Altered activities of kininase II,an angiotensin converting enzyme,prekallikrein,and nitric oxide in Kuwaiti patients with type 2 diabetes

Abstract

The current investigation was conducted to examine kininase II or angiotensin converting enzyme (ACE), plasma prekallikrein (PK), and nitric oxide (NO) concentrations in healthy Kuwaiti subjects and newly diagnosed Kuwaiti type 2 diabetic patients before and after treatment for 6 weeks with metformin hydrochloride 500 mg twice daily after meal. With the consent of volunteers, blood and urine samples were collected after an overnight fasting. Samples were collected from the diabetic patients before and after treatment for 6 weeks. Enzyme linked immunosorbent assay (ELISA) was carried out on the aliquoted samples to measure the concentration of kininase II. NO was detected via colorimetry. Plasma Kininase II or ACE levels were significantly (P <0.01) increased by 18% in untreated diabetics when compared with healthy volunteers. However, after treatment there was a significant decrease of 20% in their ACE levels. Plasma prekallikrein levels were raised significantly (P <0.01) by 28% in diabetic patients in contrast with the control subjects and the levels were significantly reduced (P <0.0001) by 44% after treatment with metformin hydrochloride. NO levels were found to be significantly decreased in plasma by 56% and in urine by 62% in untreated diabetic patients as compared with the healthy subjects. However, when the treated diabetic patients were compared with untreated diabetics, there was an increase of 50% in plasma and 37% in urine samples. The high levels of kininase II, prekallikrein, and reduced NO may be partly responsible for the induction of renal, cardiac, and hypertensive complications associated with type 2 diabetes. Reduced NO level is an indication of endothelial dysfunction resulting in increased blood pressure. Oral anti-diabetic treatment is associated with protective effects through the reduction of kininase II (ACE), prekallikrein, and elevation of NO levels.

Keywords

diabetes mellitus kininase II metformin nitric oxide prekallikrein

Introduction

Diabetes mellitus is a major risk factor in the development of cardiovascular and renal complications. Previous studies have indicated conflicting results of the bradykinin (BK)-forming components¹ in diabetic patients and experimental animals. High prevalence rates (20–25%) of type 2 diabetes have been documented in Kuwaiti population.² Type 2 diabetes can lead to hypertension, renal, and cardiac complications resulting in high rates of mortality worldwide and in Kuwait as well. BK, a pharmacologically active polypeptide, is one of the kinins which is released in the tissues and body fluids as a result of enzymatic action of kallikreins on kininogens.³ Plasma prekallikrein is activated to form kallikrein. The half-life of BK is very short (<15 s) because it is rapidly inactivated by Kininase II, present in the plasma.⁴ It coverts angiotensin I (Ang I) to angiotensin II (Ang II), a powerful vasoconstrictor, acting on vascular smooth muscles.⁵ Ang II causes volume expansion through sodium and fluid retention and renal vasoconstriction^6,7 and may be associated with nephropathy. Hence, excessive production of ACE in diabetic patients may be the cause of diabetic nephropathy. In addition to catalyzing the formation of Ang II, ACE or kininase II catalyzes the inactivation of BK⁸ and the latter promotes vasodilation and release of prostaglandins as well as NO.⁹ In the kidney, BK causes natriuresis via direct tubular effects.¹⁰ BK has renal protective effects. Therefore, kininase II regulates the balance between the vasodilatory and natriuretic activities of BK and vasoconstrictive and salt-retaining actions of Ang II, whereas, reduced endothelium-derived NO may contribute to vascular disease in diabetes. NO is a potent vasodilator released by the endothelium that plays an important role in maintaining vascular homeostasis.^11,12 Moreover, impaired NO mediated vasodilation in patients with type 2 diabetes mellitus has been reported.¹³ The current investigation, therefore, was conducted to examine kininase II (ACE), plasma prekallikrein, and NO levels in healthy subjects, newly diagnosed type 2 diabetic patients and type 2 diabetics after 6 weeks of treatment with oral metformin hydrochloride.

Methods

Selection of patients and healthy subjects

All procedures followed in this study were in accordance with the standards of the ethical committee on human experiments (Health Sciences Centre, Kuwait University and Ministry of Health, Kuwait) and with Declaration of Helsinki (1964). Written consents were obtained from all the subjects before their inclusion in the study.

The participants in this study were: 18 untreated newly-diagnosed type 2 diabetic patients, 10 treated type 2 diabetics, and 20 healthy subjects. The age of the control and patients groups is in the range of 25–50 years. Body mass index (BMI) was matched in the selected groups. Women were in the premenopausal state. The diabetic patients were all freshly diagnosed individuals with no prior treatment. Subsequently, they were treated for 6 weeks with metformin hydrochloride 500 mg twice daily after meal. The samples were again collected from these patients after 6 weeks of treatment. The healthy subjects were clinically examined and had laboratory tests conducted to rule out absence of hypertension, diabetes, and other co-morbid conditions. Diabetic patients and healthy subjects were instructed to fast for 10–12 h but were asked to drink enough water before the blood sampling and morning urine collection.

Sample collection and storage

A total of 20 mL of peripheral blood samples were collected from the patients and healthy subjects in appropriate BD vacutainer tubes (containing 3.8% sodium citrate, ethylene-diamine-tetra-acetic acid [EDTA] and clot activator and gel) and around 10 mL of midstream urine was collected in sterile containers. A total of 10 mL of blood sample was used for clinical analysis to measure glucose, glycosylated hemoglobin (HbA1c), and plasma creatinine. The remaining blood samples were used to estimate kininase II and NO levels. The collected blood samples were immediately centrifuged at 1000 rpm for 15 min at 4°C. Aliquots of the obtained plasma samples were stored at −80°C pending analysis. In total, 10 mL of urine samples was centrifuged for 30 min at 1000 rpm at 4°C and aliquots were stored at −80°C pending analysis.

Glucose and HbA1c measurements

Aliquots of the collected fasting blood samples were used for the measurement of the plasma glucose levels by means of the oxygen rate method employing a Beckman Coulter Oxygen Electrode (glucose oxidase method, Beckman Coulter Inc., Galway, Ireland). HbA1c was measured by the Beckman Synchron LX system (Bechman Instrument Inc., Fullerton, CA, USA). This utilizes two cartridges, Hb and A1c, to determine A1c concentration as a percentage of total Hb. The hemoglobin was measured by a colorimetric method and the A1c concentration by a turbidimetric immunoinhibition method.¹⁴ The normal range of the HbA1c in our lab was 4.3–5.9%. Serum creatinine was measured by Kobas Integra 400 (Roche Diagnostics, Basel, Switzerland). The test was performed according to the manufacture’s protocol.¹⁵

Estimation of kininase II

Kininase II concentration in plasma samples was estimated using ACE Human ELISA kit from Abcam (AB119577, Cambridge, USA). This assay is based on standard sandwich ELISA technology. Human ACE specific-specific polyclonal antibodies were precoated onto a 96-well plate. Aliquoted plasma samples that were stored at −80°C were used to be analyzed by enzyme linked immunosorbent assay (ELISA) system. The plasma samples were diluted 100 times with a diluent provided with the kit. The optical density (OD) of the samples were read at 450 nm using Elisys Uno from Human Diagnostics (Germany). The values obtained were multiplied by a dilution factor. The concentration of kininase II in plasma is expressed in ng/mL. The sensitivity of the assay was <5 pg/mL and precision was 4.2%.

Estimation of plasma prekallikrein (PK)

The concentration of PK in plasma was determined by ELISA employing ELISA kits from Uscn Life Science (E91801Hu, Wuhan, PR China). The plasma samples were diluted 10 times with 0.02 mol/L (pH = 7.0–7.2) phosphate buffered saline (PBS). The steps for ELISA were carried out as per the protocol described in the kit. The OD of the samples were read at 450 nm using Elisys Uno from Human Diagnostics (Germany). The OD values of the sample were read against the standard. The values obtained were multiplied by the dilution factor. The results are expressed in µg/mL.

Estimation of nitric oxide (NO)

The concentrations of NO in plasma and urine samples were determined using colorimetric detection kit from Arbor Assay (K023-H1). The plasma samples were diluted four times and urine samples were diluted eight times with 0.02 mol/L (pH = 7.0–7.2) phosphate buffered saline (PBS). The procedures were carried out as per the protocol described in the kit. The OD of the samples were read at 540 nm using a UV-VIS spectrophotometer, Multiskan Spectrum, Thermo Electron Corporation. The software used was SkanIt Research Edition, Multiskan spectrum 2.4.2. The OD values of the samples were read against the standard. The values obtained were multiplied by the dilution factor. The results are expressed in µg/mL. The sensitivity of the assay was determined as 1.02 µM and intra-assay precision of 1.8% and inter-assay precision of the assay was 4.1%.

Statistical analysis of the data

The parameter values are expressed as mean ± SEM. Non-parametric Mann-Whitney U-test and one-way ANOVA (Turkey’s multiple comparison test) were applied to determine the significance of differences. A P value of <0.05 was considered as a level of significance. Statistical analysis was done using Graphpad prism software, version 5.0.

Results

Clinical data

The patients as well as healthy volunteers’ demographic data and characteristics are presented in Table 1. The mean (± SEM) fasting plasma glucose levels were significantly high (P <0.001) in untreated diabetic patients (8.2 ± 0.89 mmol/L) in contrast to the controls (4.6 ± 0.07 mmol/L). This obviously confirmed the presence of type 2 diabetes. Similarly, the mean (± SEM) HbA1c (%) concentration was found to be significantly higher (P <0.0001) in untreated diabetic patients (7.6± 0.3% / 60 mmol/mol) in comparison with the control subjects (5.2± 0.1% / 33 mmol/mol). BMI was matched in untreated diabetic patients and control group. Their blood pressure (BP) was not significantly different (P >0.05) and there was no significant (P >0.05) change in serum creatinine between the control subjects (61.5 ± 2.78 µmol/L), diabetic patients (58.3 ± 3.9 µmol/L), and treated diabetics (54.2 ± 2.8 µmol/L).

Table 1.

Characteristics of healthy subjects and type 2 diabetic patients.

Clinical characteristics	Control (n = 20)	Untreated diabetics (n = 18)	Treated diabetics (n = 10)
Age (years)	33.3 ± 1.3	45.2 ± 3.2	43.4 ± 1.0
BMI (kg/m²)	32.05 ± 0.94	34.59 ± 0.9	32.03 ±1.3
Body weight (kg)	90.8 ± 1.9	89.6 ±3.8	80.4 ± 5.2
Blood pressure (mmHg)	118/73 ± 2.9/2.3	117/77 ± 2.6/2.3	112/70 ± 3.2/3
HbA1c (%)	5.2 ± 0.1	7.6 ± 0.3***	Not repeated
Glucose (mmol/L)	4.6 ± 0.07	8.2 ± 0.89***	6.8 ± 0.1
Serum creatinine (umol/L)	61.5 ± 2.78	58.3 ± 3.9	54.2 ± 2.8

Data are presented as mean ± SEM.

***

(P <0.0001) significantly different from healthy subjects.

Kininase II or angiotensin converting enzyme (ACE) levels

The mean (± SEM) kininase II levels were significantly increased (P <0.01) in untreated diabetic patients (401.4 ±19.5 ng/mL) in comparison with the healthy subjects (339.3 ± 11.5 ng/mL) (Figure 1). However, ACE levels in treated diabetics (321.7±23.9 ng/mL) were significantly lower (P <0.01) than those of untreated diabetics. The 95% confidence interval (CI) level of difference was in the range of −123.9 to −0.304 in control group versus untreated diabetics and the same in untreated versus treated diabetic patients (range, 12.55–146.9).

Figure 1.

Plasma kininase II level in healthy, untreated diabetic, and treated diabetic patients. Data are presented as mean and SEM. There is a * significant (P <0.05) difference between control and untreated diabetics and also the same in treated and untreated diabetics (P <0.05). n = 20 control, n = 18 untreated diabetics, and n = 10 treated diabetics.

Plasma prekallikrein levels

The mean (± SEM) plasma prekallikrein level was significantly increased (P <0.05) in diabetic patients (198.7 ± 12 µg/mL) compared to healthy subjects (54.7 ± 11.9 µg/mL) (Figure 2). After treatment with metformin the prekallikrein values reduced significantly (P <0.0001) (110.5 ± 9.7 µg/mL).

Figure 2.

Prekallikrein level in healthy, untreated diabetic, and treated diabetic patients. Data are presented as mean and SEM. There is a * significant (P <0.05) difference between control and untreated diabetics and a *** significant difference between treated and untreated. n = 20 control, n = 18 untreated diabetics, and n = 10 treated diabetics.

Nitric oxide (NO) levels

NO levels in urine and plasma samples were significantly (P <0.0001) decreased in untreated diabetic patients in comparison with the control subjects. However, NO level was found to be higher in urine than in plasma samples. In control group, the mean (± SEM) NO level detected in urine was 903.7 ± 87.5 µM while in untreated diabetics it was 341.3 ± 47.02 µM (Figure 3). In plasma, the mean (± SEM) concentration of NO in controls was 26.7 ± 1.07 µM and in untreated diabetics was 11.7 ± 0.5 µM (Figure 4). In case of urinary NO, there was no significant difference between the mean (± SEM) NO level in treated (467.8 ±85.7 µM) and untreated diabetics while plasma NO level showed a significant increase (P <0.001) in the mean (± SEM) level in the treated diabetics (17.5 ± 1.5 µM) when compared to untreated group. The 95% CI of the difference in urinary NO was 316.3–808.6 in control subjects versus untreated diabetic patients and 418.9–165.9 in the untreated versus treated diabetics. Meanwhile, the 95% CI of the difference in plasma level was 11.15–17.37 in control groups versus treated diabetics and −8.358 to −1.661 in untreated versus treated diabetics.

Figure 3.

Nitric oxide level in urine samples for control subjects, untreated diabetic, and treated diabetic patients. Data are presented as mean and SEM. *** significantly different (P <0.0001) between control and untreated diabetics and no significant difference between treated and untreated diabetic patients. n = 20 in control, n = 18 in untreated diabetic patients, and n = 10 in treated diabetic patients.

Figure 4.

Nitric oxide levels in plasma samples for control subjects, untreated diabetics, and treated diabetic patients. Data are presented as mean and SEM. There is a *** significant difference (P <0.0001) between control subjects and untreated diabetic patients and also ** significant difference (P <0.001) between treated and untreated diabetics. n = 20 in control, n = 18 in untreated diabetics, and n = 10 in treated diabetics.

Discussion

The present study was conducted to investigate the levels of kininase II or ACE and NO in Kuwaiti patients with newly diagnosed type 2 diabetes. These patients were treated for 6 weeks with oral metformin hydrochloride. These patients had both elevated fasting blood glucose and HbA1c levels than that of controls. Treated diabetic patients showed a reduction (17.5%) in fasting blood glucose level, but it did not differ significantly from untreated diabetics. However, their renal functions were normal because creatinine levels were within the normal ranges.

Plasma kininase II causes inactivation of vasodilator, BK.¹⁶ This investigation suggested that raised levels of kininase II can serve as a risk factor for myocardial infarction and hypertension as observed previously.¹⁷ This study demonstrated increased levels of kininase II or ACE activities in untreated type 2 diabetic patients compared to control subjects. High levels of ACE were associated with the development of left ventricular hypertrophy (LVH) in hypertensive patients.¹⁸ In this regard, there is an evidence to suggest that diabetes induced by streptozotocin showed the presence of LVH and hypertension in diabetic rats associated with altered BK activities.¹⁹ Thus, the present study will be a significant advancement in predicting the cardiovascular in the early stages of type 2 diabetes. In fact, the clinical benefits of ACE inhibitors are known to protect the renal function and to treat hypertension.²⁰ Moreover, changes in kallikrein and kininogen levels in type 1 and type 2 diabetes have been reported earlier.^21,22 Furthermore, treatment with oral metformin in this study caused significant reduction in kininase II levels. This finding has not been previously reported. Hence, it is possible to suggest that the reduced kininase II level may be clinically beneficial to the patients against developing cardiovascular disorders.

There was no alteration in the creatinine levels in the present study, however, high plasma prekallikrein levels may suggest an early onset of renal abnormalities. In fact, high plasma prekallikrein levels have been documented in type 1 diabetic patients.²¹ These investigators suggested that increased plasma prekallikrein level could be a risk factor for hypertension and nephropathy in type 1 diabetes. The present study may also suggest a similar mechanism for developing hypertension and nephropathy in type 2 diabetic patients. Therefore, plasma prekallikrein could be a useful tool for monitoring diabetic patients for developing hypertension and/or renal abnormalities. It is interesting to note that there was significant reduction in the prekallikrein levels after treatment with oral anti-diabetic agent (metformin hydrochloride), which may be considered as a renal protective effect.^21,22

This study demonstrates a significant reduction in the urinary and plasma NO levels in untreated Kuwaiti patients with type 2 diabetes. However, there are controversial reports regarding the levels of NO in type 2 diabetes. In this regards, Ghose et al.²³ have reported higher levels of NO in type 2 diabetes whereas the other group²⁴ has reported a reduced NO level in type 2 diabetes. Thus, the present investigation confirms this finding.²⁴ NO is a potent vasodilator released by the endothelium, which plays a pivotal role in maintaining vascular homeostasis.¹² There is an evidence that NO is reported to be reduced in experimental diabetes.²⁵ Several potential mechanisms have been proposed to explain the reduced endothelial-dependent vasodilation in patients with diabetes, such as, decreased synthesis of NO by endothelium and increased inactivation of NO.⁷ This view is further supported by the present study. Reduced activity of NO may serve as a risk factor for development of hypertension and cardiovascular disorders. Decreased synthesis of NO in subjects with diabetes and nephropathy has been reported previously.²⁶ It is suggested that reduced NO concentration in plasma and urine is an indicator of endothelial abnormalities as observed in the present investigation. Furthermore, it has been reported that treatment with oral anti-diabetic agents can improve the NO levels and vasodilation in type 2 diabetics.²⁷ In this investigation, we found that after treatment, there was a significant increase in NO levels in the plasma but in urine there was no significant increase in NO levels. These findings may support in predicting the early risk factors for diabetes and prevention of diabetic complications, in particular renal and cardiovascular.

Footnotes

Acknowledgements

We appreciate and thank the technical help of Dr Parvathy Narayanan and the OMICS Research Unit Grant SRUL02/13, Health Sciences Centre, Kuwait University, for the utilization of their equipment.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This study was supported by the clinical priority research grant No: RP01/09 from the Research Sector, Kuwait University, Kuwait.

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