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Biomedical Research 32 (2) 83-90, 2011

Left ventricular hypertrophy is associated with inflammation in sodium loaded


subtotal nephrectomized rats

Yoshiyuki Moriguchi1, Kenji Yogo1, Ken Aizawa1, Ken-ichi Serizawa1, Yoshihito Tashiro1, Keigo Yorozu1,
Nobuhiko Ishizuka1, Sadahiro Iwabuchi2, Hidemitsu Kitamura2, and Takashi Nishimura2, 3
1
Product Research Department, Chugai Pharmaceutical Co., LTD., 2 Division of Immunoregulation, Research Section of Disease Con-
trol, Institute for Genetic Medicine, Hokkaido University, and 3 Division of ROYCE Health Bioscience, Institute for Genetic Medicine,
Hokkaido University
(Received 13 October 2010; and accepted 13 December 2010)

ABSTRACT
The pathological influences of inflammation on left ventricular hypertrophy (LVH) were studied in
subtotal nephrectomized (SNx) rats after 0.3% NaCl loading for 5 weeks. We found that mild hy-
pertension, increased plasma levels of creatinine, inorganic phosphate, asymmetric dimethylargi-
nine (ADMA), and parathyroid hormone (PTH) were observed in the present SNx rats without
LVH. In the present study, the NaCl-loaded SNx (SNx + NaCl) rats were characterized by signifi-
cant LVH and hypertension with aggravated values of all the parameters. We further confirmed
that glomerular sclerosis, tubulointerstitial fibrosis, and inflammatory cell infiltration into the tubu-
lointerstitial area, observed in the SNx rats, were more severely caused in the SNx + NaCl rats. In
addition, plasma interleukin-6 (IL-6) levels in the SNx + NaCl rats were significantly increased
compared to those in the SNx rats. These findings indicated that NaCl-loaded SNx rats developed
LVH and hypertension, which were accompanied with increased plasma levels of PTH, creatinine,
inorganic phosphorus, ADMA, and IL-6. Thus, these results suggest that inflammation as well as
endothelial dysfunction would be correlated with LVH as non-traditional risk factors at the early
stage in the present renal failure model.

Compared with general population, a large propor- abetes (12). Although the prevalence of traditional
tion of patients with chronic kidney disease (CKD) risk factors in patients with CKD is high, the sever-
die from cardiovascular disease (9). The mecha- ity of cardiovascular disease does not necessarily
nisms underlying the higher risk of cardiovascular correspond to these risk factors (18), which suggests
events in CKD patients are not fully understood, the relation of non-traditional cardiac risk factors to
though they are thought to be associated with the patients with CKD. It has been demonstrated that
high incidence of both traditional and non-tradition- the prevalence of non-traditional cardiac risk factors
al risk factors for cardiovascular disease. A large such as hyperparathyroidism, endothelial dysfunc-
percentage of patients with CKD have traditional tion, albuminuria, inflammation and anemia in-
cardiovascular risk factors such as hypertension, left creased as kidney function declines (2, 19). It was
ventricular hypertrophy (LVH), dyslipidemia and di- also reported that parathyroid hormone (PTH) con-
tributed to the LVH observed in chronic renal fail-
ure, which would play roles in vascular calcification
Address correspondence to: Takashi Nishimura, Ph.D.
Division of Immunoregulation, Research Section of Dis- and the development of cardiac fibrosis via activa-
ease Control, Institute for Genetic Medicine Hokkaido tion of fibroblasts (1).
University, Sapporo 060-0815, Japan Recently, the consequences of inflammation, a
Tel & Fax: +81-11-706-7546 non-traditional risk factor, have gained attention in
E-mail: tak24@igm.hokudai.ac.jp nephrology. Chronic inflammation is characterized
84 Y. Moriguchi et al.

by the persistent effect of a causative stimulus, (SNx + NaCl) from 1 week after the second opera-
which leads to destruction of cells and tissues. Pre- tion. All animal procedures were conducted in
vious paper demonstrated that plasma levels of in- accordance with Chugai Pharmaceuticals ethical
flammatory factors such as C-reactive protein (CRP) guidelines for animal care, and all experimental pro-
and interleukin-6 (IL-6) were elevated in patients tocols were approved by the Animal Care Commit-
with CKD (17), but the relative importance of these tee of the institution.
factors in predicting cardiovascular outcomes re-
mained unresolved. Although CRP reflects systemic Measurement of blood pressure and blood biochemi-
inflammation and predicts cardiovascular risk, there cal parameters. Systolic blood pressure (SBP) was
is no direct supporting data for CRP to promote vas- measured every week from immediately before
cular disease. On the other hand, it has been report- NaCl loading, by the tail cuff method (BP-98A;
ed that IL-6 might directly act as a promoter of Softron, Tokyo, Japan) under conscious conditions.
atherosclerosis and wasting disorders by providing At 5 weeks after NaCl loading, whole blood was
oxidative stress and endothelial dysfunction (24). collected from the abdominal aorta under deep pen-
Endothelial dysfunction is also a non-traditional tobarbital anesthesia. Plasma levels of creatinine, in-
risk factor in patients with CKD which causes re- organic phosphorus and calcium were measured
duced bioavailability of nitric oxide (NO). Recent with an autoanalyzer (Hitachi 7170; Hitachi Co.
papers indicated that the accumulation of asymmet- Ltd, Tokyo, Japan). Plasma PTH levels were mea-
ric dimethylarginine (ADMA), an endogenous inhib- sured by rat intact PTH enzyme-linked immunosor-
itor of NO synthase (NOS), became not only a bent assay (ELISA) (Immutopics, Inc., CA, USA).
biomarker of endothelial dysfunction but also a new Plasma IL-6 levels were measured with a rat IL-6
potential risk factor for LVH in patients with or immunoassay kit (Invitrogen Corp., CA, USA).
without CKD (14, 26). It was also demonstrated that Plasma CRP levels were measured with a rat C-
ADMA was a potential proinflammatory factor to reactive protein (CRP) ELISA Kit (Alpha Diagnostic
induce the monocytic adhesion to endothelial cells International, TX, USA).
and the release of chemokines in vitro (4). However,
the precise mechanism of proinflammatory action of Measurement of plasma ADMA. Plasma ADMA lev-
ADMA on monocytes has not been fully understood els were determined using an API4000QTRAP (AB
in the kidney. SCIEX, CA, USA) equipped with Acquity UPLC
In the present study, we established NaCl-loaded H-Class Bio system (Waters Corp., MA, USA). An-
SNx rats as an early stage CKD model with LVH alytical chromatography of ADMA was performed
and evaluated the involvements of non-traditional on a COSMOSIL column (4.6 250 mm, type
risk factors, namely inflammation and endothelial NAP, Waters; Nacalai Tesque, Kyoto, Japan). The
dysfunction in addition to the traditional risk factors. Q1/Q3 values of analytes were 204.2/70.6 (ADMA).
Instrument control and data processing were run by
Analyst software version 1.4 (AB SCIEX, CA,
MATRIALS AND METHODS
USA). 13C6-arginine (internal standard, IS, 100 mol/
Animal models. Male Wistar rats (6 weeks old; Ja- L) was added to 50 L of plasma as an internal
pan SLC Inc, Shizuoka, Japan) were fed ordinary standard. To precipitate plasma proteins, 90 L ace-
laboratory chow (CE-2; CREA Japan, Tokyo, Japan) tonitrile was added to each plasma sample. The
and allowed free access to water under a constant samples were voltex-mixed and centrifuged at
light and dark cycle of 12 h. After 1 week of adap- 10,000 g for 10 min at 4C. Supernatants were
tation, animals were subjected to two-step surgical then collected and dried under vacuum at 60C. The
partial nephrectomy under anesthesia (22.5% iso- residue was reconstituted in 500 L mobile phase.
flurane). Laparotomy was undergone under sterile Calibration curves were plotted using the ratio of
conditions and the upper and lower side of the left peak areas of analyzed molecules to IS versus con-
kidney was resected in the first procedure. One centrations. Calibration curve was linear in the
week after the first operation, animals were re- range of 0.1 to 50 mol/L. The limit of detection
anesthetized and the right kidney was removed. was 0.1 mol/L. The intra-assay precision and accu-
Sham-operated animals (sham) underwent laparoto- racy were in the range of 0.32.9 and 1.56.1%,
my alone. Subtotal nephrectomized rats were divid- respectively. The inter-assay precision and accuracy
ed into 2 groups and received drinking water ad were in the range of 6.612.8 and 4.36.5%, re-
libitum containing either 0 (SNx) or 0.3% NaCl spectively.
IL-6 is associated with LVH in SNx 85

Table 1Changes in physiological and biochemical parameters in the sham, SNx, and
SNx + NaCl rats
Sham SNx SNx + NaCl
Body weight (g) 296 3 257 12# 243 6
LV/BW (mg/g) 1.85 0.04 2.10 0.09# 2.92 0.09*
SBP (mmHg) 142 3 168 5# 239 6*
Creatinine (mg/dL) 0.38 0.01 1.24 0.15# 1.95 0.16*
IP (mg/dL) 6.5 0.3 8.2 0.4# 10.8 0.6*
Ca (mg/dL) 11.2 0.2 11.4 0.2 10.5 0.3*
Data are expressed as mean SEM of 5 to 6 animals. #P < 0.05 comparing SNx with sham by Stu-
dents t-test. *P < 0.05 comparing SNx + NaCl with SNx by Students t-test.
LV/BW: left ventricular mass/body weight, SBP: systolic blood pressure, IP: inorganic phosphorus.

Histological analysis. At the experimental end point,


body weight and left ventricular mass were mea-
sured. The heart and kidney were excised and fixed
in 10% neutral buffered formalin, and embedded in
paraffin according to the standard procedure. Hema-
toxylin-eosin and Azan staining were used in light
microscopic evaluation. Pathophysiological changes
were assayed in a blinded manner and evaluated by
semi-quantitative score.

Statistical analysis. Data are expressed as mean


SEM. Comparison between two groups was per-
formed by Students t-test or Wilcoxon test using Fig. 1Changes in systolic blood pressures of the sham,
SAS version 8.2 software (SAS Institute, NC, USA). SNx and SNx + NaCl rats. Data are expressed as mean
SEM of 5 to 8 animals. : sham, : SNx, : SNx + NaCl.
Values with P < 0.05 were considered statistically #
P < 0.05 when comparing SNx with sham by Students
significant. Multivariate correlation analysis was t-test. *P < 0.05 when comparing SNx + NaCl with SNx by
performed to evaluate the associations between car- Students t-test.
diac abnormality and the other variables using JMP
7.0.1 software (SAS Institute).
rus: P < 0.05, Table 1).
We further confirmed that all values of the pa-
RESULTS
rameters in the SNx + NaCl rats were significantly
Traditional risk factors and left ventricular hyper- higher than those of the SNx rats (LV/BW: P <
trophy in NaCl-loaded SNx rats 0.001; inorganic phosphorus: P < 0.05; creatinine:
In order to address the involvements of traditional P < 0.05, Table 1). Plasma calcium levels in the
risk factors and non-traditional risk factors such as SNx + NaCl rats significantly decreased compared
inflammation and endothelial dysfunction, we estab- to those of the SNx rats (P < 0.05), though there
lished NaCl-loaded SNx rats as an early stage CKD were no differences between the sham and the SNx
model. There were no differences in body weight rats (Table 1). We found that SBP in the SNx + NaCl
between the SNx and SNx + NaCl rats (Table 1), rats was higher than that of the SNx rats, which sig-
though slight growth retardation was observed in the nificantly increased dependent on the period of Na-
SNx rats compared to the sham-operated control rats Cl-loading (SNx vs. sham: P < 0.01; SNx + NaCl vs.
(data not shown). We found that left ventricular SNx: P < 0.001) (Fig. 1, Table 1).
mass/body weight (LV/BW) of the SNx rats was
significantly higher than that of the sham rats (P < Non-traditional risk factors in the NaCl-loaded SNx
0.05) at five weeks after NaCl loading (Table 1). rats
Plasma creatinine and plasma inorganic phosphorus We also investigated the non-traditional cardiac risk
of the SNx rats were also higher than those of the factors in the present models and then found that
sham rats (creatinine: P < 0.001; inorganic phospho- plasma ADMA and PTH levels in the SNx rats were
86 Y. Moriguchi et al.

significantly higher than those of the sham rats IL-6 showed no difference between the sham and
(ADMA, SNx vs. sham: P < 0.01; PTH, SNx vs. the SNx rats (Table 2). On the other hand, plasma
Sham: P < 0.05, Table 2). Additionally, statistically levels of CRP did not differ among the present rat
significant increases in PTH and ADMA were ob- models (Table 2).
served in the SNx + NaCl rats compared to those of
the SNx rats (PTH, SNx + NaCl vs. SNx: P < 0.01; Histological analysis of the kidney
ADMA, SNx + NaCl vs. SNx: P < 0.05, Table 2). We then confirmed pathological changes in the kid-
We further evaluated plasma IL-6 and CRP as in- ney of the sham, SNx, and SNx + NaCl rats. Subto-
flammatory markers. As a result, plasma IL-6 levels tal nephrectomy induced significant tissue damages
in the SNx + NaCl rats increased significantly com- such as glomerular sclerosis, tubule enlargement,
pared to those of the SNx rats (P < 0.05), though urinary casts or tubulointerstitial fibrosis (Fig. 2A

Table 2Changes in inflammatory and cardiovascular biomarkers in the sham, SNx, and
SNx + NaCl rats
Sham SNx SNx + NaCl
IL-6 (pg/mL) 8.5 0.7 7.6 0.5 11.6 1.5*
CRP (mg/mL) 495 15 512 15 478 24
PTH (pg/mL) 113 19 1902 655# 5378 508*
ADMA (mol/L) 0.66 0.03 1.27 0.13# 1.61 0.07*
Data are expressed as mean SEM of 5 to 6 animals. #P < 0.05 comparing SNx group with sham
group by Students t-test. *P < 0.05 comparing SNx + NaCl with SNx by Students t-test.
IL-6: interleukin 6, CRP: C-reactive protein, PTH: parathyroid hormone, ADMA: asymmetric di-
methylarginine.

Fig. 2Representative pathological changes in the kidneys of the sham, SNx, and SNx + NaCl rats. Glomerular sclerosis
was observed in the kidney of the SNx (B) and SNx + NaCl (C) rats compared to that of the sham (A) rats. Enlargement of
tubule, urinary casts, and inflammatory cell infiltration were observed in the kidney of the SNx (E and H) and the SNx + NaCl
(F and I) rats in contrast to that of the sham (D and G) rats. A through C are Azan-stained sections (bar = 50m). D
through I are HE-stained sections (D through F: bar = 200 m; G through I: bar = 50 m).
IL-6 is associated with LVH in SNx 87

through 2F, Table 3). We confirmed that the loading ganic phosphorus (r = 0.884, P < 0.0001), SBP (r =
of NaCl into the SNx rats significantly exacerbated 0.851, P < 0.0001), ADMA (r = 0.838, P < 0.0001),
the glomerular sclerosis (Fig. 2B, 2C, and Table 3). mononuclear cell infiltration (r = 0.765, P = 0.0006)
There were no remarkable histopathological changes and IL-6 (r = 0.639, P = 0.0077). There was no cor-
such as myocardial fibrosis in the left ventricle of relation between LV/BW and CRP (r = 0.256, P =
the heart among the rats (data not shown). 0.3385). The score of mononuclear cell infiltration
We first observed that the numbers of inflamma- in the interstitial area was highly correlated with
tory cells such as neutrophils and mononuclear cells creatinine (r = 0.900, P < 0.0001), ADMA (r = 0.899,
infiltrating into the kidney of the SNx rats signifi- P < 0.0001), PTH (r = 0.827, P<0.0001), inorganic
cantly increased compared to the sham rats. Then, phosphorus (r = 0.780, P = 0.0004) and SBP (r =
we found that the mononuclear cells infiltrated espe- 0.756, P = 0.0007).
cially into the tubulointerstitial area (Fig. 2G through
2I and Table 4). Most inflammatory cells in the tu-
DISCUSSION
bulointerstitial area of the SNx and SNx + NaCl rats
were mononuclear cells comprising lymphocytes In the present study, we established LVH-developed
and macrophages. Among them, neutrophil infiltra- CKD model using NaCl-loaded SNx rats and inves-
tion into the tubulointerstitial area of the kidney in tigated the effects of the non-traditional risk fac-
the SNx + NaCl rats was significantly increased torsinflammation and endothelial dysfunctionas
compared to the SNx rats (Fig. 2H, 2I, and Table 4). well as those of the traditional risk factors on the
LVH. We confirmed that LVH was correlated with
Correlation analysis among the non-traditional risk plasma level of PTH, creatinine, inorganic phospho-
factors, traditional risk factors, and left ventricular rus, and SBP in the NaCl-loaded SNx rats. Previous
hypertrophy reports have demonstrated that non-traditional risk
We finally evaluated multivariate correlation coeffi- factors such as vascular calcification factors (PTH,
cients among the traditional, non-traditional factors inorganic phosphorus) and uremic toxin (creatinine)
and LVH in the present model, and the calculated are involved in the pathogenesis of a number of car-
data were summarized in Table 5. As a result, LV/ diovascular abnormalities including LVH in CKD
BW was highly correlated with PTH (r = 0.946, patients (6, 19, 20). These results suggest that the
P < 0.0001), creatinine (r = 0.894, P < 0.0001), inor- present rat model would provide the evidence for

Table 3Pathological changes in the kidney of the sham, SNx, and SNx + NaCl rats
Glomerular sclerosis Enlargement of tubule Urinaly casts Interstitial fibrosis
N + ++ +++ + ++ +++ + ++ +++ + ++ +++
Sham 5 5 0 0 0 0 5 0 0 0 0 5 0 0 0 0 5 0 0 0 0
SNx 5 0 4 1 0 0# 0 0 4 1 0# 0 4 1 0 0# 0 3 1 1 0#
SNx + NaCl 6 0 0 3 3 0* 0 0 3 2 1 0 2 4 0 0 0 0 3 3 0
Date are expressed as the number of animals in each score. Score was evaluated with following criteria in a blinded manner.
: negative, : minimal, +: mild, 2+: moderate, 3+: marked.
#
P < 0.05 comparing SNx with sham by Wilcoxon test. *P < 0.05 comparing SNx + NaCl with SNx by Wilcoxon test.

Table 4Changes in inflammatory cell infiltration in the kidney of the sham, SNx, and SNx + NaCl rats
Whole kidney Interstitial area
Neutrophil and mononuclear Neutrophil Mononuclear cell
cell infiltration infiltration infiltration
N + ++ +++ + ++ +++ + ++ +++
Sham 5 5 0 0 0 0 5 0 0 0 0 5 0 0 0 0
SNx 5 0 1 3 1 0# 3 2 0 0 0 0 0 4 1 0#
SNx + NaCl 6 0 0 4 2 0 0 6 0 0 0* 0 0 2 4 0
Data are expressed as the number of animals with each score. Score was evaluated with the following criteria in a blinded manner.
: negative, : minimal, +: mild, 2+: moderate, 3+: marked.
#
P < 0.05 comparing SNx with sham by Wilcoxon test. *P < 0.05 comparing SNx + NaCl with SNx by Wilcoxon test.
88 Y. Moriguchi et al.

Table 5Multivariate correlation matrix among non-traditional risk factors, traditional risk factors, and LV/BW
LV/BW IL-6 CRP ADMA PTH MNCI SBP CRE IP
Non-traditional risk factors
IL-6 0.639a
CRP 0.256 0.352
ADMA 0.838b 0.409 0.103
PTH 0.946b 0.563 0.183 0.896b
MNCI 0.765b 0.391 0.001 0.899b 0.827b
Traditional risk factors
SBP 0.851b 0.426 0.161 0.792b 0.847b 0.756b
CRE 0.894b 0.522 0.110 0.970b 0.901b 0.900b 0.819b
IP 0.884b 0.658a 0.231 0.873b 0.856b 0.780b 0.772b 0.926b
Ca 0.670a 0.558 0.045 0.573 0.638a 0.408 0.444 0.658a 0.619
a
P < 0.01, bP < 0.001 comparing each parameters by correlation analysis.
LV/BW: left ventricular mass/body weight, CRP: C-reactive protein, IL-6: interleukin 6, ADMA: asymmetric dimethylarginine, PTH:
parathyroid hormone, MNCI: mononuclear cell infiltration, SBP: systolic blood pressure, CRE: creatinine, IP: inorganic phosphorus.

the effects of non-traditional risk factors on LVH tubular epithelial cells (11). The present study might
and hypertension in the initial stage of the renal suggest that subtotal nephrectomy generated an in-
failure. creased level of urinary protein, judging from the
Although it is noted that inflammatory markers observed glomerular sclerosis and urinary casts. Si-
such as CRP and IL-6 are elevated in patients with multaneously, we speculated that the released che-
CKD, the question as to which of these markers mokines might recruit mononuclear cells into the
predict cardiovascular outcomes still remains unre- tubulointerstitial area, resulting in the amplification
solved. Previous papers reported that increased plas- of the production of IL-6 and chemokines from
ma levels of IL-6 in patients correlated with the proximal tubular epithelial cells.
severity of heart failure, which would be predictive Endothelial dysfunction, another non-traditional
of mortality in patients with or without CKD (7, cardiovascular risk factor, is related to renal failure
21). In addition, a recent report indicated that IL-6 and hypertension. It has been demonstrated that ele-
infusion in normal rats caused LVH (15). The pres- vated plasma concentration of ADMA was associat-
ent findings indicated that inflammation (plasma ed with LVH and LV dysfunction in hemodialysis
IL-6) as well as endothelial dysfunction (ADMA) patients (27). Ueda et al. reported that increased
was correlated with LVH in the early stage of the plasma levels of ADMA were correlated with the re-
CKD model. On the other hand, we found that plas- nal damages including decreased numbers of peritu-
ma CRP was not increased and might not be corre- bular capillaries, increased tubulointerstitial fibrosis,
lated with LVH in the SNx + NaCl rat model. These hypertension, and proteinuria in the SNx rats (22).
results were consistent with the previous clinical Furthermore, over-expression of the ADMA degrad-
data that CRP was not significantly associated with ing enzymedimethylarginine dimethylaminohydro-
cardiovascular outcomes after adjustment for tradi- lase (DDAH-1 and DDAH-2), which were mainly
tional risk factors (5), suggesting that CRP and IL-6 located in proximal tubules and endothelium (16)
might have different roles in the progression of decreased plasma levels of ADMA, prevented hy-
LVH. pertension, and blocked the progression of renal
It has been indicated that IL-6, a pleiotropic cyto- dysfunction in the SNx rats (13). As a mechanism
kine, is produced by proximal tubular epithelial cells of ADMA to induce renal damage, ADMA was
(8) and various inflammatory cells such as mono- shown to be involved in production of IL-6 and
cytes (3) and macrophages (10). Whereas we ob- chemokines in renal proximal tubular cells (25)
served a significant increase of inflammatory cell through the activation of NAD(P)H oxidase (23).
infiltration into the tubulointerstitial area in the SNx The present results demonstrate that plasma level of
and SNx + NaCl rats, the precise source of increased ADMA is highly correlated with the infiltration of
plasma levels of IL-6 was still unclear. Previous pa- mononuclear cells into intersitial area, suggesting
per suggested that urinary protein might induce pro- that ADMA would play a pivotal role in recruitment
duction of IL-6 and chemokines from proximal of mononuclear cells into interstitial area.
IL-6 is associated with LVH in SNx 89

In conclusion, the present study has demonstrated Fukami K, Matsuoka H, Imaizumi T and Okuda S (2007)
that NaCl-loading to SNx rats caused LVH, which Dimethylarginine dimethylaminohydrolase prevents progres-
sion of renal dysfunction by inhibiting loss of peritubular
was associated with increased plasma levels of PTH, capillaries and tubulointerstitial fibrosis in a rat model of
creatinine, inorganic phosphorus, ADMA, and IL-6 chronic kidney disease. J Am Soc Nephrol 18, 15251533.
and with hypertension. Although further studies 14. Meinitzer A, Seelhorst U, Wellnitz B, Halwachs-Baumann G,
would be necessary to clarify the detailed mecha- Boehm BO, Winkelmann BR and Marz W (2007) Asymmet-
rical dimethylarginine independently predicts total and
nisms via which such factors could contribute to
cardiovascular mortality in individuals with angiographic cor-
cardiovascular disease in the present animal model, onary artery disease (the Ludwigshafen Risk and Cardiovas-
these findings suggest that inflammation and endo- cular Health study). Clin Chem 53, 273283.
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sis, concentric hypertrophy, and diastolic dysfunction in rats.
tional risk factors. Hypertension 56, 225231.
16. Palm F, Onozato ML, Luo Z and Wilcox CS (2007) Dimeth-
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