction of MAPK
activation in obese Zucker rat cortex in the present study
might be accompanied by a parallel reduction of MCP-1 and
IL-6 expression in the ARB-treated obese Zucker rat kidney.
In confirmation of this hypothesis, ARB administration was
found to attenuate the upregulation of MCP-1 and IL-6 in the
treated obese Zucker rats (Figures 7A and 7B).
Discussion
Morbid obesity can cause glomerulosclerosis in humans and
experimental animals. In the present study, we evaluated the
potential mediators of renal injury in the obese Zucker rat, a
model of prediabetic obesity and insulin resistance. The male
obese Zucker rats used in the present study exhibited significant kidney disease (as evidenced by the presence of albuminuria and histological abnormalities) in the absence of
hypertension or frank diabetes. This was associated with
Figure 5. 12/15-LO expression in lean Zucker rat, obese Zucker
rat, and obese Zucker rat with ARB treatment (Obese⫹ARB)
after 4 months. 12/15-LO mRNA (A) and protein (B) expression
were determined by RT-PCR and Western blot, respectively.
Data are mean⫾SEM from 6 rats per group. *P⬍0.01 vs lean;
⫹P⬍0.05 vs obese Zucker rat (by ANOVA).
significant upregulation of 12/15-LO, COX-2, and the inflammatory cytokines MCP-1 and IL-6 in the kidney cortex.
Given the critical role of these proinflammatory cytokines
and products of arachidonate metabolism in the pathogenesis
of renal disease, the observed association is of considerable
interest. The upregulation of MCP-1 and IL-6 in the obese
rats was accompanied by activation of the p38 and ERK1/2
MAPK pathways. Because MAPK activation can promote the
expression of proinflammatory/profibrotic cytokines, the observed activation of MAPKs in the kidney cortex of the
untreated obese Zucker rats may have contributed to the
augmented expression of MCP-1 and IL-6.
It is well known that Ang II increases blood pressure,
induces renal cell hypertrophy, and promotes synthesis of
ECM proteins, processes linked to the progression of renal
disease.16,30 –32 The effects of Ang II are mediated by 2
plasma membrane receptors, referred to as the AT1 and AT2
subtypes.33,34 Most of the known effects of Ang II in adult
tissues are attributable to AT1R.34 In the present study, the
untreated obese Zucker rats exhibited marked upregulation of
AT1R mRNA and protein expressions. AT1R mediates not
only the hemodynamic but also the nonhemodynamic actions
of Ang II, including those that lead to oxidative stress,
cellular hypertrophy, proliferation, and tissue remodeling.
Consequently, the observed upregulation of AT1R expression
in the obese Zucker rat kidney may have contributed to the
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Figure 6. COX-1 and COX-2 expression levels in lean, obese,
and obese Zucker rat with ARB treatment (Obese⫹ARB) after 4
months. COX-1 (A) and COX-2 (B) mRNA expression determined by RT-PCR with 18S as internal control. COX-2 protein
expression (C) was assessed by Western blot. Data are
mean⫾SEM from 6 rats per group. *P⬍0.01 vs lean; ⫹P⬍0.05
vs obese Zucker rat (by ANOVA).
associated glomerulopathy by augmenting the susceptibility
of this organ to the available circulating and locally produced
Ang II. This supposition is supported by the favorable
response to ARB administration in treated obese Zucker rats
in the present study. In both clinical and experimental
studies,34 –37 ACE inhibitors and AT1R antagonists have been
shown to exert renoprotective effects that cannot be explained
entirely by their hemodynamic actions. In the present study,
chronic ARB administration prevented glomerular injury and
reversed upregulation of AT1R and several key inflammatory
mediators in obese Zucker rats.
AT1R overexpression is one potential molecular mechanism that links a variety of exogenous risk factors to cellular
events in several renal diseases. According to a recent study,
transgenic rats overexpressing human AT1R in podocytes
develop FSGS.32 The major new finding of the present study
is the upregulation of AT1R mRNA and protein expression in
untreated obese Zucker rat cortex and its normalization by
ARB treatment. Several studies have suggested that the
AT1R gene expression is influenced by various cytokines and
growth factors.35 In addition, insulin could upregulate AT1R
in mesangial cells in vitro and in vivo,35,38,39 and this
ARB Effect on Obese Zucker Rat Kidney
1967
Figure 7. MCP-1 and IL-6 mRNA levels in lean rats, obese
Zucker rats, and obese Zucker rat with ARB treatment
(Obese⫹ARB) after 4 months. Cortical expression levels of
MCP-1 (A) and IL-6 (B) mRNAs were determined by RT-PCR
with 18S as internal control. Data are mean⫾SEM from 6 rats
per group. *P⬍0.01 vs lean; ⫹P⬍0.05 vs obese Zucker rat (by
ANOVA).
upregulation of AT1R by insulin was suggested to be responsible for the additive effects of insulin and Ang II in
mesangial cells.38,39 Because hyperinsulinemia is a known
feature of metabolic syndrome in obese Zucker rats, the
observed upregulation of AT1R in the kidney cortex of obese
Zucker rats in the present study in part may be due to
hyperinsulinemia. Upregulation of AT1R in obese Zucker
rats was reversed by ARB administration. The precise mechanism responsible for this phenomenon is not clear; however,
it may be due to the compensatory rise in renin activity and
Ang II production leading to a simultaneous activation of
AT-2 receptor (AT2R) and downregulation of AT1R.40 It is
also possible that AT1 antagonists may cause hyperstimulation of the AT2 subtype.40 Because AT2R may counteract the
effects of AT1R, concomitant stimulation of AT2R and
blockade of AT1R may contribute to the beneficial effects of
AT1R antagonists.40
The 12/15-LO and COX-2 pathways have been implicated
in the pathogenesis of inflammation and DN. Products of
these pathways have potent inflammatory, vasoactive,
growth, and matrix-inducing properties.16 –18,27,41 We recently
demonstrated that the 12/15-LO pathway was enhanced in
mesangial cells cultured in high glucose and in rats with DN.
The changes in 12/15 LO correlated with expression of the
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matrix protein, fibronectin,23,26 and 12/15-LO could mediate
Ang II–induced effects.16 COX-2 metabolites have also been
implicated in the pathogenesis of functional and structural
abnormalities associated with certain glomerular and tubulointerstitial inflammatory disorders. Moreover, COX inhibitors can ameliorate proteinuria and/or structural injury.27,28,42
COX-2 has been implicated in the early stages of DN28;
however, its role in development of the overt nephropathy is
not entirely clear. In the present study, we showed for the first
time that 12/15-LO and COX-2 levels are increased in the
renal cortex of prediabetic obese Zucker rats. We also
demonstrated that an ARB can reduce 12/15-LO and COX-2
expression, thereby supporting the role of the Ang II–AT1R
pathway in regulating these enzymes. Moreover, these data
reveal an additional mechanism for renoprotective effects of
ARBs in obese Zucker rat cortex.
The chemokine MCP-1 is produced mainly by tubular
epithelial cells in kidney and contributes to inflammation and
fibrosis.43,44 Recently, urinary MCP-1 excretion was shown to
increase in proportion to the degree of albuminuria in patients
with type 2 diabetes mellitus.45,46 Blockade of the renin-angiotensin system patients with type 2 diabetes mellitus with
DN reduces urinary MCP-1 levels and preserves renal function.46,47 Because MCP-1 induces monocyte/macrophage infiltration, which promotes matrix accumulation and tubulointerstitial fibrosis, pharmacological interventions to inhibit
AT1R expression or action may exert their beneficial effects
in part by downregulating renal MCP-1. The present data
demonstrate that MCP-1 expression and IL-6 were increased
in obese Zucker rat kidney and were significantly lowered by
ARB treatment. These findings implicate the Ang II–AT1R
pathway in the regulation of proinflammatory cytokines in
the kidney of animals with obesity and insulin resistance.
Proinflammatory cytokines and enzymes of arachidonate
metabolism may represent the effectors and downstream
targets of well-known signal transduction pathways such as
protein kinase C and MAPKs in mediating renal damage.
MAPKs have been implicated in both glomerular matrix
accumulation and tubulointerstitial fibrosis.48 –50 They are
also activated by Ang II, LO, and COX products. In the
present studies, p38 and ERK1/2 MAPKs were significantly
activated in cortex of obese Zucker rats and were normalized
by chronic ARB treatment. Taken together, the present data
suggest that expressions of enzymes of arachidonate metabolism, key inflammatory genes, and AT1R and activity of
MAPKs are elevated in obese Zucker rat kidneys and are
most likely involved in the pathogenesis of nephropathy in
this model. Moreover, these changes are associated with and
mediated, at least in part, by Ang II action, as evidenced by
their reversal with ARB treatment. This reversal with ARB
was accompanied by significant amelioration of proteinuria
and histological abnormalities of nephropathy in the obese
Zucker rats. Therefore, the present results have provided
additional rationale for the use of ARBs in the treatment and
prevention of the obesity-associated FSGS.
Acknowledgments
These studies were supported by grants from the National Institutes
of Health (RO1 DK58191) and the Juvenile Diabetes Research
Foundation.
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Upregulation of Angiotensin II Type 1 Receptor, Inflammatory Mediators, and Enzymes
of Arachidonate Metabolism in Obese Zucker Rat Kidney: Reversal by Angiotensin II
Type 1 Receptor Blockade
Zhong-Gao Xu, Linda Lanting, Nosratola D. Vaziri, Zhen Li, Lili Sepassi, Bernardo
Rodriguez-Iturbe and Rama Natarajan
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Circulation. 2005;111:1962-1969
doi: 10.1161/01.CIR.0000161831.07637.63
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