# Upregulation of Monocyte  Chemoattractant Protein-1  (MCP-1) in Early

Authors: Eid El-Shafey; G. F. El-Nagar; Hasan Abu Hatab; Alaa Sabry; Hesham Elbaz
Journal: Scholarly Research Exchange
 (2008)

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## Abstract

Background. Monocyte chemoattractant protein-1 (MCP-1) is a member of the chemokine family and specifically attracts monocytes. MCP-1 can directly elicit an inflammatory response by inducing cytokine and adhesion molecule expression in the kidney. This is an important new mechanism in the pathogenesis of  tubulointerstitial  inflammation. We investigated the role of MCP-1 in the development of early nephropathy in insulin-dependent diabetes mellitus (IDDM) patients, in addition to the effect of high-dose vitamin E treatment (8 weeks) on early stages of diabetic nephropathy. Methods. This study was carried out on 30 diabetic patients subdivided into two equal groups: Group I, IDDM patients without microalbuminuria; Group II, IDDM patients with microalbuminuria; in addition to 10 healthy matched volunteers included as controls for comparison. MCP-1, glycated hemoglobin (HbA1c), and albuminuria—before and after vitamin E treatment—were measured in all studied groups. Results. Serum MCP-1 and HbA1c were significantly elevated in patients with microalbuminuria and poor glycemic control (941.67±47.03 pg/mL; 16.95±2.74%) compared to normoalbuminuric diabetic patients (622.73±103.23 pg/mL; 7.23±0.86%), and controls (366.60±129.01; 3.35±0.66) (P=.001), respectively. There was positive correlation between MCP-1 and HbA1c. Both MCP-1 and albuminuria decreased significantly after  using high-dose vitamin E treatment,  though there was no change in HbA1c in IDDM patients with early nephropathy. Conclusion. These observations suggest that MCP-1 may be involved in the pathogenesis of diabetic nephropathy. High-dose vitamin E may provide a novel form of therapy for the prevention of microvascular complications in type-1 diabetic patients.


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## Body

## 1. Introduction

Diabetic nephropathy  occurs in 20–40% of patients
with diabetes and  is the single leading cause of end-stage renal disease
(ESRD). Persistent albuminuria  in the range of 30–299 mg/24 h
(microalbuminuria) has been shown  to be the earliest stage of diabetic
nephropathy in type-1 diabetes and a marker  for the development of nephropathy
in type-2 diabetes. Microalbuminuria is also a well-established marker of
increased CVD risk. Patients with microalbuminuria who progress to
macroalbuminuria (300 mg/24 h) are likely to progress to ESRD over a period of
years. Over the past several years, a number of interventions have been demonstrated to reduce the risk and slow the progression of renal disease [1]. The accumulation of matrix
proteins resulting in glomerular sclerosis and interstitial fibrosis is a
prominent feature of human diabetic nephropathy [2]. There may be an interplay of metabolic and hemodynamic
pathways in the progression of diabetic nephropathy [3–5]. In addition to these pathways,
the infiltration of inflammatory cells such as monocyte/macrophage  into the diseased kidneys is a hallmark of the progression of diabetic
nephropathy. Infiltrated monocyte/macrophage release
lysosomal enzymes, nitric oxide, reactive oxygen intermediates, an transforming
growth factor-β, which have reported to play an essential role in renal damage [3, 6].MCP-1 belongs to
chemotactic cytokine that exhibits its most potent chemotactic activity toward
monocytes and T-cells, and promotes the transmigration of circulating
monocytes into tissues [7].
Recent studies reveal that MCP-1 plays an important role in the pathogenesis of
crescentic formation and progressive tubulointerstitial lesions via M∅
recruitment and activation in experimental
glomerulonephritis models [8–10]
and human nephritis [11–13]. We determined the serum levels of human MCP-1 in type-1 diabetic patients with
nephropathy to find out the relationship between poor glycemic control and
MCP-1 generation and to evaluate the role of vitamin E on MCP-1 expression.


## 2. Subjects and Methods

### 2.1. Subjects

Thirty ambulatory adolescents and young adults (15 males and 15 females)
with type-1 diabetes mellitus having a mean age of12±6 years were included.
All were on conventional insulin therapy having two daily subcutaneous
injections. They were divided into two equal groups, according to the presence
of persistent microalbuminuria.
Persistent microalbuminuria was
defined as an albuminuria between 30 and 299 mg/24 h: Group I, IDDM patients
without nephropathy (8 male and 7 females); Group II, IDD patients with early
nephropathy (7 males and 8 females).  In addition 10 apparent healthy volunteers, age- and sex-matched, were enrolled as reference controls.
Informed written consent was obtained from all the patients and volunteers.


### 2.2. Methods

All the patients and control group were subjected to a thorough history
taking and complete clinical examination. Fasting and postprandial blood sugar,
glycated hemoglobin, (HbA1c), blood urea, serum creatinine, microalbuminuria, and quantitative
determination of serum MCP-1 concentrations (by ELISA  technique) were  determined in patients and
controls at the beginning of the study and 8 weeks after using high-dose
vitamin E treatment (600 mg/BID).Quantitative
determination of serum MCP-1 levels was determined by an enzyme-linked immunosorbent assay (ELIZA),
using a specific murine monoclonal antihuman MCP-1 antibody as a capture, and a
biotinylated anti-MCP-1 antibody as the second antibody which binds to the solid
phase antibody-antigen complex. All assays were performed in duplicate. The
detection limits of this ELISA system were 40 pg/mL for human MCP-1 (Biosource
International, Camarillo, Calif, USA).


### 2.3. Statistics

Statistical analysis was done by SPSS V. 13. The data are expressed as
mean and standard deviation. The pairedt-test, analysis of variance [ANOVA],
and Scheffe test were used wherever applicable. Spearman's rank correlation
coefficient (r) was calculated to test the association between two
variables;  for quantitative data, the
number and percent distribution were calculated. Probability (P) values
of less than .05 were considered of statistical significance.


## 2.1. Subjects

Thirty ambulatory adolescents and young adults (15 males and 15 females)
with type-1 diabetes mellitus having a mean age of12±6 years were included.
All were on conventional insulin therapy having two daily subcutaneous
injections. They were divided into two equal groups, according to the presence
of persistent microalbuminuria.
Persistent microalbuminuria was
defined as an albuminuria between 30 and 299 mg/24 h: Group I, IDDM patients
without nephropathy (8 male and 7 females); Group II, IDD patients with early
nephropathy (7 males and 8 females).  In addition 10 apparent healthy volunteers, age- and sex-matched, were enrolled as reference controls.
Informed written consent was obtained from all the patients and volunteers.


## 2.2. Methods

All the patients and control group were subjected to a thorough history
taking and complete clinical examination. Fasting and postprandial blood sugar,
glycated hemoglobin, (HbA1c), blood urea, serum creatinine, microalbuminuria, and quantitative
determination of serum MCP-1 concentrations (by ELISA  technique) were  determined in patients and
controls at the beginning of the study and 8 weeks after using high-dose
vitamin E treatment (600 mg/BID).Quantitative
determination of serum MCP-1 levels was determined by an enzyme-linked immunosorbent assay (ELIZA),
using a specific murine monoclonal antihuman MCP-1 antibody as a capture, and a
biotinylated anti-MCP-1 antibody as the second antibody which binds to the solid
phase antibody-antigen complex. All assays were performed in duplicate. The
detection limits of this ELISA system were 40 pg/mL for human MCP-1 (Biosource
International, Camarillo, Calif, USA).


## 2.3. Statistics

Statistical analysis was done by SPSS V. 13. The data are expressed as
mean and standard deviation. The pairedt-test, analysis of variance [ANOVA],
and Scheffe test were used wherever applicable. Spearman's rank correlation
coefficient (r) was calculated to test the association between two
variables;  for quantitative data, the
number and percent distribution were calculated. Probability (P) values
of less than .05 were considered of statistical significance.


## 3. Results

There was no
significant difference between the studied groups regarding the age and sex.
However, the duration of DM and HbA1c showed statistically significant
difference between Group I (3.67±1.32;
7.63±0.86) versus Group II (9.37±2.65 years; 16.95±2.74%), respectively.Microalbuminuria was significantly higher in Group II (251.80±43.86 mg/d)
compared to Group I (22.23±5.09 mg/d) and controls (16.80±5.57 mg/d) (P=.001). Serum MCP-1
showed statistically significant higher levels in Group II (941.67±47.03 pg/mL),
in comparison to Group I (622.73±103.23 pg/mL) and controls (366.60±129.01 pg/mL)
(P=.001); see Table 1.Table 1
Comparison between the studied groups regarding different studied parameters.
ParameterPatients groupsControlsGroup IGroup IIFPN=10N=15N=15Age (yrs)13.43±2.1714.57±2.1915.01±0.972.25.25Mean ± SDDuration of DM (yrs)—3.67±1.329.37±2.65t=754.001*Mean ± SDHbA1c (%)3.35±0.607.63±0.8616.95±2.74193.57.001*Mean ± SD24 hours urine albumin (mg/d)16.80±5.5722.23±5.09251.8±43.86337.77.001*Mean ± SDMCP-1 (PG/mL)366.60±29.01622.73±103.23941.67±47.03115.41.001*Mean ± SDSingnificant association between MCP-1 and HbA1c in diabetic patients
with microalbuminuria (r=0.728, P<.0001). Controls and type-1 diabetic patients without
nephropathy (Group I) showed no  significant difference regarding HbA1c,
microalbuminuria, and serum
MCP-1 before using vitamin
E treatment versus after using it.Meanwhile, in type-1 diabetic patients with nephropathy (Group II), HbA1c,
microalbuminuria, and serum MCP-1 were significantly lower after treatment than before treatment (Table2; Figures 1, 2, and 3).Table 2
Effects of treatment with vitamin E on different studied parameters among Group I and Group II patients.
ParameterPatients groupsGroup I (N=15)Group II (N=15)Before vit. EAfter vit. EtPBefore vit. EAfter vit. EtPHbA1c (%)7.63±0.867.43±0.932.82.1416.95±2.7416.95±2.742.45.28Mean ± SD24 hr urine albumin (mg/d)22.23±5.0921.93±4.180.90.38251.8±43.86174.80±41.7450.41.001*Mean ± SDMCP-1 (PG/mL)622.73±103.23630.80±71.00.381.71941.67±47.03685.67±45.7428.96.001*Mean ± SDFigure 1
HbA1C before and after  using vitamin E therapy in different studied groups.Figure 2
Microalbuminuria before and after using vitamin E therapy in different studied groups.Figure 3
MCP-1 before and after  using  vitamin E therapy in different studied groups.


## 4. Discussion

Monocyte infiltration in the mesangium plays an important role in
glomerular diseases [14], is associated with fibroblast activation and
increased  extracellular matrix deposition in diabetic rats, and   diffuse glomerulosclerosis in patients with diabetic nephropathy [15, 16]. As MCP-1 is
a potent chemoattractant for monocytes, it is of interest  to know that the
increased glomerular expression of MCP-1 has been shown in several glomerular
diseases [17] as well as in the mesangium of rats with streptozotocin-induced
diabetes [14]. More recently, increased production of MCP-1 by blood
mononuclear cells of patients with diabetes has been demonstrated [18]. In this
prospective study, we investigated the possible role of MCP-1 in the
development of early diabetic nephropathy in patients with type-1 DM. In
addition, we studied the relationship between poor glycemic control and MCP-1
generation and the possible role of high-dose vitamin E treatment on
microalbuminuria and MCP-1 expression.The duration of IDDM has generally been mentioned as the strongest risk
factor for the development of nephropathy. In the present study, patients with
significant microalbuminuria had a mean IDDM duration of (9.3±3.7 years), 2 out
of 15 patients in Group II  had less than 5 years duration of DM.Hovind et al. [19] reported significant association between duration of
DM and occurrence of diabetic nephropathy, microalbuminuria occurs in 15–40% of patients
with type-1 diabetes, with a peak incidence around 15–20 years. Stage
II or incipient nephropathy may develop  for  2–10 years after
the initial changes are noted, microalbuminuria is present at this point and
may range between 20–200 μg/m [20]. For
patients with type-1 DM, the first screening has been recommended at 5 years
after diagnosis, however, the prevalence of  microalbuminuria  before 5 years can reach 18%,
especially in patients with poor glycemic control, lipid control, and high
normal blood pressure levels [21]. Repeated measurement of microalbuminuria
from the time of diagnosis may be useful in the early detection of patients who
will develop microalbuminuria and ultimately overt diabetic nephropathy.Mean HbA1c values were significantly higher in  microalbuminuric diabetic patients than in  normoalbuminuric  diabetic
patients (15.25±4.25 versus 7.75±1.25%, P=.001) (Table 1), and a
positive correlation was found between HbA1c and microalbuminuria in Group II,
so that the poor glycemic control is correlated with the development of early
diabetic nephropathy. This is in agreement with Chiarelli et al. [22] who
reported that mean HbA1c values were significantly higher in  microalbuminuric diabetic
patients than in  normoalbuminuric
diabetic patients (9.6±1.4 versus 7.3±0.93%, P<.05).In the present study, serum MCP-1 has significantly increased in type-1
diabetic patients with early nephropathy compared to matched patients without
nephropathy and controls. This is in agreement with Chiarelli et
al. [22] who found that Plasma MCP-1 was significantly higher (P=.004)
in the  microalbuminuric
patients with respect to the  normoalbuminuric
patients  and healthy volunteers. In
contrast, they found no significant differences between the  normoalbuminuric  and the
control groups. Cipollone et al. [23]
observed Bcl-2 downregulation in  microalbuminuric  diabetic patients as a consequence of increased
oxidant burden secondary to persistent hyperglycemia which was associated with
enhanced expression of MCP-1, and showed a strong correlation with the albumin
excretion rate. This is also in accordance with our observation of the
significant association between HbA1c  and  MCP-1 in diabetic patients with
microalbuminuria. However, it is not in accordant with Banba et al. [24] who found
that urine levels, but not serum levels, of MCP-1 increased in accordance with
the extent of GHb and albuminuria. However, in this study, only a small group
of patients (9) had microalbuminuria and so could be correctly compared with
our patients. Furthermore, eight of these nine patients had type-2 diabetes.
Again, the age (mean ± SD) was 62.210.4 years in the study from Banba et al. but
only 15.01±0.97 years in our study. Also, Kiyici et al. [25] reported that there
was no difference in serum levels of MCP-1 between type-1 diabetic patients
with  diabetic nephropathy and without it.Moreover, in our prospective study glycemic, control did not change in
diabetic patients after vitamin E treatment, however, the MCP-1 and
microalbuminuria were  significantly
reduced after vitamin E therapy in  microalbuminuric  patients, thus confirming that MCP-1 reduction
after vitamin E was specifically due to vitamin E and not to improved glycemic
control. This is in agreement with Chiarelli et al. [22] who
observed that both MCP-1 and AER decreased significantly after vitamin E
treatment in  microalbuminuric
patients, despite  there were  no changes in HbA1c percentages. In addition to Gaede et al. [26],
who reported that treatment with vitamin E plus vitamin C in patients with
micro- or macroalbuminuria significantly lowers microalbuminuria though there
is no change in HbA1c, Cipollone et al. [23] observed that prolonged
hyperglycemia may lead to higher oxidative burden, consumption of endogenous
antioxidant buffer, and overexpression of MCP-1. But
in contrast, Ishikawa et al. [27] stated that the increased circulating MCP-1
could be merely a secondary effect of diabetic nephropathy, also Cipollone et
al. [7] reported that most of the changes observed in diabetic patients after
vitamin E therapy were in response to treatment with insulin and the improvement
of glycemic control. However, this hypothesis is unlikely, because the direct
role of vitamin E in MCP-1 generation is supported by the observation that in
vivo generation of MCP-1 was reduced by the administration of high-dose vitamin
E in diabetic patients with microalbuminuria [23], and the in vitro MCP-1
generation in monocytes was exclusively downregulated by vitamin E, while
insulin or changes in osmolar conditions failed to produce any effect [22]. In
contrast, a subgroup analysis of 31654 diabetic patients participating in the
heart outcomes prevention evaluation (HOPE) study [28] demonstrated no renal
effects in patients receiving low-dose (400 iu) vitamin E. However some
characteristics of the HOPE study may contribute to explain this apparent
discrepancy, the daily dose of antioxidant administered in the HOPE study is
significantly lower with respect to both Gaede et al.’s study [26] and ours.
Thus, we can speculate that the simple administration of 400 iu of vitamin E
may not  sufficiently  restore the antioxidant supply to the level necessary to prevent the induction
of inflammatory genes, ultimately leading to renal damage and  microalbuminuria .Based on the previous observations, we concluded that upregulation of
MCP-1 gene expression by  persistent
hyperglycemia in type-1 diabetic patients results in the recruitment of  monocytes  into the
kidney, possibly contributing to the development of diabetic nephropathy.
Moreover, the causative role of poor glycemic control in diabetic nephropathy
is mediated by increased oxidative stress. These findings indicate a pathogenic
role for MCP-1 in the evolution of diabetic microvascular complications. High-dose vitamin E may provide a new form of therapy for prevention of
microvascular complications in type-1 diabetic patients in  which acceptable glycemic
control is difficult to achieve despite appropriate insulin therapy.



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