Effect of Renin Angiotensin System Blockade on Pentraxin 3 Levels in Type-2 Diabetic Patients With Proteinuria

Patients and Controls

We performed a prospective study of selected patients referred to the Department of Nephrology, Gulhane School of Medicine Outpatient Clinics during the period of January 1st, 2006 to January 1st, 2008. From these referrals, after a 24-h urine collection, we selected patients with stage 1 CKD (24-h protein excretion ≥ 500 mg/d, systolic BP ≥ 140 mmHg and/or diastolic BP ≥ 90 mmHg, respectively) but a normal estimated GFR (eGFR) (≥90 ml/min), who were older than 18 yr and characterized as having type 2 diabetes mellitus as the only cause of nephropathy (renal biopsy and medical history). A total of 266 patients fulfilled the above inclusion criteria, but from these, we then excluded patients who were previously treated using ACEI or angiotensin receptor blockers (ARBs, medical history); who were obesity (BMI > 30kg/m²); who had dyslipidemia (total cholesterol > 200 mg/dl, fasting triglycerides > 150 mg/dl), renal failure, (eGFR < 90 ml/min), nephrotic syndrome (urinary protein excretion > 3000 mg/d), or a history of CVD (medical history, abnormal electrocardiogram [ECG], see below); who smoked or had smoked within the last 3 mo and were taking either statins or ACEI or ARBs. The final cohort of eligible patients starting the study consisted of 60 individuals (29 men, age 47 ± 5 yr) (Figure 1). The duration of proteinuria and diabetic nephropathy in these patients before and after the initial diagnosis of diabetes was made was unknown.

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Figure 1.

Flow chart of patients enrolled in the trial.

We also recruited 32 healthy subjects (16 men, age 46.9 ± 5.4 yr) to serve as controls. These individuals had no known diseases and were not currently taking any drugs. The control subjects were subject to the same inclusion and exclusion criteria as the patients. Informed consent to participate in the study was obtained from both patients and controls. The drug ethical committee of Gulhane School of Medicine approved the study, which was also registered at clinicaltrials.gov (NCT00674596: “The Effect of Renin Angiotensin System Blockage (RAS) On PTX3 Levels In Diabetic Patients With Proteinuria”).

Baseline Characterization

Recruited patients were evaluated by standard physical examination; chest x-ray; baseline ECG; two-dimensional echocardiography; and routine clinical laboratory tests, including liver and kidney function tests and 24-h urinary protein measurements. Arterial BP was measured in the right arm by mercury sphygmomanometer three times in a resting condition in the morning, and mean values were calculated for diastolic and systolic pressures. The exclusion criteria were as follows: nephrotic syndrome, coronary heart disease (patients with ischemic ST-T wave changes in electrocardiogram alterations and voltage criteria for LVH and a history of revascularization or myocardial infarction), elevated liver enzymes (aspartate amino transferase or alanine amino transferase levels ≥ 40 U/L), and renal failure (serum creatinine levels > 1.3 mg/dl).

Intervention and Follow-Up Measurements

In an open-label trial, patients were given an ACE inhibitor (ramipril, 5 mg once per day) for 12 wk immediately after baseline measurements. During the study period, serum creatinine and potassium concentrations were measured every 2 wk, and the dose of ramipril was titrated to achieve a serum potassium concentration of <5.5 mEq/L. The average dose of ramipril treatment was one tablet a day (5 mg/d). The medications were given with meals, and the doses were increased as needed to a maximum of 10 mg/d. After this period, blood samples were obtained for measurements as shown below. Urine samples were also collected over 24 h to determine the degree of proteinuria.

Blood Chemistry

Morning blood samples were collected from patients and control subjects after 12 h of fasting. Subjects were asked to refrain from physical activity for at least 30 min before the blood draw. In addition to routine clinical laboratory tests, serum PTX3 concentrations and basal insulin levels were analyzed from all patients. After the intervention period, blood samples were obtained for the measurement of serum PTX3 concentration, high-sensitivity C-reactive protein (hsCRP) levels, and insulin levels, as well as for determining the insulin resistance index. The measurement of total cholesterol (TC), triglyceride (TG), HDL cholesterol, and fasting plasma glucose (FPG) was performed by the enzymatic colorimetric method with an Olympus AU 600 auto analyzer, using reagents from Olympus Diagnostics, GmbH (Hamburg, Germany). LDL cholesterol was calculated by Friedewald's formula (13). The serum basal insulin value was determined by the coated tube method. In particular, the insulin resistance index homeostasis model assessment-insulin resistance (HOMA-IR) was computed with the formula: HOMA-IR = fasting plasma glucose(mg/dl) × immunoreactive insulin (μIU/ml)/405 (14). All samples were run in triplicate.

Plasma PTX-3 Measurements

In both patients and controls, plasma PTX-3 concentration was measured a posteriori from frozen samples by using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Perseus Proteomics Inc, Japan).

hsCRP Assessment

Serum samples were diluted at a ratio of 1:101 with the diluent solution. Calibrators, kit controls, and serum samples were all added on each micro well with an incubation period of 30 min. After three washing intervals, 100 μl enzyme conjugate (peroxidase-labeled anti-CRP) was added on each micro well for additional 15 min incubation at room temperature in the dark. The reaction was stopped with a stop solution, and photometric measurement was performed at the 450-nm wavelength. The amount of serum samples was calculated in milligrams per liter with a graphic that was made by noting the absorbance levels of the calibrators.

GFR Assessment

GFR was calculated according to the simplified version of the Modification of Diet in Renal Disease (MDRD) Study prediction equation formula, GFR = 186 × PCR^−1.154 × age^−0.203 × 1.212 (if black) × 0.742 (if female), as defined by Levey (15).

Assessment of Endothelial Dysfunction

The determination of endothelial dysfunction was performed according to the method described by Celemajer et al. (16). Measurements were made by a single observer using an ATL 5000 ultrasound system (Advanced Technology Laboratories Inc., Bothell, WA) with a 12-Mhz probe. All vasoactive medications were withheld for 24 h before the procedure. The subjects remained at rest in a supine position for at least 15 min before the examination started. The subject's right arm was comfortably immobilized in an extended position to allow consistent recording of the brachial artery 2 to 4 cm above the antecubital fossa. Three adjacent measurements of end-diastolic brachial artery diameter were made from single two-dimensional frames. All ultrasound images were recorded on S-VHS videotape for subsequent blinded analysis. The maximum FMD diameters were calculated as the average of the three consecutive maximum diameter measurements after hyperemia and nitroglycerin, respectively. The FMD levels were then calculated as the percent change in diameter compared with baseline resting diameters.

Statistical Analyses

All of the statistical analyses were performed by using SPSS 11.0 (SPSS Inc., Chicago, IL) statistical package. Non-normally distributed variables were expressed as median (range) and normally distributed variables were as mean ± SD as appropriate. A P value of <0.05 was considered to be statistically significant. One-sample Kolmogorov-Smirnov test was used for analysis distribution of data. One-way ANOVA, t test, and paired-sample t test were used to compare numeric data. Spearman's rank correlation was used to determine correlations with continuous variables. Stepwise multivariate regression analysis was used to assess the predictors for flow-mediated dilation levels.

Baseline Characteristics

Baseline clinical and laboratory characteristic and vascular measurements for the study population are shown in Table 1. There were no differences between diabetic patients and the control group with respect to age, sex, eGFR, and BMI. As expected, serum PTX3, 24-h proteinuria, HbA1c, serum albumin, HOMA, SBP, DBP, and hs-CRP levels were higher in diabetic patients. Moreover, FMD levels were lower in diabetic patients than in controls (Table 1).

Effect of ACEI Treatment

Table 1 shows the longitudinal changes of selected parameters in the 49 patients who completed the study. After ACEI treatment, PTX3, HOMA index, hsCRP, eGFR, SBP, DBP, HbA1c, serum albumin, and proteinuria levels were significantly decreased, and FMD levels were significantly increased (Figure 2). However, the lipid parameters and BMIs of the patients did not change significantly during the study period. During the study period, 11 patients were excluded because of adverse drug reactions, including cough (n = 3) and hyperkalemia (n = 4), or because of noncompliance with the study protocol (n = 4).

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Figure 2.

Long pentraxin 3 (PTX3), proteinuria, and flow-mediated dilation (FMD) levels in the group receiving ramipril before and after 12 wk of treatment.

Univariate Correlations

At baseline, FMD was negatively correlated with serum PTX3, 24-h protein excretion, and hsCRP levels and positively correlated serum albumin and eGFR levels. The negative correlations between FMD levels and PTX3, 24-h proteinuria, serum albumin, and hsCRP were present after the 12-wk treatment period as well (Table 2). PTX3 levels were positively correlated with HOMA, 24-h proteinuria, hsCRP, and duration of diabetes and negatively correlated with FMD and HbA1c levels before the treatment period. After the treatment period, PTX3 levels were positively correlated with 24-h proteinuria and hsCRP levels, whereas PTX3 levels were negatively correlated with serum albumin and FMD levels. The percent increase in FMD was negatively correlated with the percent reduction in serum PTX3 concentration (ρ = −0.51, P < 0.001) (Figure 3). In parallel, the reduction in hs-CRP levels correlated with the percent reduction in serum PTX3 concentration (ρ = 0.35, P = 0.015).

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Figure 3.

Scatter plot showing the significant negative relationship between the percent changes in long pentraxin 3 (PTX3) and flow-mediated dilation (FMD) during the 12-wk ramipril intervention.

Multivariate Regression Analysis

We next investigated the independence of the observed correlations (Table 2) with FMD using a multiple regression model incorporating sex and age, as well as variables significantly associated with FMD at basal (sex, age, 24-h proteinuria, eGFR, PTX3, serum albumin, HOMA-IR, HbA1c, and hs-CRP). Briefly, PTX3 levels were independently related to FMD both before (P = 0.02) and after (P = 0.03) ACEI treatment (Table 2). In addition, in a third and fourth model, we investigated independent predictors of the change in PTX3 and FMD after ACEI therapy (Table 3). The models included changes in 24-h protein excretion, eGFR, SBP, DBP, serum albumin, and hs-CRP, as well as change in FMD or PTX3, respectively. Briefly, change in serum PTX3 levels was independently related only to changes in proteinuria (P < 0.001) and FMD (P = 0.009). Changes in FMD were independently related to changes in serum PTX3 (P = 0.002), hsCRP (P = 0.02), and serum albumin (P = 0.04).