Plasma endothelin-1 level, measured glomerular filtration rate and effective renal plasma flow in diabetic nephropathy

Abstract

Background: Endothelin-1 (ET-1) is potent vasoconstrictor peptide which is able to contribute to the functional and structural renal changes. The aim of this study was to investigate the relationship between plasma concentration of ET-1 and indices of renal function in patients with diabetic nephropathy. Methods: We measured plasma ET-1 levels in 99 patients with type 2 diabetes, divided into two groups according to the values of their glomerular filtration rate (GFR): group I (GFR ≥ 60 mL/min/1.73 m²; n = 50), group II (GFR ≥ 60 mL/min/1.73 m², n = 49), and the control group (n = 30) with clinically healthy subjects who were matched by age and sex. GFR and effective renal plasma flow (ERPF) were measured by the radioisotopic clearance. Other renal function parameters, such as serum concentrations of cystatin C, urea, creatinine, uric acid, 24-h albuminuria and proteinuria were additionally measured. Results: There were significant differences in plasma concentration of ET-1 among groups I, II and the control group (1.45 vs. 2.40 vs. 0.80 pg/mL, p < 0.001). The correlation between ET-1 and mGFR (r = −0.52, p < 0.001), ERPF (r = −0.42, p < 0.001), albuminuria and proteinuria (r = 0.36, p < 0.001; r = 0.48, p < 0.001) and cystatin C (r = 0.42, p < 0.001) was significant. In multiple regression analyses, only plasma concentration of ET-1 (p < 0.001) and duration of hypertension (p < 0.05) were independently and significantly associated with mGFR. Conclusion: A higher plasma concentration of ET-1 is independently associated with a decreased value of GFR in patients with diabetic nephropathy.

• Type 2 diabetes
• diabetic nephropathy
• endothelin-1
• glomerular filtration rate
• effective renal plasma flow

Introduction

Diabetic nephropathy (DN) is one of the most serious microvascular complications of diabetes, recognized today as the leading cause of end-stage renal diseases and it approximately affects 30–40% of type 2 diabetic patients.1^,2 During the past two decades, numerous research papers have lightened a few new paths explaining the development and progression of DN. Systemic hypertension, as well as intraglomerular hypertension, and the activation of vasoactive systems including renin-angiotensin system and endothelin system are well known hemodynamic factors that contribute to the development of DN. On the other hand, an elevated glucose level leads to increased oxidative stress and the activation of alternative metabolic pathways followed by the formation of polyols and the advanced glycation end products.3–5 Combining the pathological mechanisms mentioned above results in an increased permeability of renal capillaries to plasma proteins and enhanced extracellular matrix (EM) accumulation, which consequently leads to proteinuria, glomerulosclerosis and tubulointerstitial fibrosis.6

While the pathophysiology of diabetic nephropathy is complex, endothelin-1 (ET-1), a potent vasoconstrictor with proliferative, profibrotic and proinflammatory properties, may contribute to many aspects of diabetic microvascular diseases.7 A scientist Paul Michel Vanhoutte was the first to report endothelial, cell-derived vasoconstriction in 1981, which was later identified as the endothelin peptide by Yanagisawa et al. in 1988.8^,9 Endothelins are a group of endogenous peptides with a similar structure which have a powerful and long-lasting vasoconstrictive effect. The endothelin family, which is widely distributed in various cells and tissues, along with the ET-1 consists of two other members: endothelin 2 and endothelin 3, both with very similar structure. Endothelin receptor A and endothelin receptor B, two subtypes of ET receptor, have been shown to mediate ET-1 action.10^,11

Hyperglycemia and impaired metabolic control may enhance vascular production of ET-1 in diabetes, which leads to glomerular damage by increasing vasoconstriction, mesangial cell proliferation and glomerular permeability.12 Increased plasma concentration of ET-1 in patients with diabetes has been reported,13^,14 and a significant correlation has also been observed between plasma ET-1 levels and diabetic complications15^,16 but the relationship between ET-1 and indices of renal function is conflictive. It is known that patients with advanced chronic kidney disease or on dialysis have higher plasma concentrations of ET-1.17–20 A small number of clinical studies have examined the relationship of plasma ET-1 to GFR across different stages of renal impairment. Therefore, the aim of this study was to evaluate the correlation between serum concentrations of ET-1 and renal functional parameters (glomerular filtration rate and effective renal plasma flow) in patients with diabetic nephropathy.

Materials and methods

This cross sectional study was done at the Clinical Center of Vojvodina (CCV), Novi Sad, Serbia, from June 2012 to July 2013. The study was conducted according to the principles of the Helsinki Declaration and it was approved by the Ethics Committee of CCV. Informed consent for participating in the study was obtained from all the participants prior to their inclusion in the study.

Study group

A total of 99 outpatients with type 2 diabetes who were secondary insulin-dependent, treated at the Clinic of Endocrinology, Diabetes and Metabolic Disorders, CCV were included in the study. The control group was formed from 30 healthy subjects matched by age and sex. Diabetic patients were divided into two groups according to their values of measured GFR (mL/min/1.73 m²): the first group (GFR ≥ 60 mL/min/1.73 m²) was formed from 50 diabetic patients (20 male/30 female) and the second group (GFR < 60 mL/min/1.73 m²) consisted of 49 patients (24 male/25 female).

All subjects were physically examined, their height and weight were obtained and their body mass index was calculated. Laboratory evaluation was performed in the morning, after 12-hour fasting and a 30-min resting period. Their blood samples and 24-hour urine samples were taken on the same day, and GFR and ERPF were also measured for all subjects. Blood samples were taken just before carrying out the isotopic clearance method. All respondents were previously informed on how to collect their 24-hour urine samples both orally and in writing. Analyses were performed immediately after blood sampling, except for ET-1 (the samples were kept frozen at −70 °C no longer than one month before the determination of ET-1).

The basic criterion for inclusion of patients in the study was the presence of diabetic nephropathy based on relevant parameters (persistent urinary albumin excretion (UAE) >30 mg/24 h or/and decreased value of GFR). The excluding factors were if the level of measured GFR was less than 15 mL/min/1.73 m², any cardiovascular event (AMI, percutaneous coronary angioplasty or revascularization, stroke and peripheral arterial disease) during the 6 months preceding the enrollment; any malignancy, presence of liver disease, or any acute inflammatory or infectious process.

Assessment of renal function

GFR measurement

Assessment of GFR (by using a single-spaced model)20 was determined by the isotopic clearance of ^99mTc-labeled diethylene triamine pentaacetic acid (^99mTc-DTPA), using a single injection at a dose of 37 MBq, in two blood samples, taken after 180 and 240 min.

Normal GFR values were calculated using regression equations by Schernthaner et al.21 as follows:

• For women: GFR = 144.1 − (0.99 × years of age)

• For men: GFR = 160.5 − (1.16 × years of age)

The GFR values were expressed as mL/min/1.73 m² and variations in GFR values compared to reference values (expected values for the sex and age) as mL/min and percentage.

ERPF measurement

Effective renal plasma flow was determined by the isotopic clearance of ¹³¹I-labeled orthoiodo hippuric acid (hippuran) in two blood samples, collected after 20 and 30 min using the Blaufox method.22 Normal ERPF values were calculated using regression equations by Schernthaner et al.21 as follows:

• For women: ERPF = 673.3 − (2.92 × years of age)

• For men: ERPF = 854.2 − (5.4 × years of age)

The ERPF values were expressed as mL/min/1.73 m² and variations in ERPF values compared to reference values (expected values for the sex and age) as mL/min and percentage.

24-h Proteinuria and albuminuria

Proteinuria was determined by using a pyrogallol red biochemical analyzer ADVIA 1800 and Siemens commercial kits (Siemens Healthcare Diagnostics, Tarrytown, NY) with a reference range of <150 mg/24 h. Albuminuria was determined using the immunometric assay sandwich method with the use of commercial NycoCard tests (Oslo, Norway) on the NycoCard reader, Norway, with a reference range of <30 mg/24h. Microalbuminuria was defined as UAE between 30 and 299 mg/24 h and macroalbuminuria was defined as UAE ≥300 mg/24 h.

Serum concentration of cystatin C was determined by the immunoturbidimetric method using a Dyazime commercial kit on the biochemical analyzer Olympus AU400 (Tokyo, Japan). The reference range for cystatin C was from 0.5 to 1.03 mg/L. Serum concentration of creatinine, urea and uric acid was determined by standard biochemical methods, on the same analyzer.

ET-1 measurements

To measure plasma concentration of ET-1, venous blood was drawn and stored in a refrigerated tube containing EDTA. The samples were centrifuged and stored for subsequent blind analyses performed simultaneously for all samples. ET-1 measurements were taken by indirect enzyme linked immunosorbent assay (R&D Systems, Minneapolis, MN) on the biochemical analyzer RAYTO (Shenzhen, Guangdong, China). The antibody specific to ET-1 was pre-coated on a microplate. Standards, samples, control and conjugates were pipetted into the wells. Any ET-1 present in the sample is sandwiched by the immobilized antibody and enzyme linked antibody specific to ET-1. Then the washing substrate is added for color development. This process is stopped by adding sulphuric acid and the color is measured spectrophotometrically. The absorbance is inversely proportional to the concentration of ET-1 in the sample. The results were expressed as pg/mL.

Furthermore, in all respondents serum concentration of glucose was measured by the glucose oxidase method with a reference range: 4.0–5.9 mmol/L and glycated hemoglobin A1c (HbA1c) by the immuno-inhibitory test (Beckman-Coulter, Kildare, Ireland) with reference range: 4.7–6.0%.

Statistical analysis

The distribution of numeric variables was tested using the Kolmogorov-Smirnov test.23 The data were presented as mean ± standard deviation (SD) or median and interquartile range for continuous variables. Categorical data were summarized as numbers and percentages. Independent-samples one-way analysis of variance parametric test, and Kruskal–Wallis H non-parametric tests were used for the comparison of continuous variables and a chi-square analysis was used for categorical variables. Pearson’s and Spearman’s coefficient of linear correlation was used to study the correlation between the variables. Multiple regression analysis was performed to estimate the independent contribution of plasma ET-1 concentration to GFR. We included variables that met statistical significance in univariate analysis, plasma concentration of ET-1, albuminuria, proteinuria and the duration of HT. A statistical analysis was performed using SPSS version 12.0 (StatSosft inc., Tulsa, OK) for Windows. A difference was considered significant if the p value was less than 0.05.

Results

The main characteristics of the study subjects are shown in Table 1.

Table 1. Clinical and laboratory characteristics of the study population.

	mGFR ≥ 60 mL/min/1.73 m^2	mGFR < 60 mL/min/1.73 m^2	CG
Number (f/m)	30/20	25/24	17/13
Age (year)	61 (40–77)	62 (47–78)	60 (43–76)
BMI (kg/m^2)	28.3 ± 5.4^b1	28.9 ± 4.2^b2	25.5 ± 2.8
Diabetes duration (year)	13 (7–17)	15 (10–20)	–
Insulin therapy (year)	3 (1–20)	4.5 (1–16)	–
HT – n	37 (50)	43 (49)	–
ACEI use – n	33 (37)	34 (43)	–
HT duration (year)	8.5 (1–20)^a1	11 (1–38)	–
Fasting glucose (mmol/L)	8.7 (4–15.6)^b3	8.8 (4–16.9)^b3	5.3 (3.8–6.1)
HbA1c (%)	7.7 (5.4–10.5)^b3	7.9 (6–11.5)^b3	5.3 (4.5–6.1)

Notes: CG – control group, BMI – body mass index, ACEI – angiotensin-converting enzyme inhibitor, HT – hypertension, HbA1c – glycated hemoglobin, ^a1p < 0.05 compared to II group; ^b1p < 0.05, ^b2p < 0.01, ^b3p < 0.001 compared to the control group.

Duration of diabetes, duration of insulin therapy, metabolic regulation of diabetes (fasting glucose and HbA1c), the presence of hypertension (HT) and the use of ACE inhibitors, as well as the value of body mass index, were similar in both groups of diabetic patients with a different degree of reduction in mGFR. Diabetic patients in group II had a significantly longer duration of HT [8.5 (120) vs. 11 (1–38) years, p < 0.05).

Renal function parameters of the study subjects are shown in Table 2.

Table 2. Renal function parameters of diabetic patients with mGFR > 60 mL/min/1.73 (I group), mGFR < 60 mL/min (II group), and the control group (CG).

	I Group	II Group	CG
mGFR (mL/min/1.73 m^2)	73 ± 8	44 ± 11	89 ± 12
Deviation of GFR from expected (%)	16 ± 5	47 ± 14	5 ± 2
ERPF (mL/min/1.73 m^2)	428 ± 37	257 ± 64	513 ± 51
Deviation of ERPF from expected (%)	17 ± 4	48 ± 13	6 ± 3
Creatinine (µmol/L)	79 ± 9.6	124 ± 59	71 ± 7.6
Urea (mmol/L)	6 ± 1.1	9 ± 2.5	5.6 ± 1.6
Uric acid (µmol/L)	295 ± 78	348 ± 78	273 ± 58
Cystatin C (mg/L)	1 ± 0.1	1.5 ± 0.4	0.8 ± 0.1
Albuminuria (mg/24 h)	13 (10–288)	60 (10–860)	9 (5–12)
Microalbuminuria – n	27 (50)	48 (49)	–
Macroalbuminuria – n	2 (50)	27 (49)	–
Proteinuria (mg/24 h)	123 (12–640)	310 (40–3000)	67 (40–90)

Note: mGFR – measured glomerular filtration rate, ERPF – effective renal plasma flow.

Diabetic patients in group II with a greater degree of reduction mGFR had a significantly lower level of ERPF compared to group I and the control group (257 ± 64 vs. 428 ± 37 vs. 513 ± 51 mL/min/1.73 m², p < 0.001) and a significantly higher serum concentration of creatinine (124 ± 59 vs. 79 ± 9.6 vs. 71 ± 7.6 µmol/L, p < 0.001), urea (9 ± 2.5 vs. 6 ± 1.1 vs. 5.6 ± 1.6 mmol/L, p < 0.001), uric acid (348 ± 78 vs. 295 ± 78 vs. 273 ± 58 µmol/L, p < 0.001), cystatin C (1.5 ± 0.4 vs. 1 ± 0.1 vs. 0.8 ± 0.1 mg/L, p < 0.001), as well as albuminuria (60 vs. 19 vs. 9 mg/24 h, p < 0.001) and proteinuria (310 vs. 123 vs. 67 mg/24 h, p < 0.001) compared to group I and the control group.

A higher plasma concentration of ET-1 was measured in diabetic patients than in control subjects. A significant difference in the median value of ET-1 was registered between the groups (p < 0.001). Plasma concentration of ET-1 was significantly lower in the control subjects than in group I (0.80 vs. 1.45 pg/mL) and group II (0.80 vs. 2.40 pg/mL). Significantly higher plasma concentrations of ET-1 were noted in diabetic patients with a higher degree of reduction in mGFR compared to diabetic patients with a lower degree of reduction in mGFR (1.45 vs. 2.40 pg/mL, p < 0.001).

Figure 1 shows the distribution of the ET-1 values in the examined groups.

Figure 1. The values of the plasma endothelin-1 in the subjects groups.

In the group of patients with diabetic nephropathy there was a statistically significant correlation between the plasma concentration of ET-1 and the measured values of GFR and ERPF (r = −0.52; p < 0.001; r = −0.42; p < 0.001). Diabetic patients with a higher plasma concentration of ET-1 had higher degree of reduction in mGFR and ERPF. Cystatin C (r = 0.42, p < 0.001), albuminuria (r = 0.36, p < 0.001) and proteinuria (r = 0.48, p < 0.001) showed a direct correlation with the plasma concentration of ET-1. Multiple regression analyses were performed to estimate the independent contribution of plasma ET-1 concentration to mGFR among the variables albuminuria, proteinuria and the duration of HT. In this model (F = 44.174, R²= 0.609) only the plasma concentration of ET-1 (p < 0.001) and the duration of HT (p < 0.05) remained independently and significantly associated with mGFR.

Discussion

Diabetes is not only an endocrine but also a vascular disease. Endothelial dysfunction, defined as an imbalance of endothelium-derived vasoconstrictor and vasodilator substances, is a common denominator in the pathogenesis and progression of microvascular complications.7^,24 ET-1 is a potent vasoconstrictor with proliferative, profibrotic and proinflammatory properties, with high expression in the renal vasculature and parenchyma. Therefore, ET-1 except being a marker of endothelial dysfunction in diabetic patients, when elevated is also potentially harmful to renal function which can further contribute to the progression of DN.25

In this study, we measured the plasma concentration of ET-1 in patients with diabetic nephropathy and a different degree of GFR reduction, as well as in the control group of healthy subjects. The results of our study showed that the plasma concentration of ET-1 is significantly higher in the group of diabetic patients than in the controls. The evidence of an increased activity of ET-1 in diabetic patients is derived from a number of experimental and clinical studies.26–29 A higher plasma concentration of ET-1 in patients with diabetes than in healthy population can partially be explained by a stimulated production of ET-1 caused by hyperglycemia and hyperinsulinemia. In an experimental study by Hargrove et al.30 it was observed that hyperglycemia increases transcriptional activity of ET-1 gene, leading to an increase in the glomerular level of ET-1 mRNA and protein in diabetic rats. These findings support the direct effect of glucose on the activity of the gene for ET-1. On the other hand, insulin increases the expression of a renal ET which is considered to be the evidence of an important pathophysiological role of endothelin system in the development of DN.31^,32

The results of our study are concordant with the significant influence of plasma ET-1 on hemodynamic and renal function status in diabetic patients, with a significant correlation between plasma concentration of ET-1 and mGFR, as well as plasma concentration of ET-1 and ERPF. In support of these results some authors emphasize that the vasoconstrictor effect of ET-1 is greater than that of angiotensin II, while the renal blood vessels are 10 times more sensitive to the action of ET-1 compared to the rest of the systemic vasculature. After an in vivo administration, ET-1 increases systemic blood pressure as a result of both increased resistance in the afferent and efferent renal arterioles and the mesangial contraction and proliferation. As a result, GFR and ERPF decrease.33 Experimental studies performed on the kidneys of rats and rabbits have demonstrated that intrarenal infusion of ET-1 leads to a decrease in ERPF and an increase in resistance in the afferent and efferent arterioles of the same intensity as the decrease in GFR.34^,35

A small number of clinical studies concerning the relationship between ET-1 and renal function parameters in patients with different stages of renal insufficiency, indicated a significant correlation of GFR with the fractional excretion of ET-1 in relation to the plasma level of ET-1. This result was expected considering the high endogenous production of ET in renal disease.36 The studies on endothelin receptor antagonists have shown that renal hemodynamic and microcirculation are under the influence of both endogenous and exogenous ET-1.37 On the other hand, in some studies there was no significant relationship between plasma concentration of ET-1 and GFR.38 These results can partially be explained by the fact that the diabetic patients included in the studies had no significant difference in the GFR level.

The results of our study showed a significant correlation between the plasma concentration of ET-1 and the severity of albuminuria and proteinuria. Some studies showed a positive correlation between the plasma concentration of ET-1 and UAE in normotensive diabetic patients with microalbuminuria.39 There are studies confirming this correlation among patients with macroalbuminuria.40 Morigi et al.41 explained how ET-1 may cause glomerular damage in their experimental in vitro study, where they observed that podocytes are subjected to phenotypic changes under the influence of dedifferentiation or autocrine and paracrine effects of ET-1, which contributes to the damage of the glomerular filtration membranes.41 There is strong evidence that ET-1 plays a central role in the pathogenesis of proteinuria and glomerulosclerosis. This process is mediated through the ETA receptor activation, which is confirmed by clinical studies with the ETA receptor antagonists leading to proteinuria reduction.42

In conclusion, the results we obtained demonstrated that a higher plasma concentration of ET-1 is independently associated with the levels of GFR in patients with diabetic nephropathy.

Declaration of interest

The work of Velibor Čabarkapa was supported by the Ministry of Science and Education of the Republic of Serbia on the basis of contract No III46005.