Urinary Platelet-Derived Growth Factor-BB as an Early Marker of Nephropathy in Patients with Type 2 Diabetes: An Egyptian Study

Abstract

Background: Diabetic nephropathy (DN) is one of the most serious complications of diabetes worldwide. Strong evidence suggests that several growth factors may contribute to the initiation and progressive fibrosis of DN. Recently, there is an overexpression of platelet-derived growth factor (PDGF) in renal biopsies from patients with DN. This study aimed to investigate the clinical significance of urinary PDGF-BB level in type 2 diabetic patients with and without nephropathy and to evaluate its relationship with various clinical and laboratory parameters. Methods: Urinary levels of PDGF-BB were measured in 60 Egyptian type 2 diabetic patients categorized into three equal groups (normo-, micro-, and macroalbuminuria), according to urinary albumin level. In addition, 20 healthy subjects were selected to serve as controls. Results: The urinary PDGF-BB levels were significantly increased in type 2 diabetic patients as compared to controls (p < 0.001). Moreover, diabetics with micro- and macroalbuminuria had significantly higher levels than in those with normoalbuminuria (p < 0.001). Urinary PDGF-BB correlated positively with disease duration, low-density lipoprotein (LDL)-cholesterol, and urinary albumin and negatively with creatinine clearance in diabetic patients. In a multiple regression model, urinary PDGF-BB was strongly and independently associated with nephropathy in diabetic patients (β = −0.03, p < 0.001). Conclusions: PDGF-BB may play an important role in the initiation and progression of DN. It is considered as a good predictor for early deterioration of renal function in DN. Thus, measurement of urinary PDGF-BB in type 2 diabetic patients could be used for early detection of diabetic renal disease.

• platelet-derived growth factor-BB
• early detection
• albuminuria
• diabetic nephropathy
• type 2 diabetes

INTRODUCTION

Diabetic nephropathy (DN) is one of the major microvascular complications of diabetes and is an important cause of increased morbidity and mortality.¹ It is characterized by thickening of glomerular and tubular basement membranes, and increased amount of mesangial matrix, which ultimately progress to glomerulosclerosis and tubulointerstitial fibrosis.²

The pathogenetic mechanisms responsible for progressive renal impairment of DN are still poorly understood, despite its growing incidence. Increasing evidence suggests that growth factors may contribute to the initiation and progressive fibrosis of DN.^3,4 Several growth factors have been implicated in the pathogenesis of DN such as transforming growth factor-beta (TGF-β),⁵ connective tissue growth factor,⁶ vascular endothelial growth factor,⁷ growth hormone, and insulin-like growth factors.

Platelet-derived growth factor (PDGF) is a polypeptide that was originally purified from human platelets as a potent mitogen for fibroblasts, osteoblasts, smooth muscle, and mesangial cells.^8–11 Three disulfide-linked isoforms of PDGF (PDGF-AA, PDGF-BB, and PDGF-AB) and two classes of PDGF receptors (PDGFR-α and PDGFR-β) have been identified.¹² Moreover, PDGF-C and PDGF-D are growth factors belonging to the PDGF family.^13–15 The PDGF-B chain exerts its biological effects by specifically binding to both classes of the PDGFR and is a more powerful factor than PDGF-A for mesangial cell proliferation.^16,17 It can induce the synthesis of TGF-β which has a fibrogenic action and plays a key role in the development of renal hypertrophy and accumulation of extracellular matrix in diabetes.^18,19

Recently, PDGF-B mRNA expression was upregulated in renal biopsies from type 2 diabetic patients with overt DN.^20,21 However, the predictive value of urinary PDGF-BB for early diagnosis of DN is still limited. This study was conducted to investigate the clinical significance of urinary PDGF-BB level in type 2 diabetic patients with and without nephropathy and to evaluate its relationship with various clinical and laboratory parameters.

MATERIALS AND METHODS

Subjects

Sixty Egyptian type 2 diabetic patients were selected from those attending the Internal Medicine Diabetic Clinic at Tanta University Hospital. Type 2 diabetes was diagnosed based on the World Health Organization criteria. Twenty healthy age- and sex-matched volunteers (10 men and 10 women) with a mean age of 53.3 ± 8.5 years were recruited for participation as control subjects. They were selected from medical and paramedical staff and from blood donors in Tanta University Hospital. The study was approved by the scientific and ethics committees of the Tanta University Hospital, Tanta University, Tanta, Egypt, and informed consent was obtained from each subject prior to the start of this study. Patients were divided into three groups according to their level of urinary albumin excretion. Group 1 included 20 diabetic patients, 8 men and 12 women, with normoalbuminuria (albuminuria <30 mg/24 h); their mean age was 52.5 ± 10.6 years, while the mean disease duration was 8.15 ± 1.3 years. Group 2 included 20 diabetic patients, 7 men and 13 women, with microalbuminuria (albuminuria 30–300 mg/24 h); their mean age was 54.9 ± 9.7 years, while the mean disease duration was 9.2 ± 2.4 years. Group 3 included 20 diabetic patients, 9 men and 11 women, with macroalbuminuria (albuminuria >300 mg/24 h); their mean age was 57.4 ± 11.3 years, while the mean disease duration was 10.3 ± 6.5 years. Patients with cardiac, hepatic, chronic obstructive pulmonary disease, or other kidney disease were excluded from the study. Also subjects with no evidence of diabetic retinopathy by fundus examination but with evidence of micro- or macroalbuminuria were excluded from this study as we considered very likely that the albuminuria was not because of diabetic renal disease. All cases included in this study were subjected to careful history taking, complete clinical examination, laboratory investigations, and abdominal ultrasonography.

Sample Collection and Laboratory Analyses

An early morning urine sample was collected from each subject for complete urine analysis and detection of microalbuminuria with a Micral test.²² Twenty-four-hour urine collections were performed for estimation of creatinine and quantitative assessment of albuminuria using an immunoturbidimetric assay.²³ The urine samples were centrifuged at 2000 × g for 10 min and 2 mL supernatant was taken and stored at −70°C until measurement of PDGF-BB level. A fasting blood sample was collected aseptically from all subjects and a 2-h postprandial blood sample was also collected for postprandial blood glucose estimation. An aliquot of fasting blood sample was collected into ethylenediaminetetraacetic acid tube and analyzed within a few hours to estimate the percentage of glycosylated hemoglobin (HbA1_c) using a fast ion exchange resin separation method,²⁴ with kits supplied by Stanbio Laboratory (Ambion, Austin, TX, USA). The remaining fasting blood sample was allowed to clot at room temperature for about 1 h; it was then centrifuged for at least 10 min at 2000 × g, and the serum was aspirated and divided into aliquots in small plastic tubes for measurement of fasting blood glucose, triglycerides, cholesterol, blood urea, and serum creatinine using commercial assay kits supplied by Bicon Co. (Mannheim, Germany) and the creatinine clearance was calculated.

Measurement of Urinary PDGF-BB Level

Urinary PDGF-BB levels were measured with a solid-phase sandwich enzyme-linked immunosorbent assay, according to the manufacturer’s instructions²⁵ (Quantikine, Human PDGF-BB Immunoassay Kit, R&D Systems, DBB00, Minneapolis, MN, USA). In brief, standards of recombinant human PDGF-BB and undiluted urine samples were added to microtiter plates precoated with human PDGF Rβ/Fc chimera and incubated for 2 h at room temperature. Wells were then washed with the washing buffer and incubated for 1.5 h at room temperature with a polyclonal antibody against PDGF-BB conjugated to horseradish peroxidase. After four washes, substrate solution was added, the plate was incubated for 30 min at room temperature in the dark, and the reaction was stopped with the stop solution before measuring the optical density of each well at 450 nm with a microtiter plate reader. The concentration of PDGF-BB was determined by interpolation from the standard curve.

Statistical Analysis

Results were expressed as mean ± SD. Comparisons between groups were made using Student’s t-test for continuous variables. Correlation between two parameters was determined by Pearson’s correlation coefficient (r). Multiple regression analysis was used to assess the predictors of nephropathy among diabetic patients; p-value of less than 0.05 was considered as statistically significant.

RESULTS

The main clinical and laboratory parameters of type 2 diabetic patients are shown in Table 1. There were no statistically significant differences among the three diabetic groups (normo-, micro-, and macroalbuminuria) and controls regarding age, sex, and high-density lipoprotein (HDL)-cholesterol levels. Diabetics with macroalbuminuria had significantly higher HbA1_c (%), total cholesterol, low-density lipoprotein (LDL)-cholesterol, and triglyceride levels (p < 0.001) and significantly lower creatinine clearance (p < 0.001) than in those with microalbuminuria. The urinary PDGF-BB levels were significantly higher in the diabetic groups with normo-, micro-, and macroalbuminuria than those in healthy controls (p < 0.001 for all). Moreover, diabetics with micro- and macroalbuminuria had significantly higher levels of urinary PDGF-BB than in those with normoalbuminuria (p < 0.001 for both). The urinary PDGF-BB levels were increased in diabetics with macroalbuminuria as compared to those with microalbuminuria, but there were no significant differences between them, as shown in Table 1 and Figure 1.

Table 1.  Clinical and laboratory parameters of type 2 diabetic patients with and without nephropathy.

Parameters	Controls (n = 20)	DM with normoalbuminuria (n = 20)	DM with microalbuminuria (n = 20)	DM with macroalbuminuria (n = 20)
Age (years)	53.3 ± 8.5	52.5 ± 10.6	54.9 ± 9.7	57.4 ± 11.3
Sex (M/F)	10/10	8/12	7/13	9/11
Disease duration (years)	—	8.15 ± 1.3	9.2 ± 2.4	10.3 ± 6.5
Fasting glucose (mg/dL)	78.2 ± 8.4	122.8 ± 7.9^a	164.5 ± 10.8^b	240.6 ± 14.2^c,d
Postprandial glucose (mg/dL)	115.4 ± 10.9	179.8 ± 13.6^a	268.4 ± 12.8^b	360.3 ± 20.5^c,d
HbA1c (%)	4.8 ± 0.4	6.2 ± 0.9^a	7.5 ± 0.7^b	9.4 ± 1.5^c,d
Total cholesterol (mg/dL)	170.6 ± 16.9	182.5 ± 28.4	217.3 ± 36.8^e	336.2 ± 26.5^c,d
HDL-cholesterol (mg/dL)	48 ± 5.6	45.3 ± 4.2	44.6 ± 6.6	41.8 ± 8.5
LDL-cholesterol (mg/dL)	109 ± 13.2	115 ± 14.9	163 ± 22^b	266 ± 41^c,d
Triglycerides (mg/dL)	85.5 ± 15.9	96.8 ± 23.6	118.2 ± 42.2	157.1 ± 20.4^c,d
Blood urea (mg/dL)	36.9 ± 6.2	40.8 ± 8.6	44.5 ± 10.2	60.3 ± 13.6^c,d
Serum creatinine (mg/dL)	0.8 ± 0.3	1.0 ± 0.4	1.3 ± 0.5^f	1.8 ± 0.6^c,g
Creatinine clearance (mL/min)	106.3 ± 11.4	100.0 ± 14.2	82.6 ± 13.8^b	58.9 ± 17.3^c,d
Urinary albumin (mg/24 h)	13.5 ± 3.8	16.2 ± 5.3	174.7 ± 45.3^b	690.2 ± 111.3^c,d
Urinary PDGF-BB (pg/mL)	84.3 ± 41.8	278.5 ± 230.9^a	1680 ± 885.9^b	2255 ± 992.2^c

Notes: Data are expressed as mean ± SD. HDL, high-density lipoprotein; LDL, low-density lipoprotein; PDGF-BB, platelet-derived growth factor-BB; DM, diabetes mellitus.

^ap < 0.001 normoalbuminuria versus controls.

^bp < 0.001 microalbuminuria versus normoalbuminuria.

^cp < 0.001 macroalbuminuria versus normoalbuminuria.

^dp < 0.001 macroalbuminuria versus microalbuminuria.

^ep < 0.01 microalbuminuria versus normoalbuminuria.

^fp < 0.05 microalbuminuria versus normoalbuminuria.

^gp < 0.01 macroalbuminuria versus microalbuminuria.

Figure 1.  Urinary platelet-derived growth factor-BB (PDGF-BB) levels in type 2 diabetic patients with and without nephropathy and controls.

In this study, urinary PDGF-BB in diabetic patients was positively correlated with disease duration (r = 0.294; p < 0.05), LDL-cholesterol (r = 0.393; p < 0.01), and urinary albumin levels (r = 0.711; p < 0.001) and negatively correlated with creatinine clearance (r = −0.615; p < 0.001), while had no significant correlations with HbA1_c, total cholesterol, HDL-cholesterol, and triglyceride levels, as shown in Table 2.

Table 2.  Correlation between urinary PDGF-BB and other parameters in type 2 diabetic patients.

	Disease duration (years)	HbA1c (%)	Total cholesterol (mg/dL)	HDL-cholesterol (mg/dL)	LDL-cholesterol (mg/dL)	Triglycerides (mg/dL)	Creatinine clearance (mL/min)	Urinary albumin (mg/24 h)
PDGF-BB (pg/mL)	r = 0.294	r = 0.124	r = 0.206	r = 0.196	r = 0.393	r = 0.212	r = −0.615	r = 0.711
	p < 0.05	p > 0.05	p > 0.05	p > 0.05	p < 0.01	p > 0.05	p < 0.001	p < 0.001

Notes: PDGF-BB, platelet-derived growth factor-BB; HDL, high-density lipoprotein; LDL, low-density lipoprotein.

p-Value of <0.05 was considered as significant.

Table 3.  Multiple regression analysis of predictors of nephropathy among diabetic patients.

Independent variables	β	Standard error	Partial r^2	p-Value
Disease duration (years)	0.002	0.015	0.08	<0.05
HbA1c (%)	0.05	0.003	0.06	>0.05
Total cholesterol (mg/dL)	0.006	0.04	0.003	>0.05
HDL-cholesterol (mg/dL)	−0.06	0.03	0.010	>0.05
LDL-cholesterol (mg/dL)	0.07	0.012	0.07	<0.05
Triglycerides (mg/dL)	−0.003	0.100	0.001	>0.05
Urinary albumin (mg/24 h)	−0.04	0.025	0.17	<0.01
Urinary PDGF-BB (pg/mL)	−0.03	0.004	0.10	<0.001

Notes: HDL, high-density lipoprotein; LDL, low-density lipoprotein; PDGF-BB, platelet-derived growth factor-BB.

p-Value of <0.05 was considered as significant.

Table 3 represents multiple regression analysis to identify independent predictors of nephropathy among diabetic patients. It was found that disease duration (β = 0.002, p < 0.05), LDL-cholesterol (β = 0.07, p < 0.05), urinary albumin (β = −0.04, p < 0.01), and urinary PDGF-BB (β = −0.03, p < 0.001) were independently associated with nephropathy.

DISCUSSION

DN is one of the most serious complications of diabetes worldwide. Elucidation of the mechanisms involved in its pathogenesis and/or progression is therefore very important. The histopathological hallmarks of DN are increased thickness of the glomerular basement membrane and mesangial expansion, which has been reported to be the critical lesion of DN because glomerulosclerosis and the decline in renal function correlate well with the degree of mesangial expansion.²⁶

Considerable evidence suggests that growth factors may contribute to the initiation and progressive fibrosis of DN.^3,4 Exploration of PDGF in the human kidney has mostly focused on its role in mesangial cell proliferation with PDGF upregulation reported in lupus nephritis,²⁷ IgA nephropathy,²⁸ chronic allograft nephropathy,²⁹ and proliferative glomerulonephritis.³⁰ PDGF-BB is one of the classical isoforms of PDGF, compared to the other forms of PDGF such as PDGF-AA, PDGF-AB, PDGF-CC, and PDGF-DD, the relationship between which and DN is recently explored in some studies.¹⁷ However, clinical evidence for the role of PDGF-BB in the pathogenesis of DN is limited.

This study has investigated the clinical significance of urinary PDGF-BB level in type 2 diabetic patients with and without nephropathy and demonstrated that the urinary levels of PDGF-BB were significantly increased in diabetic patients as compared to controls. Moreover, diabetics with micro- and macroalbuminuria had significantly higher levels than in those with normoalbuminuria. However, no significant difference was found between diabetics with micro- and macroalbuminuria. These results are in agreement with the findings of Wang et al.³¹ who observed that the levels of urinary PDGF-BB in the group of macroalbuminuria were higher than those in the group of microalbuminuria, with no significant differences between them.

In addition, Fagerudd et al.³² studied the urinary excretion of PDGF-BB in patients with insulin-dependent diabetes mellitus of a long duration and controls and found that excretion of PDGF-BB was significantly higher in patients with micro- and macroalbuminuria compared to normoalbuminuria patients, but there was a considerable overlap between the groups. Their results were similar to this study. It is thus evident that the excretion rates of PDGF-BB in type 1 diabetes are similar to that in type 2 diabetes. The possible mechanism may be that the histopathological hallmarks in the early stage of DN are mainly the proliferation of mesangial cells which secrete PDGF-BB, while that in the later stage are mainly the accumulation of extracellular matrix and the thickening of glomerular basement membrane. Therefore, the extent of urinary excretion of PDGF-BB in the early stage of DN is large and that in the later stage is small.

In this study, urinary PDGF-BB correlated positively with urinary albumin and negatively with creatinine clearance in diabetic patients. These data suggest that detecting the urinary levels of PDGF-BB can be a marker for early structural change of DN, and it is a sensitive indicator for early deterioration of renal function in DN.

On the other hand, Lev-Ran and Hwang³³ observed elevated serum PDGF levels in diabetics as compared with controls. Similarly, Guillausseau et al.³⁴ suggested that PDGF release might be increased in diabetic subjects, and this may account in part for the cell proliferation observed in diabetic angiopathy. Furthermore, there were a lot of studies indicating that the expression of PDGF-BB in DN was increased. Nakagawa et al.³⁵ conducted immunohistochemical analysis for PDGF-BB and PDGFR-β in the glomeruli of streptozotocin-induced diabetic rats and found that the protein expression of PDGF-BB and PDGFR-β was actually increased. Uehara et al.²¹ determined the expression of PDGF and PDGFR mRNA in the renal tissues of type 2 diabetic patients with DN and found that the percentages of PDGF-B and PDGFR-β mRNA-positive cells in DN were significantly higher than those in normal human kidneys. Moreover, Langham et al.²⁰ demonstrated that the gene and protein expression of PDGF-BB in renal biopsies from patients with DN was increased, which implied a potential role for PDGF-BB in the development of the progressive fibrosis that characterizes human diabetic kidney disease.

Interestingly, it has been suggested that hyperglycemia increased the expression of PDGF-BB and PDGFR-β in macrophage, mesangial cell, and vascular endothelial cell by the activation of protein kinase C signaling pathways.³⁶

With respect to lipid profile, this study revealed that urinary PDGF-BB correlated positively with LDL-cholesterol in diabetic patients. Of interest, Nishida et al.³⁷ reported that LDL and triglyceride-rich lipoproteins (very low-density lipoprotein and intermediate density lipoprotein) caused proliferation of mesangial cells, and oxidized LDL had a cytotoxic effect on mesangial cells. It is possible that these actions are mediated via cytokines such as interleukin-6, tumor necrosis factor-alpha, TGF-β, and PDGF.

It is worth noting that Tang et al.³⁸ examined the effect of daily PDGF-BB administration on renal tubulointerstitial architecture in rats and found that PDGF-BB induced dose-dependent renal tubulointerstitial cell proliferation and fibrosis and it may be an important mediator of tubulointerstitial hyperplasia and fibrosis. In addition, Wang and Hirschberg³⁹ reported that proteinuria induced renal interstitial fibrosis and accelerated progression of renal impairment in DN, and PDGF-BB took part in this pathological change. Thus, PDGF-BB is a highly interesting therapeutic target for treatment of renal fibrosis in DN.

The cytokine TGF-β has emerged as having a key role in the development of renal hypertrophy and accumulation of extracellular matrix in diabetes. Fraser et al.⁴⁰ confirmed that PDGF-BB could increase the expression of TGF-β in tubular cell. Glucose sensitized the PDGFR-α, which lowered the threshold at which a stimulus such as PDGF-BB stimulates TGF-β protein synthesis.

In view of these results, possible factors which can decrease the expression of PDGF-BB and its signal transduction system should be investigated in order to provide new approaches to prevent the development and retard the progression of DN. Importantly, Vieitez et al.⁴¹ confirmed that administration of angiotensin receptor blockers such as irbesartan decreased glomerular expression of PDGF-BB, due to a direct blockade of glomerular renin–angiotensin system. It would be very interesting if the delay of progression of DN provided by angiotensin receptor blockers or angiotensin-converting enzyme inhibitors can be seen in the urine of patients by a reduced excretion of PDGF-BB particularly in patients with newly detected type 2 diabetes. Therefore, measuring urinary excretion of PDGF-BB would give the clinician a helpful tool to watch their therapeutic strategy.

Indeed, other novel strategies such as specific inhibitors of growth factors, particularly PDGF-BB, will need to be developed to open new and exciting avenues of research with possible future therapeutic implications that would be more efficacious in arresting or even reversing DN in order to improve the quality of life of patients afflicted with diabetes.

In conclusion, PDGF-BB may play an important role in the initiation and progression of DN. It is considered as a good predictor for early deterioration of renal function in DN. Thus, measurement of urinary PDGF-BB in type 2 diabetic patients could be used for early detection of diabetic renal disease. However, large-scale future prospective studies are required to better clarify the exact role of PDGF-BB in the pathogenesis of DN and to explore the potential benefit of specific PDGF inhibitors as a promising novel therapeutic approach to DN.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.