Clinical significance of urinary Monocyte Chemoattractant Protein-1 (uMCP-1) in Indian type 2 diabetic patients at different stages of diabetic nephropathy

1. Introduction 

Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus and an important cause of increased mortality and morbidity in diabetic patients. Both metabolic and hemodynamic pathways play a crucial role in the progression of diabetic nephropathy [1], [2], [3]. Several pathogenic mechanisms such as presence of glomerular hypertension, hyperglycemia, advanced glycation end products (AGE’s), activation of polyol pathway, high activity of Protein Kinase C (PKC) and genetic susceptibility have been found to be associated with the development and progression of renal impairment related to diabetes [2].

Further, infiltration of the diseased kidneys by inflammatory cells such as monocytes/macrophages is a hallmark of diabetic nephropathy. In DN, similar to the other glomerulonephropathies, infiltration and activation of monocytes/macrophages in the mesangium plays an important role in glomerular injury. Macrophages are thought to be playing a role in tubulo interstitial damage in DN [4], [5], [6], [7]. Recent evidence has highlighted the role of Monocyte Chemoattractant Protein-1 (MCP-1) in DN and showed it as a major factor influencing macrophage accumulation in renal disease. MCP-1 is a member of the CC chemokine family which is produced by endothelial cells, vascular smooth cells, keratinocytes, fibroblasts, mesangial cells, tubular epithelial cells, lymphocytes and monocytes/macrophages in response to a variety of proinflammatory stimuli. It is the strongest known chemotactic factor for monocytes and is upregulated in DN. Its expression has been identified in kidney diseases which involve significant inflammation [2], [8], [9], [10], [11].

Current evidence from both human and animal studies suggests that kidney MCP-1 production plays a critical role in the development and progression of diabetic nephropathy. It was also suggested that increased urinary MCP-1 expression appears earlier than microalbuminuria in diabetes and several studies have reported that urinary MCP-1 levels are increased in inflammatory renal diseases and diabetic nephropathy [5], [11], [12]. A recent study in Chinese population, suggested that MCP-1 may be involved in the development and progression of DN and showed the importance of measuring uMCP-1 [13].

It was also reported that blockade of the Renin Angiotensin system using ACE inhibitors in type 2 diabetic patients with diabetic nephropathy improved renal function by suppressing renal MCP-1 levels [14]. So measuring urinary MCP-1 is of great significance in the diagnosis and intervention of early diabetic nephropathy. There are no studies defining the levels of urinary MCP-1 at different stages of diabetic nephropathy from Indian population, hence this study was planned with the aim of determining the levels of urinary MCP-1 at different stages of diabetic nephropathy and its correlation with other parameters in Indian type 2 diabetic subjects.

2. Research design and methods 

3. Results 

Table 1 shows the demographic, anthropometric and hemodynamic details of the study subjects. The study group (normoalbuminuric, microalbuminuric and proteinuric) subjects were older than control group. Age and BMI was similar among the study subjects in all the groups. Percentage of subjects with hypertension was significantly higher in microalbuminuric and proteinuric groups compared to the normoalbuminuric group.

Table 1. Demographic, anthropometric and hemodynamic details of the study groups.

	Variables	Control	Study groups			p value between study groups by ANOVA
		Group 1 n=20	Group 2 n=16	Group 3 n=23	Group 4 n=25
	M:F	13:7	10:6	13:10	17:8
	Values are mean±SD
	Age (years)	34.4±7.4	56.0±11.0⁎	56.7±10.8⁎	58.1±11.5⁎	0.83
	BMI (kg/m2)	24.0±3.2	26.2±4.1	28.0±4.1⁎	26.6±3.4⁎	0.26
	Blood pressure (mmHg)
	Systolic	114.0±8.8	128.1±11.1⁎	135.2±13.4⁎	142.0±18.9⁎,⁎⁎	0.02
	Diastolic	76.5±6.7	79.4±6.8	79.7±6.7	82.2±10.4	0.49
	Dur DM (years)	–	8.3±7.3	10.0±6.5	14.4±6.7⁎⁎,#	0.013
	Values are n (%); p value by Trend χ2-test
	HTN (%)	–	5 (31.2)	15 (65.2)	21 (84)	0.003
	FH of DM (%)	5 (25)	10 (62.5)	14 (60.9)	17 (68)	0.02
	FH of HTN (%)	1 (5)	3 (13.7)	1 (4.3)	2 (8)	0.302
	Smoking (%)	–	–	2 (8.7)	3 (12)	0.24
	Alcohol (%)	–	1 (6.3)	3 (13)	5 (20)	0.16

FH: family history; Dur DM: duration of diabetes.

⁎ p<0.05 vs Group 1. ⁎⁎ p<0.05 vs Group 2. # p<0.05 vs Group 3.

Table 2 shows the biochemical details of the study subjects. Fasting and 2h plasma glucose values and HbA1c % was similar among the study groups. Mean levels of uMCP-1 in subjects with type 2 diabetes were significantly higher than those in the control group (p<0.05). The levels of uMCP-1 were higher in patients with proteinuria as compared with normoalbuminuria (p<0.05), microalbuminuria (p=0.07) and healthy controls (p<0.05). uMCP-1 levels in Group 3 (microalbuminuric) were significantly higher than Group 1 (normoalbuminuric). There was a significant difference seen in the urinary MCP-1 levels between the normoalbuminuric patients and controls (p<0.001). The levels of uMCP-1 in type 2 diabetic subjects increased gradually with deteriorating renal status (p=0.006). As expected, the urea and creatinine levels were higher in Group 4 compared to Groups 2 and 3. The lipid profile was similar among the study group subjects.

Table 2. Biochemical details of the study groups.

	Variables	Control	Study groups			p value between study groups by ANOVA
		Group 1 n=20	Group 2 n=16	Group 3 n=23	Group 4 n=25
	Plasma glucose (mmol/l)
	Fasting	4.7±0.38	9.1±3.7⁎	9.5±2.7⁎	9.5±4.1⁎	0.93
	2h	5.4±0.5	14.6±6.6⁎	12.9±5.4⁎	13.6±5.9⁎	0.67
	HbA1c (%)	5.5±0.28	8.6±2.1⁎	9.2±2.4⁎	9.0±2.2⁎	0.71
	uMCP-1/U. Cr (pg/mg)	9.8±6.7	70.0±65.3⁎	125±93.4⁎,⁎⁎	196±163.1⁎,⁎⁎	0.006
	Urea (μmol/l)	56.0±10.4	60.2±11.5	66.1±24.4	119±66.4⁎⁎,#	<0.0001
	Sr. Crea (mmol/l)	52.8±8.8	63.4±15.8	76.6±51	123.2±85.4⁎⁎,#	0.006
	T. Chol (mmol/l)	3.6±0.5	4.6±1.1	4.5±1.3	4.5±1.7	0.97
	TG (mmol/l)	0.97±0.37	1.93±0.57	1.61±0.80	2.07±1.03	0.175
	HDL-C (mmol/l)	1.13±0.19	1.04±0.20	1.09±0.16	1.06±0.29	0.788
	LDL-C (mmol/l)	2.25±0.48	2.99±0.81	2.64±0.84	2.62±1.29	0.486
	VLDL-C (mmol/l)	0.24±0.08	0.96±1.04	0.56±0.4	0.80±0.57	0.19

Values are mean±SD.

⁎ p<0.05 vs Group 1. ⁎⁎ p<0.05 vs Group 2. # p<0.05 vs Group 3.

Table 3 shows the levels of uMCP-1 according to eGFR stage wise as per KDOQI guidelines. The levels of uMCP-1 were significantly higher in subjects with eGFR value of <60ml/min compared to the subjects with eGFR values of >60ml/min (p=0.008). Serum protein and serum albumin levels were significantly different among the study subjects according to eGFR stages (p=0.001 and 0.029), respectively.

Table 3. Biochemical details of the study subjects stage wise as per KDOQI guidelines.

	Variable	eGFR (ml/min)				p value between groups by ANOVA
		>90 n=31	60–89 n=19	30–59 n=11	<30 n=3
	Plasma glucose (mmol/l)
	Fasting	9.4±3.4	10.6±4.1	8.4±2.3	5.9±1.6	0.107
	2h	12.9±5.7	16.4±6.3	12.3±4	7.5±2.6	0.031
	HbA1c (%)	8.8±2.1	9.7±2.4	8.5±2.1	7.4±0.8	0.23
	uMCP-1/U. Cr (pg/mg)	110.7±95.7	111±67.7	226.8±203	285.3±221	0.008
	Total protein (g/dl)	7.6±0.6	7.2±0.7	6.9±0.8	6.2±0.98	0.001
	Serum albumin (g/dl)	4.2±0.6	4.0±0.48	3.8±0.4	3.2±0.29	0.029
	T. Chol (mmol/l)	4.5±1.2	4.7±2.6	4±1.4	5.7±3.9	0.54
	TG (mmol/l)	1.6±0.7	1.9±0.6	2.3±1.3	2.1±1.4	0.112
	HDL-C (mmol/l)	1.13±0.22	1.06±0.24	0.93±0.19	1.0±0.15	0.079
	LDL-C (mmol/l)	2.9±0.9	2.6±0.8	2.07±0.8	3.6±2.7	0.04
	VLDL-C (mmol/l)	0.6±0.7	0.9±0.6	0.9±0.6	1.2±1.2	0.25

Values are mean±SD.

Among the study subjects, uMCP-1 levels correlated positively with urinary ACR or PCR (protein to creatinine ratio) (r=0.551, p<0.0001), serum urea (r=0.43, p<0.0001), serum creatinine (r=0.478, p<0.0001), triglycerides (r=0.302, p=0.015) and VLDL cholesterol (r=0.273, p=0.031). A negative correlation between uMCP-1, eGFR (r=−0.338, p=0.006) and HDL cholesterol (r=−0.101, p=0.43) was observed, but HDL cholesterol did not show the statistical significance. No significant correlation was found between urinary MCP-1 and age, BMI, duration of diabetes, systolic and diastolic blood pressure, fasting and post-prandial glucose values, HbA1c, total cholesterol and LDL cholesterol.

4. Discussion 

MCP-1 being a potent chemoattractant for monocytes, increased glomerular expression of MCP-1 has been shown in several glomerular diseases like diabetic nephropathy [17]. Recently, it has been considered that diabetic nephropathy is an inflammatory disease [18]. Several studies have shown the associations of MCP-1 and renal injury among diabetic patients [5], [11], [19]. The levels of uMCP-1 at different stages of DN among Indian type 2 diabetic subjects are unknown. The current study findings showed that urinary MCP-1 levels were increased in diabetic patients with proteinuria compared to normoalbuminuric, microalbuminuric and control subjects. Since the results of our study also showed the increasing uMCP-1 levels with the progression of diabetic nephropathy, inflammation can be considered as one of the major cause and the increased levels indicates the extent of renal tubular damage. Proteinuria itself may contribute to this damage thereby increasing the expression of MCP-1 in renal tubule which further accelerate the progression of DN. Similar findings were also observed in a study conducted by Wang and Chen [13] which showed the increasing urinary MCP-1 levels with deteriorating renal function and found that uMCP-1 along with other risk factors is associated with diabetic nephropathy.

The levels of uMCP-1 in normoalbuminuric group were significantly higher than those in healthy controls in our study. Similarly, several studies reported that MCP-1 appeared earlier than urine microalbumin [13], [19], [20]. The above findings of our study suggested that some changes would have occurred early in the pathogenesis of DN and MCP-1 may play an important role in the progression and development of DN. In our study, uMCP-1 levels correlated positively with urinary ACR or PCR, serum urea, serum creatinine, triglycerides and VLDL cholesterol and negatively with eGFR. Banba et al. [11] showed a highly significant correlation between urinary levels of albumin and MCP-1 in diabetic subjects. Similarly many studies showed that uMCP-1 levels positively correlated with albumin excretion rate [13], [21]. Banba et al. also found that urine levels but not serum levels of MCP-1 increased in accordance with the extent of HbA1c and albuminuria [11] but in our study we did not find a significant correlation with HbA1c and more over we have not estimated MCP-1 in serum or plasma samples.

Urinary MCP-1 levels inversely correlated with eGFR values in our study. Our finding agrees with a recent report, which showed an inverse correlation with eGFR [21]. The inverse correlation between MCP-1 and eGFR define the use of MCP-1 as a marker which reflects the degree of kidney damage as estimated by glomerular filtration rate. Earlier reports [13], [21], [22] demonstrated a significant positive correlation between MCP-1 and glycemic control as indicated by HbA1c levels but we did not find a significant correlation either with glucose levels or with HbA1c in our study. This suggests that hyperglycemia alone may not be sufficient in determining increased MCP-1 expression and other factors may be involved in the process which needs to be confirmed in a large sample size. There was no significant correlation found between uMCP-1 and age, duration of diabetes, BMI, systolic and diastolic blood pressure, plasma glucose values, HbA1c, total cholesterol and LDL-cholesterol in our study. Another report also showed no correlation between MCP-1 and age, BMI and duration of diabetes [21].

As far as treatment regimen is concerned, current treatment for diabetic nephropathy includes glycemic control, use of an appropriate antihypertensive to keep blood pressure under control, use of statins, diuretics and diet management but still many patients progress rapidly with a deteriorating renal function. This proves the role of inflammation in the pathogenesis of diabetic nephropathy and also suggesting a need for additional immunotherapy as an adjunct to the existing treatment regime. Some studies showed [14], [22] that intervention with ACE inhibitor or vitamin E supplement decreased the level of MCP-1 and improved the renal function among diabetic subjects with diabetic nephropathy. The limitations of our study are that being a cross-sectional study we could not define if reducing MCP-1 levels improves renal function in our population. We could not correlate urinary MCP-1 with actual measurement of 24h protein which is a gold standard method. However, a good correlation between ACR in a spot urine sample and 24h urine collection has been reported earlier [23]. Long term clinical trials are required in determining the importance of reduction of MCP-1 level in improving renal status. The findings of our study certainly lay a foundation to consider uMCP-1 as a new and non-invasive diagnostic marker for early diagnosis of diabetic nephropathy. It may be useful in evaluating and monitoring the renal inflammation in patients with DN.

In conclusion, our study showed the increasing levels of uMCP-1 in type 2 diabetic subjects with deteriorating renal function and it is significantly associated with the other risk factors of diabetic nephropathy. The diagnostic value of urinary MCP-1 to assess the effectiveness of therapy needs to be studied.