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Abstract

We aimed to find the relationship between serum transforming growth factor beta 1(TGF-β₁) and urinary monocyte chemoattractant protein-1 (MCP-1) throughout the course of diabetic nephropathy (DN) and to assess the relationship between both levels and other parameters of renal injury such as albumin/creatinine ratio and estimated glomerular filtration rate (eGFR). Serum TGF-β₁, urinary MCP-1, eGFR, and glycosylated hemoglobin (HbA_1c) were measured in 60 patients with type II diabetes mellitus with different degrees of nephropathy (20 patients with normoalbuminuria, 20 patients with microalbuminuria, and 20 patients with macroalbuminuria) and compared with 20 matched healthy control subjects. Both the levels of serum TGF-β₁ and urinary MCP-1 were significantly higher in patients with micro- and macroalbuminuria (137.8 ± 69.5 and 329.25 ± 41.46 ng/dl, respectively, for TGF-β₁ and 167.41 ± 50.23 and 630.87 ± 318.10 ng/g creatinine, respectively, for MCP-1) compared with normoalbuminuric patients and healthy controls (33.25 ± 17.5 and 29.64 ± 10.57 ng/dl, respectively, for TGF-β₁ and 63.85 ± 21.15 and 61.50 ± 24.81 ng/g creatinine, respectively, for MCP-1; p < 0.001). There was a positive significant correlation between the levels of serum TGF-β₁ and those of urinary MCP-1 (r = 0.73, p < 0.001). Also, serum TGF-β₁ and urinary MCP-1 correlated positively with HbA_1c (r = 0.49 and 0.55, respectively, p < 0.05 for both) and inversely with eGFR (r = −0.69 and −0.60, respectively, p < 0.001 for both). We can conclude that serum TGF-β₁ and urinary MCP-1 can be used as the markers for detection of progression of DN. Antagonizing TGF-β₁ and MCP-1 might be helpful in attenuating the progression of nephropathy in diabetic patients.

Keywords

Diabetic nephropathy MCP-1

Introduction

Diabetic nephropathy (DN) is the most common cause of end-stage renal failure (ESRF) in the Western world. DN follows a well defined clinical course, starting with microalbuminuria through proteinuria, azotaemia, and culminating in ESRF.¹ DN is thought to result from the interactions between metabolic and hemodynamic factors. Specific metabolically driven, glucose-dependent pathways are activated within diabetic renal tissues. Hemodynamic factors are also implicated in the pathogenesis of DN and include elevations of systemic and intraglomerular pressure and the activation of various vasoactive hormone pathways. These altered hemodynamics act independently and in concert with metabolic pathways to activate intracellular secondary messengers such as protein kinase C (PKC) and mitogen-activated protein kinase, nuclear transcription factors such as nuclear factor-kappa B (NF-κB), and various growth factors such as connective tissue growth factor (CTGF). Ultimately, these molecular mechanisms lead to increased renal albumin permeability and extracellular matrix accumulation, which results in increasing proteinuria, glomerulosclerosis, and tubulointerstitial fibrosis.²

The manifestations of DN may be a consequence of the actions of certain cytokines and growth factors. Prominent among these is the transforming growth factor beta 1 (TGF-β₁) because it promotes the renal cell hypertrophy and stimulates the extracellular matrix accumulation, the two hallmarks of diabetic renal disease.³ There are three isoforms of TGF-β in the mammalian cells: TGF-β₁, TGF-β₂, and TGF-β₃. Among these three isoforms, TGF-β₁ is the only one that circulates in the blood stream and the most highly expressed isoform in the kidney.⁴ In cell culture, high ambient glucose increases TGF-β₁ messenger RNA (mRNA) and protein in proximal tubular, glomerular epithelial, and mesangial cells.⁵ Although the kidney of a nondiabetic subject extracts TGF-β₁ from the blood, the kidney of a diabetic patient actually elaborates TGF-β₁ protein into the circulation.³

A chemokine, monocyte chemoattractant protein (MCP-1), also termed monocyte chemotactic and activating factor, is secreted by mononuclear cells and various nonleukocytic cells including renal resident cells. In human nephritis, it is thought to play an important role in the pathogenesis of crescent formation and progressive tubulointerstitial lesions by monocyte recruitment and activation.⁶ Recent studies had demonstrated that protein overload in renal tubular cells upregulates the MCP-1 gene and its protein expression.⁷

There is a controversy about the relationship between TGF-β₁ and MCP-1 in the kidney tubules in the course of progressive nephropathies. Some studies reported that TGF-β₁ in the proximal tubule cells of the kidney stimulates the production of proinflammatory cytokines including interleukin-8 (IL-8), MCP-1, and MCP-4, which is thought to promote renal damage.^8,9 Other studies had reported that TGF-β₁ downregulates MCP-1 in human proximal tubular epithelial cells.¹⁰

The aim of this study is to estimate the levels of serum TGF-β₁ and MCP-1 in variable degrees of DN and to find the possible relationship between them. Also, to assess the correlation between both levels and other parameters of renal injury such as albumin/creatinine ratio (ACR) and estimated glomerular filtration rate (eGFR).

Patients and methods

The study was conducted on 60 patients with type II diabetes mellitus (DM) and 20 healthy volunteers who were followed-up in the internal medicine outpatient clinic in Cairo University Hospital. All participants gave their informed consent before participation in the study, and the study protocol was approved by Cairo University Hospital research committee. All the subjects were divided into four groups as follows: group I (n = 20, control group): age and gender matched healthy volunteers (11 males and nine females). The 60 patients with type II DM were divided into three groups according to the degree of nephropathy, which was measured by urinary ACR. Group II (n = 20, diabetic patients with normoalbuminuria): ACR <30 μg/mg creatinine, comprising ten males and ten females. Group III (n = 20, diabetic patients with microalbuminuria): ACR 30–299 μg/mg creatinine, comprising nine males and 11 females. Group IV (n = 20, diabetic patients with macroalbuminuria): ACR ≥300 μg/mg creatinine, comprising ten males and ten females.

From all the diseased and control subjects, relevant medical history data were recorded, including age, sex, and duration of diabetes. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured in the sitting position and the median of three successive measurements was noted. From each subject, venous blood samples (10 ml) and three overnight first-voided urine samples were collected. Blood samples were divided into two parts: 5 ml were collected into EDTA tubes to estimate glycosylated hemoglobin (HbA_1c), the remaining 5 ml were collected into an empty tube and allowed to clot in water bath for 30 min, then centrifuged at 4000 r/min for 10 min to obtain the serum separately; one part of the serum was used for the estimation of creatinine, and the other part was kept frozen at −70°C until used for TGF-β₁ estimation. Freshly voided urine samples were stored at −20°C until used to measure urinary albumin, creatinine, and MCP-1.

Laboratory investigations

HbA_1c was measured by a colorimetric method using kit provided by Stanbio laboratory, TX, USA.¹¹ Serum TGF-β₁ was measured by enzyme-linked immunosorbent assay (ELISA) technique using kit provided by BioSource Europe S.A. (Nivelles, Belgium)¹² Serum and urinary creatinine were measured using colorimetric commercial kit.¹³ Urinary albumin and creatinine concentrations were measured by radioimmunoassay commercial kit (Diagnostic Products Corporation, Apeldoorn, The Netherlands) and colorimetric commercial kit¹³, respectively. Then the ACR was calculated to assess the degree of nephropathy: normoalbuminuric patients have ACR below 30 μg/mg creatinine; microalbuminuric patients have ACR between 30 and 300 μg/mg creatinine; macroalbuminuric patients have ACR more than 300 μg/mg creatinine. Urinary MCP-1 was measured with solid-phase ELISA using kit provided by R&D Systems (Minneapolis, Minnesota, USA).¹⁴ The eGFR was measured using the equation derived by the Modification of Diet in Renal Disease Study (MDRD) Group^15
–17 as follows:

\begin{aligned} e G F R (m l / m i n / 1 . 73 m^{2}) = 186.3 \\ \times {s e r u m c r e a t i n i n e}^{- 1.154} \\ \times {a g e}^{- 0. 203} (\times 0. 742 f o r w o m e n) \end{aligned}

Statistical analysis

The results were analyzed using Statistical Package for the Social Sciences (SPSS) computer software package, version 10.0 (Chicago, Illinois, USA). Data were presented as mean ± SD. Differences among the four groups were compared using one-way analysis of variance. To study the relationship between the measured parameters, Pearson’s correlation was performed. The results were considered statistically significant at p < 0.05.

Results

The demographic data were shown in Table 1. There is a significant increase (p < 0.001) in the serum levels of TGF-β₁ in microalbuminuric (group III) and macroalbuminuric patients (group IV) when compared with normoalbuminuric patients (group II) and healthy controls (group I). Also, patients with macroalbuminuria had significantly higher levels of serum TGF-β₁ than those with microalbuminuria (p < 0.001). But, no significant difference in serum TGF-β₁ levels was found between normoalbuminuric patients and healthy controls (p = 0.41; Table 2).

Table 1.

Demographic data of studied groups^a

	Group I (control) n = 20	Group II (normoalbuminuria) n = 20	Group III (microalbuminuria) n = 20	Group IV (macroalbuminuria) n = 20
Age (years)	57.40 ± 4.65	56.37 ± 6.83	52.15 ± 7.73	53.41 ± 9.89
Gender (M/F)	11/9	10/10	9/11	10/10
Diabetes duration (years)	NO	4.07 ± 2.05	5.76 ± 3.67	6.83 ± 3.45^b
SBP (mmHg)	126.12 ± 5.23	131.72 ± 3.11	139.57 ± 6.13^a	145.62 ± 6.29^a
DBP (mmHg)	74.52 ± 6.5	75.43 ± 2.46	83.81 ± 5.31^a	92.24 ± 5.71^a
Serum creatinine (mg/dl)	0.72 ± 0.13	0.97 ± 0.13	1.45 ± 0.36^a	2.98 ± 1.19^a,b,c
ACR (μg/mg creatinine)	2.07 ± 1.88	10.27 ± 16.219	140.09 ± 67.484^a,b	366.66 ± 64.44^a,b,c
HbA_1c (%)	5.11 ± 0.96	7.22 ± 0.862^a	7.31 ± 1.37^a	8.66 ± 1.13^a,b,c
eGFR (ml/min/1.73 m²)	98.07 ± 12.72	93.83 ± 27.27	68.72 ± 14.90^a,b	55.25 ± 22.31^a,b

ACR: albumin/creatinine ratio; eGFR: glomerular filtration rate; HbA1c: glycosylated hemoglobin; SBP: systolic blood pressure; DBP: diastolic blood pressure. Data are represented as mean + SD. Values are statistically significant at p < 0.05.

^aSignificant p when compared with Group I.

^bSignificant p when compared with Group II.

^cSignificant p when compared with Group III.

Table 2.

Serum TGF-β₁ and urinary MCP-1 levels in the studied groups^a

	Group I (control) n = 20	Group II (normoalbuminuria) n = 20	Group III (microalbuminuria) n = 20	Group IV (macroalbuminuria) n = 20
Serum TGF-β₁ (ng/dl)	29.64 ± 10.57	33.25 ± 17.5	137.8 ± 69.5^a,b	329.25 ± 61.46^a,b,c
Urinary MCP-1 (ng/g creatinine)	61.50 ± 24.81	63.85 ± 21.15	167.41 ± 50.23^a,b	630.86 ± 318.10^a,b,c

TGF-b1: transforming growth factor-beta 1; MCP-1: monocyte chemoattractant protein-1. Data are represented as mean + SD. Values are statistically significant at p < 0.05.

^aSignificant p when compared with Group I.

^bSignificant p when compared with Group II.

^cSignificant p when compared with Group III.

Urinary MCP-1 levels were significantly higher in both micro- and macroalbuminuric patients when compared with normoalbuminuric patients and healthy controls (p < 0.001). Patients with macroalbuminuria had significantly higher levels of urinary MCP-1 than microalbuminuric patients (p < 0.001). There was no significant difference in urinary MCP-1 level between the normoalbuminuric patients and healthy controls (Table 2).

HbA_1c levels were significantly higher in macroalbuminuric group when compared with microalbuminuric, normoalbuminuric, and control groups (p < 0.05). Levels of eGFR were significantly reduced in micro- and macroalbuminuric groups when compared with normoalbuminuric and control groups (p < 0.05; Table 2).

Studying the correlation between serum TGF-β₁ and urinary MCP-1, this work showed a positive correlation between the levels of serum TGF-β₁ and those of urinary MCP-1 in diabetic patients (Figure 1, r = 0.73, p < 0.001). Both serum TGF-β₁ and urinary MCP-1 showed a nonsignificant correlation with age, gender, and duration of diabetes (p > 0.05); and also showed positive significant correlation (Table 3) with ACR (r = 0.69 and r = 0.75, respectively, p < 0.001), HbA_1c (r = 0.49 and 0.55, respectively, p < 0.05), SBP (r = 0.51 and 0.56, respectively, p < 0.05), and DBP (r = 0.48 and r = 0.47, respectively, p < 0.05). Meanwhile, both are inversely correlated with eGFR (r = −0.69 and −0.60, respectively, p < 0.001).

Figure 1.

Correlation between serum TGF-β₁ and urinary MCP-1 levels in diabetic patients (r = 0.73, p < 0.001). TGF-β₁: transforming growth factor-beta 1; MCP-1: monocyte chemoattractant protein-1.

Table 3.

Correlations of serum TGF-β₁ and urinary MCP-1 levels with some studied parameters in diabetic patients^a

	TGF-β₁		MCP-1
	r	p	r	p
ACR	0.69	<0.001	0.75	<0.001
HbA_1c	0.49	<0.05	0.55	<0.05
SBP	0.51	<0.05	0.56	<0.05
DBP	0.48	<0.05	0.47	<0.05
eGFR	−0.69	<0.001	−0.60	<0.001

TGF-β₁: transforming growth factor-beta 1; MCP-1: monocyte chemoattractant protein-1; ACR: albumin/creatinine ratio; eGFR: glomerular filtration rate; HbA_1c: glycosylated hemoglobin; SBP: systolic blood pressure; DBP: diastolic blood pressure.

^aValues at p < 0.05 are statistically significant. Bold values are statistically significant.

There was a significant positive correlation between serum TGF-β₁ and serum creatinine (r = 0.542, p < 0.01), while urinary MCP-1 did not correlate with serum creatinine (r = 0.23, p = 0.09). Among the studied groups, ACR was directly correlated with HbA_1c (r = 0.65, p < 0.001) and inversely correlated with eGFR (r = −0.60, p < 0.001). HbA_1c did not correlate with eGFR (r = −0.31, p = 0.06).

Discussion

TGF-β₁ and MCP-1 appear to play a pivotal role in the pathogenesis of DN. TGF-β₁ promotes the renal cell hypertrophy, regulates the production of extracellular matrix molecules, and induces the chemokines production in proximal tubules of the kidney.^1,3 MCP-1 is a specific chemokine to recruit and activate monocytes from the circulation to inflammatory site.⁷

One of the main findings of the current study was increased levels of serum TGF-β₁ in diabetic patients with micro- and macroalbuminuria compared with normoalbuminuric diabetics and healthy controls. This increase in the levels of TGF-β₁ in diabetic patients was due to the fact that increased glucose levels stimulates de novo synthesis of diacylglycerols (DAGs) generated from glycolytic intermediates through the polyol pathway; elevated DAG leads to the activation of PKC, which increases TGF-β₁ synthesis and matrix protein synthesis in mesangial and tubular cells.¹⁸ Restoration of normoglycemia by insulin attenuates the renal cortical and glomerular expression of these molecules.¹⁹

The present results were in accordance with the results of Bordin et al.²⁰ who demonstrated increased glomerular and circulating levels of TGF-β₁ in patients having type II DM with mild and advanced nephropathy. They explained their results by elevated TGF-β₁ production that plays an important role in stimulating excessive extracellular matrix production in fibrotic sites of target tissues, such as in DN. However, other factors are also likely to be necessary for matrix overproduction to progress the point of clinical significance. Such factors may include a persistently elevated glomerular capillary pressure, glomerular angiotensin II production, the formation of early and advanced glycation products (AGEs), and the complex interactions of other growth factors and cytokines. As many of these processes have been found to stimulate TGF-β₁ production itself, one may speculate that the cumulative effect is a pronounced increase in TGF-β₁ production leading to excessive matrix deposition and clinical DN.

Also, the results of this study were in agreement with the results reported by Hellmich et al.²¹ who proposed that the activation of circulating TGF-β₁ is markedly increased in patients having type II DM with DN compared with patients having type II DM without renal involvement and healthy controls. They suggested that glucose may be a direct activator of latent TGF-β₁. Mesangial and proximal tubular cells cultured in media containing high glucose concentrations exhibit increased production of TGF-β₁ mRNA and protein followed by enhanced collagen biosynthesis, suggesting an increased bioactivity of TGF-β₁. Another study showed that the mRNA and protein levels of tissue and serum TGF-β₁ were increased in the incipient stage of DN with further increase at later stages of the disease.²²

Another important finding of this study was the increase in the urinary MCP-1 levels in the micro- and macroalbuminuric patients compared with the normoalbuminuric patients and healthy controls. Increased urinary excretion of MCP-1 in the patients with established DN (micro- and macroalbuminuric patients) is probably due to the enhanced production of MCP-1 in renal tubuli, presumably induced by excessive exposure to plasma protein filtered from the damaged glomeruli. Protein overload in renal tubular cells upregulates the MCP-1 gene and its protein expression.⁷ Recent studies on diabetic type II patients had suggested that MCP-1 synthesis in mesangial cells is induced by elevated glucose and AGEs, tubular reabsorbed protein, mechanical stretch, angiotensin II, and aldosterone with NF-κB activation.^23,24,25

In consistence with the findings of the current study, Tashiro et al.²⁶ reported that the measurement of urinary MCP-1 and IL-8 may be useful for evaluating the degree of renal injuries in patients with type II DN. Levels of urinary MCP-1 in all the patients with DN were significantly higher than those in healthy adults. The levels of urinary MCP-1 in patients with DN increased gradually according to the clinical stage of this disease, with significant rise in the advanced stage of this disease. In contrast, the levels of urinary IL-8 increased in the early stage of DN.

A recent study confirmed the association of the development of DN with increased kidney MCP-1 production, which occurred mostly in tubules. The elements of the diabetic milieu, as high glucose and AGEs, directly stimulate the tubular MCP-1 secretion.²³

Banba et al.²⁷ and Morii et al.⁷ suggested that MCP-1 production by mesangial cells in diabetic milieu contributes to the initiation and progression of DN. Urinary levels of MCP-1 increased in accordance with the extent of albuminuria. The combination of glycated albumin and hyperglycemia showed the greatest stimulation in more than an additive manner on MCP-1 production.

Interestingly, the higher levels of serum TGF-β₁ and urinary MCP-1 in our patients with macroalbuminuria than those with microalbuminuria could be due to the significantly poorer glycemic control (HbA_1c)²⁸ observed in our study. HbA_1c is regarded as the gold standard for the measurement of glycemic control and is important for long-term management of diabetic patients, as measures that reduce HbA_1c correspondingly reduces the risk of complications.²⁹

In contrast to micro- and macroalbuminuric patients, the present study showed that normoalbuminuric patients had normal levels of serum TGF-β₁ and urinary MCP-1. This could be attributed to the effect of other additive factors that might be present in advanced DN as prolonged hyperglycemia, poor glycemic control, AGEs, high oxidative burden, and local activation of rennin–angiotensin system. All these factors together can induce cytokine and chemokine production in genetically predisposed patients.^20,23,30

A positive correlation between the levels of serum TGF-β₁ and those of urinary MCP-1 was found in this study. TGF-β₁ induces the production of certain cytokines in various cells. Prasad et al.³¹ reported that TGF-β₁ induces the tumor necrosis factor-alpha (TNF-α) and IL-1 in various cells. Over expression of TNF-α and IL-1 stimulates the expression of chemokines like MCP-1 in human mesangial cells. Also, Weier et al.⁴ demonstrated that TGF-β₁, in the proximal tubule cells of the kidney, is known to stimulate the production of proinflammatory cytokines including IL-8, MCP-1, and MCP-4 by the CTGF-independent pathway.

In contrast to the current results, some studies reported that TGF-β₁ downregulates the MCP-1 in human proximal tubular epithelial cells.^10,32

In the current study, there was a decrease in the eGFR in micro- and macroalbuminuric patients compared with normoalbuminuric patients and healthy controls, which was naturally considered due to the progression of renal damage as an effect of advanced DN.³³ The inverse association between the levels of serum TGF-β₁ and urinary MCP-1, on the one hand, and eGFR, on the other hand, could suggest the use of TGF-β₁ and MCP-1 as markers that reflect accurately the degree of renal damage.

This work showed a significant increase in serum TGF-β₁ in patients having DN with hypertension compared with those without hypertension. These results were in agreement with Li et al.³⁴ who found that the circulating levels of TGF-β₁ correlate with the blood pressure levels. Moreover, the results were similar to that of Suthanthiarn et al.³⁵ who reported that TGF-β₁ is hyperexpressed in hypertensive patients compared with normotensive patients and they concluded that the TGF-β₁ hyperexpression is a risk factor for hypertension and its complications.

Also, the present work showed significant correlation between urinary MCP-1 level and blood pressure. This was in agreement with Jana et al.³⁶ who found that MCP-1 levels were elevated in patients with hypertension. These results were confirmed by Tucci et al.³⁷ who reported that overexpression of MCP-1 is predominant in hypertensive patients.

It seems most probably that demographic parameters as age, gender, or duration of diabetes have no major effect on both TGF-β₁ and MCP-1. No significant correlation was found in this study between each of serum TGF-β₁ and MCP-1 and age, gender, or duration of diabetes. These results were in agreement with Sharma et al.³⁸ and Stefoni et al.³⁹ who found that the production of TGF-β₁ did not correlate with the duration of diabetes, age, or sex of the patients. Similar results were also reported concerning urinary MCP-1.⁴⁰

In conclusion, the present results suggest that the progression of DN is associated with increased levels of serum TGF-β₁ and urinary MCP-1 that are closely linked to renal damage and the degree of glycemic control. Thus, it seems possible that, in addition to adequate glycemic control, strategies aiming at antagonizing TGF-β₁ and MCP-1 might be beneficial in attenuating the progression of nephropathy in diabetic patients. Long-term studies concerning these strategies are needed to further clarify this issue.

Footnotes

Acknowledgments

We gratefully acknowledge Internal Medicine Department, Faculty of Medicine, Cairo University.

Funding

This work was financially supported by Faculty of Medicine, Cairo University, Cairo, Egypt.

Conflict of Interest

The authors declare no conflicts of interest.

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Transforming growth factor beta 1 and monocyte chemoattractant protein-1 as prognostic markers of diabetic nephropathy

Abstract

Keywords

Introduction

Patients and methods

Laboratory investigations

Statistical analysis

Results

Discussion

Footnotes

Acknowledgments

Funding

Conflict of Interest

References