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Poor glycemic control and decreased renal function are associated with increased intrarenal RAS activity in Type 2 diabetes mellitus
DIAB-6057; No. of Pages 7 diabetes research and clinical practice xxx (2014) xxx–xxx
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Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Poor glycemic control and decreased renal function are associated with increased intrarenal RAS activity in Type 2 diabetes mellitus S. Nakatani a,b,1, E. Ishimura b,1,*, T. Naganuma c, A. Nakatani a, M. Ichii a,b, S. Fukumoto a, K. Mori a, M. Emoto a, T. Nakatani c, M. Inaba a a
Departments of Metabolism, Endocrinology, and Molecular Medicine Osaka City University Graduate School of Medicine, Osaka, Japan b Departments of Nephrology, Osaka City University Graduate School of Medicine, Osaka, Japan c Department of Urology, Osaka City University Graduate School of Medicine, Osaka, Japan
article info
abstract
Article history:
Aims: The renin–angiotensin system (RAS) plays an important role in the pathogenesis of
Received 20 December 2013
diabetic nephropathy. The aim of the present study was to investigate intrarenal RAS
Received in revised form
activity in patients with type 2 diabetes (T2DM).
14 March 2014
Methods: We measured urinary angiotensinogen, a reliable biomarker of intrarenal RAS
Accepted 19 April 2014
activity, in 14 controls without T2DM, 25 T2DM patients without nephropathy, 11 chronic
Available online xxx
kidney disease (CKD) patients without T2DM and 46 CKD patients with T2DM. Associations between urinary angiotensinogen and clinical parameters were examined.
Keywords:
Results: Compared with the controls, urinary [angiotensinogen:creatinine] were significant-
Urinary angiotensinogen
ly higher in T2DM patients without nephropathy (4.70 2.22 vs. 8.31 5.27 mg/g, p = 0.037).
Renin–angiotensin system
Age, hemoglobin A1c (HbA1c) and fasting plasma glucose correlated significantly and
Type 2 diabetes mellitus
positively with the log{urinary [angiotensinogen:creatinine]} (r = 0.632, p = 0.007; r = 0.405,
Diabetic nephropathy
p = 0.027; r = 0.583, p = 0.003, respectively) in T2DM patients without nephropathy. In contrast, the urinary [angiotensinogen:creatinine] were not significantly different between CKD patients with and without T2DM (22.7 27.8 vs. 33.5 40.8 mg/g, p = 0.740); although they were significantly higher when compared with non-CKD patients. In the CKD patients with T2DM systolic blood pressure, serum creatinine, estimated glomerular filtration rate and urinary [albumin:creatinine] correlated significantly with the log{urinary [angiotensinogen:creatinine]} (r = 0.412, p = 0.004; r = 0.308, p = 0.037; r = 0.382, p = 0.001; r = 0.648, p < 0.001, p < 0.001, respectively). Conclusions: Our findings indicate that poor glycemic control is significantly associated with intrarenal RAS activity in T2DM patients without nephropathy, and that decreased renal function is significantly associated with intrarenal RAS activity in CKD patients with T2DM. # 2014 Elsevier Ireland Ltd. All rights reserved.
* Corresponding author. Tel.: +81 6 6645 3806; fax: +81 6 6645 3808. E-mail address: [email protected] (E. Ishimura). 1
Contributed equally to this work. http://dx.doi.org/10.1016/j.diabres.2014.04.019 0168-8227/# 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Nakatani S, et al. Poor glycemic control and decreased renal function are associated with increasedUrinary angiotensinogen in type 2 diabetes mellitus–> intrarenal RAS activity in Type 2 diabetes mellitus. Diabetes Res Clin Pract (2014), http://dx.doi.org/ 10.1016/j.diabres.2014.04.019
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1.
Introduction
Diabetic nephropathy is the most frequent cause of endstage renal disease, worldwide, and currently accounts for 40% of patients requiring renal replacement therapy [1,2]. According to the U.S. Renal Data System report, diabetes mellitus (DM) accounted for 54% of new patients in 2007. The incidence of end-stage renal disease in patients with DM is currently 155 patients per million per year [2]. Thus, prevention of the development and progression of diabetic nephropathy is clinically important in patients with DM. It has been reported that the intrarenal renin–angiotensin system (RAS) plays an important role in the pathogenesis of diabetic nephropathy [3]. Indeed, based on numerous clinical trials, RAS inhibition is currently the first line treatment for diabetic nephropathy [4,5]. Recently, the focus of interest on the RAS has shifted to the investigation of its role in specific tissues, such as the brain, heart, blood vessels, and kidney [6– 11]. In addition to the liver, the kidney produces angiotensinogen, the main component of the RAS [12]. Kobori et al. reported that the urinary excretion of angiotensinogen provides a specific index of intrarenal RAS status in angiotensin II-dependent hypertensive rats [13–17]. Moreover, Kobori et al. developed an enzyme-linked immunosorbent assay (ELISA) specific for human angiotensinogen, and they showed that the log{urinary [angiotensinogen:creatinine]} correlated positively with the urinary [albumin:creatinine], and correlated inversely with the estimated glomerular filtration rate (eGFR) in chronic kidney disease (CKD) patients. [18,19]. In their previous study, urinary angiotensinogen levels were measured in 80 CKD patients; however, there were only 14 patients with type 2 diabetes mellitus (T2DM) in their study [18]. There are only a few studies that have investigated urinary angiotensinogen levels in patients with T2DM [18]. It has been suggested that hyperglycemia activates the intrarenal renin angiotensin system [3,20–22]. We hypothesize that the hyperglycemic state affects the levels of urinary angiotensinogen in patients with T2DM, and that these levels are associated with decreased renal function.
2.
Subjects, materials and methods
2.1.
Study design and sample collections
The Institutional Review Board of Osaka City University Graduate School of Medicine approved the use of the clinical data in accordance with the Declaration of Helsinki and guidelines of Osaka City University Graduate School of Medicine (approval no. 2554).
2.1.1. Protocol 1: Investigation of urinary [angiotensinogen:creatinine] in T2DM patients without nephropathy We recruited 25 T2DM patients without nephropathy (eGFR 60 ml/min/1.73 m2 and urinary [albumin:creatinine] < 30 mg/g), and 14 controls without T2DM (eGFR 60 ml/
min/1.73 m2 and [albumin:creatinine] < 30 mg/g). T2DM patients without nephropathy were admitted to Osaka City University Hospital for a medical check-up for T2DM, and controls were planned donors for living kidney transplantation. T2DM patients and controls who provided us with written informed consent were enrolled consecutively from January to October in 2013 and were examined in the present study. Spot urine and blood samples were collected from all participants in the morning after fasting.
2.1.2. Protocol 2: Investigation of urinary [angiotensinogen:creatinine] in CKD patients with T2DM We recruited 46 CKD patients with T2DM and 11 CKD patients without T2DM, including hypertensive nephrosclerosis (n = 5), IgA glomerular nephritis (n = 2), and unknown cause (n = 4). CKD was defined as eGFR < 60 ml/ min/1.73 m2 or the presence of albuminuria (urinary [albumin:creatinine] 30 mg/g). Consecutively enrolled CKD patients, who provided us with written informed consent in 2013, were admitted to Osaka City University Hospital for a medical check-up for CKD. Serum and urine samples were collected in the morning in the fasting state. Spot urine and blood samples were collected from all participants in the morning after fasting. The urine samples were kept on ice for 1 h and then centrifuged at 1500 rpm for 10 min, as described previously [23].
2.2.
Measurements
Urinary concentrations of angiotensinogen were measured using a commercially available ELISA kit (IBL, Gunma, Japan), as described in detail previously [18,24]. In brief, the ELISA plates, coated with the polyclonal antibody against human angiotensinogen, were incubated with urine samples (100 ml/ well) at 37 8C for 1 h. After washing the plates with washing buffer (PBS containing 0.05% Tween 20, pH 7.5), the plates were incubated with horseradish peroxidase-labelled monoclonal antibody against human angiotensinogen at 37 8C for 30 min. After washing the plates, plates were incubated with tetramethylbenzidine sodium for 30 min. The reaction was stopped by treatment with sulfuric acid (0.5 mol/l), and the absorbance values were measured at 450 nm [18,24]. Intraand interassay coefficients of variation ranged from 4.4 to 5.5% and from 4.3 to 7.0%, respectively [24]. The measurements of urea nitrogen, serum creatinine and plasma glucose were performed using routine assays with automated methods. Hemoglobin A1c was measured using high performance liquid chromatography. Urinary albumin was measured by immunoturbidimetry (TIA MicroAlb Kit; Nittobo, Tokyo, Japan), and urinary creatinine was measured by the enzymatic method. eGFR was calculated using the new Japanese coefficient for the abbreviated Modification of Diet in Renal Disease Study equation, including a correction factor of 0.739 for women [25], according to the following formula. eGFRcr ðmL=min=1:73 m2 Þ ¼ 194 Cr 1:094 Age 0:287 ðI f female 0:739Þ Height, body weight, systolic blood pressure and diastolic blood pressure were recorded on the day of sample collection.
Please cite this article in press as: Nakatani S, et al. Poor glycemic control and decreased renal function are associated with increasedUrinary angiotensinogen in type 2 diabetes mellitus–> intrarenal RAS activity in Type 2 diabetes mellitus. Diabetes Res Clin Pract (2014), http://dx.doi.org/ 10.1016/j.diabres.2014.04.019
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2.3.
Statistical analysis
Statistical analyses were performed using Graphpad Prism version 6.0 for Windows (Graphpad Software, San Diego, CA) or JMP software version 10 (SAS Institute, Inc., Cary, NC). Values are expressed as the mean SD. Comparisons between patients with and without T2DM were made using unpaired Student’s t-test or Mann–Whitney U test for continuous variables and chi-square test for categorical variables. Correlations between log{urinary [angiotensinogen:creatinine]} and clinical data were examined by Pearson’s analyses and Spearman’s analyses for parametric data and nonparametric data, respectively. Independent associations of the variables with log{urinary [angiotensinogen:creatinine]} in patients with T2DM were assessed by multiple regression analysis. In the analysis, variables from the univariate analysis with p-values up to 0.1, either in protocol 1 or protocol 2, and factors possibly affecting intrarenal RAS activity, i.e., urinary [albumin:creatinine], systolic blood pressure and eGFR [14,18,26], were included. p-values <0.05 were considered statistically significant.
3.
creatinine, eGFR and the urinary [albumin:creatinine] were not significantly different between the two groups. Compared with the controls without T2DM, the urinary [angiotensinogen:creatinine] were significantly higher in T2DM patients without nephropathy (4.70 2.22 vs. 8.31 5.27 mg/g, p = 0.037) (Table 1). The correlations between the clinical factors and the log{urinary [angiotensinogen:creatinine]} in T2DM patients without nephropathy were examined by simple linear regression analyses. While age, HbA1c and fasting plasma glucose correlated significantly and positively with the log{urinary [angiotensinogen:creatinine]} (r = 0.632, p = 0.007; r = 0.405, p = 0.027; r = 0.583, p = 0.003, respectively) (Fig. 1A–C), the systolic blood pressure, diastolic blood pressure, body mass index, urea nitrogen, serum creatinine, eGFR, or urinary [albumin:creatinine] did not (all, p > 0.1). As explanatory variables for multiple regression analysis for log{urinary [angiotensinogen:creatinine]}, the variables of age, hemoglobin A1c, urinary [albumin:creatinine], systolic blood pressure and eGFR were included. As shown in Table 2, age and hemoglobin A1c were significantly and independently associated with log{urinary [angiotensinogen:creatinine]}(R2 = 0.614, p = 0.003).
Results
3.1. Protocol 1: Investigation of urinary [angiotensinogen:creatinine] in T2DM patients without nephropathy The clinical characteristics of controls without T2DM and T2DM patients without nephropathy are shown in Table 1. The mean body mass index, hemoglobin A1c and fasting plasma glucose were significantly higher in T2DM patients without nephropathy compared with the controls without T2DM. Age, systolic blood pressure, diastolic blood pressure, urea nitrogen, serum
3.2. Protocol 2: Investigation of urinary [angiotensinogen:creatinine] in the CKD patients with T2DM The clinical characteristics of the CKD patients with and without T2DM are shown in Table 1. HbA1c and fasting plasma glucose were significantly higher in the CKD patients with T2DM, compared with those CKD patients without T2DM. Age, body mass index, systolic blood pressure, diastolic blood pressure, urea nitrogen, serum creatinine, eGFR and urinary [albumin:creatinine] were not significantly different between the two groups.
Table 1 – Comparison of the clinical characteristics between controls without T2DM and T2DM patients without nephropathy (Protocol 1), and CKD patients without T2DM and CKD patients with T2DM (Protocol 2). Variables
N Gender (male/female) Age (years) Systolic BP (mmHg) Diastolic BP (mm Hg) BMI (kg/m2) HbA1c (%) (Hb A1c (mmol/mol)) moledmolmol) FPG (mg/dl) Urea nitrogen (mg/dl) Serum creatinine (mg/dl) eGFR (ml/min/1.73 m2) UACR (mg/gCr) UAGT/UCre (mg/g)
Protocol 1: non-CKD patients Controls
T2DM
14 3/11 56.0 21.3 123 25 69 13 19 3 5.6 0.2 (37.7 2.5) 85.6 10 16.8 9.0 0.70 0.11 73.5 14.3 9.69 7.63 4.70 2.22
25 14/11 63.3 14.8 121 11 66 11 26 6 8.3 1.7 (67.2 18.3) 132 38 14.6 4.1 0.67 0.14 82.0 16.9 13.0 9.26 8.31 5.27
Protocol 2: CKD patients
p-Values
Non-T2DM
T2DM
p-Values
0.037 0.380 0.920 0.700 0.003 <0.001 <0.001 <0.001 0.460 0.450 0.140 0.250 0.037
11 5/6 61.6 18.1 139 26 71 12 22 3 5.3 0.4 (34.4 4.1) 101 15 33.2 25.2 2.16 1.87 36.5 21.4 1330 712 35.5 40.8
46 27/19 66.7 11.8 127 19 66 12 24 4 7.9 1.8 (62.8 19.3) 122 32 25.9 10.7 1.57 0.93 44.6 22.7 557 864 22.7 27.8
0.430 0.480 0.200 0.200 0.110 <0.001 <0.001 <0.039 0.990 0.600 0.440 0.078 0.740
CKD: chronic kidney disease, T2DM: type 2 diabetes mellitus, BMI: Body mass index, BP: blood pressure, HbA1c: hemoglobin A1c, FPG: fasting plasma glucose, eGFR: estimated glomerular filtration, UACR: urinary [albumin:creatinine], UAGT/UCre: urinary [angiotensinogen:creatinine]. Data are expressed as the mean SD. Unpaired Student’s t-test was used for continuous variables that exhibited a normal distribution, Mann– Whitney U test was used for continuous variables with a skewed distribution and chi-square test was used for comparison of categorical variables.
Please cite this article in press as: Nakatani S, et al. Poor glycemic control and decreased renal function are associated with increasedUrinary angiotensinogen in type 2 diabetes mellitus–> intrarenal RAS activity in Type 2 diabetes mellitus. Diabetes Res Clin Pract (2014), http://dx.doi.org/ 10.1016/j.diabres.2014.04.019
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Fig. 1 – Investigation of Log (urinary [angiotensinogen:creatinine]) (Log(UAGT/Ucre)) in patients with type 2 diabetes mellitus without nephropathy. Age, hemoglobin A1c (HbA1c), and fasting plasma glucose (FPG) correlated significantly and positively with Log (UAGT/Ucre) ((A)–(C), respectively).
Table 2 – Multiple regression analysis for log{urinary [angiotensinogen:creatinine]} in T2DM patients without nephropathy (Protocol 1), and CKD patients with T2DM (Protocol 2). Term
Age HbA1c UACR SBP eGFR
Protocol 1: T2DM patients without nephropathy
Protocol 2: CKD patients with T2DM
b-Value
p-Values
b-Value
p-Values
0.513 0.432 0.238 0.070 0.011
0.007 0.009 0.142 0.649 0.950
0.199 0.038 0.346 0.321 0.178
0.129 0.796 0.036 0.035 0.238
R2 = 0.614 p = 0.003
R2 = 0.490 p < 0.001
CKD: chronic kidney disease, T2DM: type 2 diabetes mellitus, HbA1c: hemoglobin A1c, UACR: urinary [albumin:creatinine], SBP: systolic blood pressure, eGFR: estimated glomerular filtration. b-value: standardized coefficient values, R2: multiple coefficient of determinant.
The urinary [angiotensinogen:creatinine] was not significantly different between the CKD patients with and without T2DM (22.7 27.8 vs. 33.5 40.8 mg/g, p = 0.740). The correlations between the clinical factors and the log{urinary [angiotensinogen:creatinine]} in the CKD patients with T2DM were examined by simple linear regression analyses. Systolic blood pressure, serum creatinine and urinary [albumin:creatinine] correlated significantly and positively with the log{urinary [angiotensinogen:creatinine]} (r = 0.412, p = 0.004; r = 0.308, p = 0.037; r = 0.648, p < 0.001, respectively) (Fig. 2A–C). While eGFR correlated significantly and inversely with the log{urinary [angiotensinogen:creatinine]} (r = 0.382, p = 0.0010) (Fig. 2D), age, diastolic blood pressure, body mass index, HbA1c, fasting plasma glucose and urea nitrogen did not (all, p > 0.1). As explanatory variables for multiple regression analysis for log{urinary [angiotensinogen:creatinine]}, variables of age, hemoglobin A1c, urinary [albumin:creatinine], systolic blood pressure and eGFR were included. As shown in Table 2, urinary [albumin:creatinine] and systolic blood pressure were significantly and independently associated with log{urinary [angiotensinogen:creatinine]} (R2 = 0.490, p < 0.001).
4.
Discussion
The result of protocol 1 in the present study showed that urinary [angiotensinogen:creatinine] was significantly higher in T2DM patients without nephropathy, compared with
controls without T2DM. In addition, log{urinary [angiotensinogen:creatinine]} was significantly associated with poor glycemic control. These associations were independent of the established risk factors for diabetic nephropathy, including blood pressure, eGFR and albuminuria. Previous studies have suggested that urinary angiotensinogen is a reliable marker for intrarenal RAS activity [11,17,26]. Our findings suggest that poor glycemic control may play an important role in the activation of intrarenal RAS in T2DM patients without nephropathy. Several studies have provided compelling support for the existence of an independent mesangial cell RAS, whose activation appears to be critical in the development of diabetic nephropathy [3,22,27]. High glucose increase the gene expression of angiotensinogen in proximal tubule cells and angiotensin II production in primary mesangial cell cultures, indicating that high glucose alone can stimulate RAS activity [22,27–29]. In the present study, poor glycemic control was significantly associated with intrarenal RAS activation in T2DM patients without nephropathy. Therefore, strict glycemic control in T2DM patients without nephropathy, which may suppress intrarenal RAS activity, may be important in preventing the progression of diabetic nephropathy, with respect to prevention of the increase in proteinuria and/or decreased glomerular filtration rate [30–33]. The result of protocol 2 in the present study showed that the urinary [angiotensinogen:creatinine] was not significantly different between CKD patients with and without T2DM. In CKD patients with T2DM, log{urinary [angiotensinogen:
Please cite this article in press as: Nakatani S, et al. Poor glycemic control and decreased renal function are associated with increasedUrinary angiotensinogen in type 2 diabetes mellitus–> intrarenal RAS activity in Type 2 diabetes mellitus. Diabetes Res Clin Pract (2014), http://dx.doi.org/ 10.1016/j.diabres.2014.04.019
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Fig. 2 – Investigation of Log(urinary [angiotensinogen:creatinine]) (Log(UAGT/Ucre)) in patients with type 2 diabetes mellitus and chronic kidney disease. Systolic blood pressure (SBP), serum creatinine, and urinary [albumin:creatinine] (UACR) correlated significantly and positively with Log (UAGT/Ucre) ((A)–(C), respectively). Estimated glomerular filtration rate (eGFR) correlated significantly and inversely with the Log (UAGT/Ucre) (D).
creatinine]} was not significantly associated with glycemic control. In contrast, established risk factors for CKD, such as high blood pressure, reduced eGFR and the presence of albuminuria were significantly associated with increased urinary log{urinary [angiotensinogen:creatinine]} in CKD patients with T2DM. These associations between established risk factors for CKD and the log{urinary [angiotensinogen:creatinine]} are consistent with previous studies of the urinary [angiotensin:creatinine] in CKD patients, including various kidney diseases [18,19]. In CKD patients with T2DM, our findings suggest that these risk factors of CKD, i.e., high blood pressure, reduced eGFR and the presence of albuminuria, are more important in the activation of intrarenal RAS than poor glycemic control. There are some limitations to the present study. First, the number of the subjects and patients examined in the present study was relatively small. This was mainly due to the fact that we consecutively enrolled the study subjects and patients in a single institute. Second, this study was cross-sectional, and does not demonstrate causality of the factors that correlated with intrarenal RAS activity, i.e., poor glycemic control, high blood pressure, increased urinary [albumin:creatinine], or decreased renal function. Further studies are required to explore whether a reduction of urinary angiotensinogen is induced by strict glycemic control in diabetic patients without nephropathy and by strict blood pressure control in CKD patients with T2DM. Third, the effect of RAS inhibitors, such as angiotensin-converting enzyme inhibitors and angiotensin type 1 receptor blockers, could not be fully examined in the present study. There are many clinical reports supporting the use of RAS inhibitors to prevent the progression of diabetic
nephropathy in patients with both early and chronic kidney disease [30–34]. The present study was a cross-sectional study and many patients have already used RAS inhibitors in these previous studies. Thus, it was difficult to evaluate the effect of RAS inhibitors on the excretion of urinary angiotensinogen in the present study. Indeed, in study protocol 1, there were 3 and 11 subjects with and without RAS inhibitors among the controls without T2DM, respectively, compared with 14 and 11 with and without RAS inhibitors, among the T2DM patients without nephropathy, respectively. The numbers of these patients were too small to allow us to compare the effect of RAS inhibitors on the urinary [angiotensinogen:creatinine]. However, among those without RAS inhibitors, urinary [angiotensinogen:creatinine] was significantly higher in T2DM patients without nephropathy (n = 11) compared with the controls without T2DM (n = 11) (9.62 5.28 vs. 5.09 2.29 mg/g, p = 0.029). This result may also suggest that diabetes itself is associated with increased intrarenal RAS. In protocol 2 of the present study, 7 and 30 patients were treated with RAS inhibitors in the CKD patients with and without T2DM, respectively. Urinary [angiotensinogen:creatinine] was not significantly different between the CKD patients with and without T2DM, treated with RAS inhibitors (50.1 45.4 vs. 28.8 29.9 mg/g, p = 0.26). This result may also suggest that the presence of T2DM is not associated with intrarenal RAS activity in CKD patients. The effect of RAS inhibitors on intrarenal RAS activity must be investigated further in a prospective study with a large number of patients. In conclusion, this is the first study to show that urinary angiotensinogen levels are significantly higher in T2DM
Please cite this article in press as: Nakatani S, et al. Poor glycemic control and decreased renal function are associated with increasedUrinary angiotensinogen in type 2 diabetes mellitus–> intrarenal RAS activity in Type 2 diabetes mellitus. Diabetes Res Clin Pract (2014), http://dx.doi.org/ 10.1016/j.diabres.2014.04.019
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patients without nephropathy compared with controls without T2DM. We also demonstrated that poor glycemic control was significantly associated with intrarenal RAS activity in T2DM patients without nephropathy. In the chronic stage of diabetic nephropathy, decreased renal function and higher blood pressure were significantly associated with increased urinary angiotensinogen.
Funding sources None.
Conflict of interest statement The authors declare that they have no conflict of interest.
Acknowledgments The author thanks Ms. Marie Kawamura for measuring urinary angiotensinogen levels, in the study.
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Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Poor glycemic control and decreased renal function are associated with increased intrarenal RAS activity in Type 2 diabetes mellitus S. Nakatani a,b,1, E. Ishimura b,1,*, T. Naganuma c, A. Nakatani a, M. Ichii a,b, S. Fukumoto a, K. Mori a, M. Emoto a, T. Nakatani c, M. Inaba a a
Departments of Metabolism, Endocrinology, and Molecular Medicine Osaka City University Graduate School of Medicine, Osaka, Japan b Departments of Nephrology, Osaka City University Graduate School of Medicine, Osaka, Japan c Department of Urology, Osaka City University Graduate School of Medicine, Osaka, Japan
article info
abstract
Article history:
Aims: The renin–angiotensin system (RAS) plays an important role in the pathogenesis of
Received 20 December 2013
diabetic nephropathy. The aim of the present study was to investigate intrarenal RAS
Received in revised form
activity in patients with type 2 diabetes (T2DM).
14 March 2014
Methods: We measured urinary angiotensinogen, a reliable biomarker of intrarenal RAS
Accepted 19 April 2014
activity, in 14 controls without T2DM, 25 T2DM patients without nephropathy, 11 chronic
Available online xxx
kidney disease (CKD) patients without T2DM and 46 CKD patients with T2DM. Associations between urinary angiotensinogen and clinical parameters were examined.
Keywords:
Results: Compared with the controls, urinary [angiotensinogen:creatinine] were significant-
Urinary angiotensinogen
ly higher in T2DM patients without nephropathy (4.70 2.22 vs. 8.31 5.27 mg/g, p = 0.037).
Renin–angiotensin system
Age, hemoglobin A1c (HbA1c) and fasting plasma glucose correlated significantly and
Type 2 diabetes mellitus
positively with the log{urinary [angiotensinogen:creatinine]} (r = 0.632, p = 0.007; r = 0.405,
Diabetic nephropathy
p = 0.027; r = 0.583, p = 0.003, respectively) in T2DM patients without nephropathy. In contrast, the urinary [angiotensinogen:creatinine] were not significantly different between CKD patients with and without T2DM (22.7 27.8 vs. 33.5 40.8 mg/g, p = 0.740); although they were significantly higher when compared with non-CKD patients. In the CKD patients with T2DM systolic blood pressure, serum creatinine, estimated glomerular filtration rate and urinary [albumin:creatinine] correlated significantly with the log{urinary [angiotensinogen:creatinine]} (r = 0.412, p = 0.004; r = 0.308, p = 0.037; r = 0.382, p = 0.001; r = 0.648, p < 0.001, p < 0.001, respectively). Conclusions: Our findings indicate that poor glycemic control is significantly associated with intrarenal RAS activity in T2DM patients without nephropathy, and that decreased renal function is significantly associated with intrarenal RAS activity in CKD patients with T2DM. # 2014 Elsevier Ireland Ltd. All rights reserved.
* Corresponding author. Tel.: +81 6 6645 3806; fax: +81 6 6645 3808. E-mail address: [email protected] (E. Ishimura). 1
Contributed equally to this work. http://dx.doi.org/10.1016/j.diabres.2014.04.019 0168-8227/# 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Nakatani S, et al. Poor glycemic control and decreased renal function are associated with increasedUrinary angiotensinogen in type 2 diabetes mellitus–> intrarenal RAS activity in Type 2 diabetes mellitus. Diabetes Res Clin Pract (2014), http://dx.doi.org/ 10.1016/j.diabres.2014.04.019
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1.
Introduction
Diabetic nephropathy is the most frequent cause of endstage renal disease, worldwide, and currently accounts for 40% of patients requiring renal replacement therapy [1,2]. According to the U.S. Renal Data System report, diabetes mellitus (DM) accounted for 54% of new patients in 2007. The incidence of end-stage renal disease in patients with DM is currently 155 patients per million per year [2]. Thus, prevention of the development and progression of diabetic nephropathy is clinically important in patients with DM. It has been reported that the intrarenal renin–angiotensin system (RAS) plays an important role in the pathogenesis of diabetic nephropathy [3]. Indeed, based on numerous clinical trials, RAS inhibition is currently the first line treatment for diabetic nephropathy [4,5]. Recently, the focus of interest on the RAS has shifted to the investigation of its role in specific tissues, such as the brain, heart, blood vessels, and kidney [6– 11]. In addition to the liver, the kidney produces angiotensinogen, the main component of the RAS [12]. Kobori et al. reported that the urinary excretion of angiotensinogen provides a specific index of intrarenal RAS status in angiotensin II-dependent hypertensive rats [13–17]. Moreover, Kobori et al. developed an enzyme-linked immunosorbent assay (ELISA) specific for human angiotensinogen, and they showed that the log{urinary [angiotensinogen:creatinine]} correlated positively with the urinary [albumin:creatinine], and correlated inversely with the estimated glomerular filtration rate (eGFR) in chronic kidney disease (CKD) patients. [18,19]. In their previous study, urinary angiotensinogen levels were measured in 80 CKD patients; however, there were only 14 patients with type 2 diabetes mellitus (T2DM) in their study [18]. There are only a few studies that have investigated urinary angiotensinogen levels in patients with T2DM [18]. It has been suggested that hyperglycemia activates the intrarenal renin angiotensin system [3,20–22]. We hypothesize that the hyperglycemic state affects the levels of urinary angiotensinogen in patients with T2DM, and that these levels are associated with decreased renal function.
2.
Subjects, materials and methods
2.1.
Study design and sample collections
The Institutional Review Board of Osaka City University Graduate School of Medicine approved the use of the clinical data in accordance with the Declaration of Helsinki and guidelines of Osaka City University Graduate School of Medicine (approval no. 2554).
2.1.1. Protocol 1: Investigation of urinary [angiotensinogen:creatinine] in T2DM patients without nephropathy We recruited 25 T2DM patients without nephropathy (eGFR 60 ml/min/1.73 m2 and urinary [albumin:creatinine] < 30 mg/g), and 14 controls without T2DM (eGFR 60 ml/
min/1.73 m2 and [albumin:creatinine] < 30 mg/g). T2DM patients without nephropathy were admitted to Osaka City University Hospital for a medical check-up for T2DM, and controls were planned donors for living kidney transplantation. T2DM patients and controls who provided us with written informed consent were enrolled consecutively from January to October in 2013 and were examined in the present study. Spot urine and blood samples were collected from all participants in the morning after fasting.
2.1.2. Protocol 2: Investigation of urinary [angiotensinogen:creatinine] in CKD patients with T2DM We recruited 46 CKD patients with T2DM and 11 CKD patients without T2DM, including hypertensive nephrosclerosis (n = 5), IgA glomerular nephritis (n = 2), and unknown cause (n = 4). CKD was defined as eGFR < 60 ml/ min/1.73 m2 or the presence of albuminuria (urinary [albumin:creatinine] 30 mg/g). Consecutively enrolled CKD patients, who provided us with written informed consent in 2013, were admitted to Osaka City University Hospital for a medical check-up for CKD. Serum and urine samples were collected in the morning in the fasting state. Spot urine and blood samples were collected from all participants in the morning after fasting. The urine samples were kept on ice for 1 h and then centrifuged at 1500 rpm for 10 min, as described previously [23].
2.2.
Measurements
Urinary concentrations of angiotensinogen were measured using a commercially available ELISA kit (IBL, Gunma, Japan), as described in detail previously [18,24]. In brief, the ELISA plates, coated with the polyclonal antibody against human angiotensinogen, were incubated with urine samples (100 ml/ well) at 37 8C for 1 h. After washing the plates with washing buffer (PBS containing 0.05% Tween 20, pH 7.5), the plates were incubated with horseradish peroxidase-labelled monoclonal antibody against human angiotensinogen at 37 8C for 30 min. After washing the plates, plates were incubated with tetramethylbenzidine sodium for 30 min. The reaction was stopped by treatment with sulfuric acid (0.5 mol/l), and the absorbance values were measured at 450 nm [18,24]. Intraand interassay coefficients of variation ranged from 4.4 to 5.5% and from 4.3 to 7.0%, respectively [24]. The measurements of urea nitrogen, serum creatinine and plasma glucose were performed using routine assays with automated methods. Hemoglobin A1c was measured using high performance liquid chromatography. Urinary albumin was measured by immunoturbidimetry (TIA MicroAlb Kit; Nittobo, Tokyo, Japan), and urinary creatinine was measured by the enzymatic method. eGFR was calculated using the new Japanese coefficient for the abbreviated Modification of Diet in Renal Disease Study equation, including a correction factor of 0.739 for women [25], according to the following formula. eGFRcr ðmL=min=1:73 m2 Þ ¼ 194 Cr 1:094 Age 0:287 ðI f female 0:739Þ Height, body weight, systolic blood pressure and diastolic blood pressure were recorded on the day of sample collection.
Please cite this article in press as: Nakatani S, et al. Poor glycemic control and decreased renal function are associated with increasedUrinary angiotensinogen in type 2 diabetes mellitus–> intrarenal RAS activity in Type 2 diabetes mellitus. Diabetes Res Clin Pract (2014), http://dx.doi.org/ 10.1016/j.diabres.2014.04.019
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2.3.
Statistical analysis
Statistical analyses were performed using Graphpad Prism version 6.0 for Windows (Graphpad Software, San Diego, CA) or JMP software version 10 (SAS Institute, Inc., Cary, NC). Values are expressed as the mean SD. Comparisons between patients with and without T2DM were made using unpaired Student’s t-test or Mann–Whitney U test for continuous variables and chi-square test for categorical variables. Correlations between log{urinary [angiotensinogen:creatinine]} and clinical data were examined by Pearson’s analyses and Spearman’s analyses for parametric data and nonparametric data, respectively. Independent associations of the variables with log{urinary [angiotensinogen:creatinine]} in patients with T2DM were assessed by multiple regression analysis. In the analysis, variables from the univariate analysis with p-values up to 0.1, either in protocol 1 or protocol 2, and factors possibly affecting intrarenal RAS activity, i.e., urinary [albumin:creatinine], systolic blood pressure and eGFR [14,18,26], were included. p-values <0.05 were considered statistically significant.
3.
creatinine, eGFR and the urinary [albumin:creatinine] were not significantly different between the two groups. Compared with the controls without T2DM, the urinary [angiotensinogen:creatinine] were significantly higher in T2DM patients without nephropathy (4.70 2.22 vs. 8.31 5.27 mg/g, p = 0.037) (Table 1). The correlations between the clinical factors and the log{urinary [angiotensinogen:creatinine]} in T2DM patients without nephropathy were examined by simple linear regression analyses. While age, HbA1c and fasting plasma glucose correlated significantly and positively with the log{urinary [angiotensinogen:creatinine]} (r = 0.632, p = 0.007; r = 0.405, p = 0.027; r = 0.583, p = 0.003, respectively) (Fig. 1A–C), the systolic blood pressure, diastolic blood pressure, body mass index, urea nitrogen, serum creatinine, eGFR, or urinary [albumin:creatinine] did not (all, p > 0.1). As explanatory variables for multiple regression analysis for log{urinary [angiotensinogen:creatinine]}, the variables of age, hemoglobin A1c, urinary [albumin:creatinine], systolic blood pressure and eGFR were included. As shown in Table 2, age and hemoglobin A1c were significantly and independently associated with log{urinary [angiotensinogen:creatinine]}(R2 = 0.614, p = 0.003).
Results
3.1. Protocol 1: Investigation of urinary [angiotensinogen:creatinine] in T2DM patients without nephropathy The clinical characteristics of controls without T2DM and T2DM patients without nephropathy are shown in Table 1. The mean body mass index, hemoglobin A1c and fasting plasma glucose were significantly higher in T2DM patients without nephropathy compared with the controls without T2DM. Age, systolic blood pressure, diastolic blood pressure, urea nitrogen, serum
3.2. Protocol 2: Investigation of urinary [angiotensinogen:creatinine] in the CKD patients with T2DM The clinical characteristics of the CKD patients with and without T2DM are shown in Table 1. HbA1c and fasting plasma glucose were significantly higher in the CKD patients with T2DM, compared with those CKD patients without T2DM. Age, body mass index, systolic blood pressure, diastolic blood pressure, urea nitrogen, serum creatinine, eGFR and urinary [albumin:creatinine] were not significantly different between the two groups.
Table 1 – Comparison of the clinical characteristics between controls without T2DM and T2DM patients without nephropathy (Protocol 1), and CKD patients without T2DM and CKD patients with T2DM (Protocol 2). Variables
N Gender (male/female) Age (years) Systolic BP (mmHg) Diastolic BP (mm Hg) BMI (kg/m2) HbA1c (%) (Hb A1c (mmol/mol)) moledmolmol) FPG (mg/dl) Urea nitrogen (mg/dl) Serum creatinine (mg/dl) eGFR (ml/min/1.73 m2) UACR (mg/gCr) UAGT/UCre (mg/g)
Protocol 1: non-CKD patients Controls
T2DM
14 3/11 56.0 21.3 123 25 69 13 19 3 5.6 0.2 (37.7 2.5) 85.6 10 16.8 9.0 0.70 0.11 73.5 14.3 9.69 7.63 4.70 2.22
25 14/11 63.3 14.8 121 11 66 11 26 6 8.3 1.7 (67.2 18.3) 132 38 14.6 4.1 0.67 0.14 82.0 16.9 13.0 9.26 8.31 5.27
Protocol 2: CKD patients
p-Values
Non-T2DM
T2DM
p-Values
0.037 0.380 0.920 0.700 0.003 <0.001 <0.001 <0.001 0.460 0.450 0.140 0.250 0.037
11 5/6 61.6 18.1 139 26 71 12 22 3 5.3 0.4 (34.4 4.1) 101 15 33.2 25.2 2.16 1.87 36.5 21.4 1330 712 35.5 40.8
46 27/19 66.7 11.8 127 19 66 12 24 4 7.9 1.8 (62.8 19.3) 122 32 25.9 10.7 1.57 0.93 44.6 22.7 557 864 22.7 27.8
0.430 0.480 0.200 0.200 0.110 <0.001 <0.001 <0.039 0.990 0.600 0.440 0.078 0.740
CKD: chronic kidney disease, T2DM: type 2 diabetes mellitus, BMI: Body mass index, BP: blood pressure, HbA1c: hemoglobin A1c, FPG: fasting plasma glucose, eGFR: estimated glomerular filtration, UACR: urinary [albumin:creatinine], UAGT/UCre: urinary [angiotensinogen:creatinine]. Data are expressed as the mean SD. Unpaired Student’s t-test was used for continuous variables that exhibited a normal distribution, Mann– Whitney U test was used for continuous variables with a skewed distribution and chi-square test was used for comparison of categorical variables.
Please cite this article in press as: Nakatani S, et al. Poor glycemic control and decreased renal function are associated with increasedUrinary angiotensinogen in type 2 diabetes mellitus–> intrarenal RAS activity in Type 2 diabetes mellitus. Diabetes Res Clin Pract (2014), http://dx.doi.org/ 10.1016/j.diabres.2014.04.019
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Fig. 1 – Investigation of Log (urinary [angiotensinogen:creatinine]) (Log(UAGT/Ucre)) in patients with type 2 diabetes mellitus without nephropathy. Age, hemoglobin A1c (HbA1c), and fasting plasma glucose (FPG) correlated significantly and positively with Log (UAGT/Ucre) ((A)–(C), respectively).
Table 2 – Multiple regression analysis for log{urinary [angiotensinogen:creatinine]} in T2DM patients without nephropathy (Protocol 1), and CKD patients with T2DM (Protocol 2). Term
Age HbA1c UACR SBP eGFR
Protocol 1: T2DM patients without nephropathy
Protocol 2: CKD patients with T2DM
b-Value
p-Values
b-Value
p-Values
0.513 0.432 0.238 0.070 0.011
0.007 0.009 0.142 0.649 0.950
0.199 0.038 0.346 0.321 0.178
0.129 0.796 0.036 0.035 0.238
R2 = 0.614 p = 0.003
R2 = 0.490 p < 0.001
CKD: chronic kidney disease, T2DM: type 2 diabetes mellitus, HbA1c: hemoglobin A1c, UACR: urinary [albumin:creatinine], SBP: systolic blood pressure, eGFR: estimated glomerular filtration. b-value: standardized coefficient values, R2: multiple coefficient of determinant.
The urinary [angiotensinogen:creatinine] was not significantly different between the CKD patients with and without T2DM (22.7 27.8 vs. 33.5 40.8 mg/g, p = 0.740). The correlations between the clinical factors and the log{urinary [angiotensinogen:creatinine]} in the CKD patients with T2DM were examined by simple linear regression analyses. Systolic blood pressure, serum creatinine and urinary [albumin:creatinine] correlated significantly and positively with the log{urinary [angiotensinogen:creatinine]} (r = 0.412, p = 0.004; r = 0.308, p = 0.037; r = 0.648, p < 0.001, respectively) (Fig. 2A–C). While eGFR correlated significantly and inversely with the log{urinary [angiotensinogen:creatinine]} (r = 0.382, p = 0.0010) (Fig. 2D), age, diastolic blood pressure, body mass index, HbA1c, fasting plasma glucose and urea nitrogen did not (all, p > 0.1). As explanatory variables for multiple regression analysis for log{urinary [angiotensinogen:creatinine]}, variables of age, hemoglobin A1c, urinary [albumin:creatinine], systolic blood pressure and eGFR were included. As shown in Table 2, urinary [albumin:creatinine] and systolic blood pressure were significantly and independently associated with log{urinary [angiotensinogen:creatinine]} (R2 = 0.490, p < 0.001).
4.
Discussion
The result of protocol 1 in the present study showed that urinary [angiotensinogen:creatinine] was significantly higher in T2DM patients without nephropathy, compared with
controls without T2DM. In addition, log{urinary [angiotensinogen:creatinine]} was significantly associated with poor glycemic control. These associations were independent of the established risk factors for diabetic nephropathy, including blood pressure, eGFR and albuminuria. Previous studies have suggested that urinary angiotensinogen is a reliable marker for intrarenal RAS activity [11,17,26]. Our findings suggest that poor glycemic control may play an important role in the activation of intrarenal RAS in T2DM patients without nephropathy. Several studies have provided compelling support for the existence of an independent mesangial cell RAS, whose activation appears to be critical in the development of diabetic nephropathy [3,22,27]. High glucose increase the gene expression of angiotensinogen in proximal tubule cells and angiotensin II production in primary mesangial cell cultures, indicating that high glucose alone can stimulate RAS activity [22,27–29]. In the present study, poor glycemic control was significantly associated with intrarenal RAS activation in T2DM patients without nephropathy. Therefore, strict glycemic control in T2DM patients without nephropathy, which may suppress intrarenal RAS activity, may be important in preventing the progression of diabetic nephropathy, with respect to prevention of the increase in proteinuria and/or decreased glomerular filtration rate [30–33]. The result of protocol 2 in the present study showed that the urinary [angiotensinogen:creatinine] was not significantly different between CKD patients with and without T2DM. In CKD patients with T2DM, log{urinary [angiotensinogen:
Please cite this article in press as: Nakatani S, et al. Poor glycemic control and decreased renal function are associated with increasedUrinary angiotensinogen in type 2 diabetes mellitus–> intrarenal RAS activity in Type 2 diabetes mellitus. Diabetes Res Clin Pract (2014), http://dx.doi.org/ 10.1016/j.diabres.2014.04.019
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Fig. 2 – Investigation of Log(urinary [angiotensinogen:creatinine]) (Log(UAGT/Ucre)) in patients with type 2 diabetes mellitus and chronic kidney disease. Systolic blood pressure (SBP), serum creatinine, and urinary [albumin:creatinine] (UACR) correlated significantly and positively with Log (UAGT/Ucre) ((A)–(C), respectively). Estimated glomerular filtration rate (eGFR) correlated significantly and inversely with the Log (UAGT/Ucre) (D).
creatinine]} was not significantly associated with glycemic control. In contrast, established risk factors for CKD, such as high blood pressure, reduced eGFR and the presence of albuminuria were significantly associated with increased urinary log{urinary [angiotensinogen:creatinine]} in CKD patients with T2DM. These associations between established risk factors for CKD and the log{urinary [angiotensinogen:creatinine]} are consistent with previous studies of the urinary [angiotensin:creatinine] in CKD patients, including various kidney diseases [18,19]. In CKD patients with T2DM, our findings suggest that these risk factors of CKD, i.e., high blood pressure, reduced eGFR and the presence of albuminuria, are more important in the activation of intrarenal RAS than poor glycemic control. There are some limitations to the present study. First, the number of the subjects and patients examined in the present study was relatively small. This was mainly due to the fact that we consecutively enrolled the study subjects and patients in a single institute. Second, this study was cross-sectional, and does not demonstrate causality of the factors that correlated with intrarenal RAS activity, i.e., poor glycemic control, high blood pressure, increased urinary [albumin:creatinine], or decreased renal function. Further studies are required to explore whether a reduction of urinary angiotensinogen is induced by strict glycemic control in diabetic patients without nephropathy and by strict blood pressure control in CKD patients with T2DM. Third, the effect of RAS inhibitors, such as angiotensin-converting enzyme inhibitors and angiotensin type 1 receptor blockers, could not be fully examined in the present study. There are many clinical reports supporting the use of RAS inhibitors to prevent the progression of diabetic
nephropathy in patients with both early and chronic kidney disease [30–34]. The present study was a cross-sectional study and many patients have already used RAS inhibitors in these previous studies. Thus, it was difficult to evaluate the effect of RAS inhibitors on the excretion of urinary angiotensinogen in the present study. Indeed, in study protocol 1, there were 3 and 11 subjects with and without RAS inhibitors among the controls without T2DM, respectively, compared with 14 and 11 with and without RAS inhibitors, among the T2DM patients without nephropathy, respectively. The numbers of these patients were too small to allow us to compare the effect of RAS inhibitors on the urinary [angiotensinogen:creatinine]. However, among those without RAS inhibitors, urinary [angiotensinogen:creatinine] was significantly higher in T2DM patients without nephropathy (n = 11) compared with the controls without T2DM (n = 11) (9.62 5.28 vs. 5.09 2.29 mg/g, p = 0.029). This result may also suggest that diabetes itself is associated with increased intrarenal RAS. In protocol 2 of the present study, 7 and 30 patients were treated with RAS inhibitors in the CKD patients with and without T2DM, respectively. Urinary [angiotensinogen:creatinine] was not significantly different between the CKD patients with and without T2DM, treated with RAS inhibitors (50.1 45.4 vs. 28.8 29.9 mg/g, p = 0.26). This result may also suggest that the presence of T2DM is not associated with intrarenal RAS activity in CKD patients. The effect of RAS inhibitors on intrarenal RAS activity must be investigated further in a prospective study with a large number of patients. In conclusion, this is the first study to show that urinary angiotensinogen levels are significantly higher in T2DM
Please cite this article in press as: Nakatani S, et al. Poor glycemic control and decreased renal function are associated with increasedUrinary angiotensinogen in type 2 diabetes mellitus–> intrarenal RAS activity in Type 2 diabetes mellitus. Diabetes Res Clin Pract (2014), http://dx.doi.org/ 10.1016/j.diabres.2014.04.019
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patients without nephropathy compared with controls without T2DM. We also demonstrated that poor glycemic control was significantly associated with intrarenal RAS activity in T2DM patients without nephropathy. In the chronic stage of diabetic nephropathy, decreased renal function and higher blood pressure were significantly associated with increased urinary angiotensinogen.
Funding sources None.
Conflict of interest statement The authors declare that they have no conflict of interest.
Acknowledgments The author thanks Ms. Marie Kawamura for measuring urinary angiotensinogen levels, in the study.
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Please cite this article in press as: Nakatani S, et al. Poor glycemic control and decreased renal function are associated with increasedUrinary angiotensinogen in type 2 diabetes mellitus–> intrarenal RAS activity in Type 2 diabetes mellitus. Diabetes Res Clin Pract (2014), http://dx.doi.org/ 10.1016/j.diabres.2014.04.019
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