Biochemical Evaluation of Selected Renal Indices in Women with Metabolic Syndrome in Ilesa Metropolis South Western Nigeria

Aim: The aim of this study was to see the effects of metabolic syndrome on some selected renal indices for kidney biological were significantly raised in women with metabolic syndrome when compared to normotensive women while significant decreases were observed in Urine (creatinine, potassium and urea) and creatinine clearance rates respectively in same comparison. However, a non-significant increase in Urine (volume and protein), as well as a non-significant decrease in urea clearance rates and urine sodium were seen in women with metabolic syndrome when compared to normotensive women. Conclusion: This work revealed that metabolic syndrome has negative effects on the renal indices investigated which were attributed to the characterizing high blood pressure and vessel dysregulation to possibly result in kidney dysfunction in its sufferer. This work thus showed that renal indices can be employed with other investigations to diagnose metabolic syndrome, prevents its medical complications and monitor treatment progress.


INTRODUCTION
Metabolic syndrome is defined as group of conditions that come together in a single individual for increased risk of heart disease, diabetes and stroke [1]. These group of conditions include insulin resistance for high blood glucose levels, hypertension (high blood pressure), cholesterol and triglyceride abnormalities, an increased risk for blood clotting and too much fat around the waist [1]. Metabolic syndrome also known as syndrome X, insulin resistance syndrome, or dysmetabolic syndrome is considered a multiplying risk factor for cardiovascular diseases, type 2 diabetes and stroke due to insulin resistance and an abnormal function and pattern of body fat [2].
Approximately 20 -25 percent of the world's adult population has the cluster of risk factors of metabolic syndrome. They are twice as likely to die from and three times as likely to have a heart attack or stroke compared with people without the syndrome [3]. In addition, people with metabolic syndrome have a fivefold greater risk of developing type 2 diabetes. They would add to the 230 million people worldwide who already have diabetes, one of the most common chronic diseases worldwide and the fourth or fifth leading cause of death in the developed world. The clustering of cardiovascular disease (CVD) risk factors found in metabolic syndrome is now considered to be the driving force for a new CVD epidemic [4]. This syndrome is a serious health condition that affects about 34 percent of adults in USA and places them at higher risk of cardiovascular disease, diabetes, stroke and diseases related to fatty build ups in artery walls [5]. The number of people with metabolic syndrome increases with age, affecting more than 40 percent of people in their 60s and 70s. According to World Health Organisation in 2004, more than one in five Americans has metabolic syndrome. Studies also show that 34% of overall African adults were affected with 43.3% in Ethiopia, 35.9% in Ghana, and 31.9% in Nigeria to mention a few [6].
The underlying cause of the metabolic syndrome continues to challenge the experts but both insulin resistance and central obesity are considered significant factors [7]. Abdominal obesity, hypertriglyceridemia, deranged cholesterol level, elevated blood pressure and blood sugar are prevalent signs and symptoms of metabolic syndrome [8]. Nevertheless, stress, overweight, sedentary lifestyle, aging, coronary heart disease, lipidystrophy, psychiatry illness, diet, genetics, physical inactivity, ageing, a proinflammatory state and hormonal changes may also have a potential risk factor effect, but the role of these may vary depending on ethnic group [9][10][11]. Possible complications of metabolic syndrome include mitochondrial dysfunction and insulin resistance, vascular and neurological disorders as well as diabetes mellitus type 2 to mention a few [12,13].
Pathophysiologically, in metabolic syndrome there is development of visceral fat, after which the adipocytes (fat cells) of the visceral fat increase plasma levels of TNFα and alter levels of a number of other substances (e.g.adiponectin, resistin, and PAI-1). TNFα has been shown not only to cause the production of inflammatory cytokines, but also possibly to trigger cell signaling by interaction with a TNFα receptor that may lead to insulin resistance [14,15].
Several management methods can be applied to prevent and treat metabolic syndrome. Treating metabolic syndrome requires addressing several risk factors together for improving the quality and length of your life. This will improve the overall cardiovascular health and greatly improve the individual risk factors that make up metabolic syndrome [16]. Lifestyle change had been considered to be potent way of preventing and treating this syndrome, as this has to do with eating a better and balanced diet, avoiding processed food, which often contains partially hydrogenated vegetable oils, and is high in salt and added sugar. Moreover, refraining from alcohol and smoking habits can do a lot to curb this syndrome. Since physical inactivity and excess weight are the few underlying contributors to the development of metabolic syndrome, getting more exercise and losing weight can help reduce or prevent the complications associated with this condition [17]. Health providers may also prescribe medications like antihypertensives and antidiabetics to manage some of the underlying problems [18].
Renal indices are substances (like chemicals, enzymes, molecules, indicators or signals) produced by kidney into body fluids [19]. These substances exhibit some characteristics such as specificity and sensitivity which makes them significant in monitoring the glomerular filtration rates and tubular functions of the kidneys and also detect alterations in renal functions [20].
Indices of use for kidney's specific functions include: Plasma creatinine, plasma urea, blood and urine electrolytes, clearance rates (of creatinine and urea), and 24 hour urine profile (volume, quantitative protein). However they aid in early diagnosis of renal dysfunction, assessment of nephrons, glomerular and tubular functions of the kidneys long before permanent damage will set-in to the kidneys [21].
This study investigated the biochemical evaluation of selected renal indices in women with metabolic syndrome in Ilesa Metropolis, South Western Nigeria. This was to see the effects of metabolic syndrome on some selected renal indices for kidney biological functions.

Experimental design and grouping of subjects
Quasi-experimental design method was utilized in this study and subjects were divided into two (2) groups: i. Group 1 was Normotensive Women (Control); ii. Group 2 was Women with Metabolic Syndrome (Tests);

Sampling areas
The hospital selected for the purpose of this research in ilesa metropolis was Obafemi Awolowo University Teaching Hospitals Complex, Wesley Guild Hospital Unit, Ilesa, Osun State, Nigeria. This hospital is recognised as referral centres for metabolic syndrome cases in ilesa metropolis.

Recruitment of subjects
A total of eighty (80) subjects of both normotensive women (n=40), and women with metabolic syndrome (n=40) were recruited for this research and their samples were processed for analysis within 72 hours of collection employing standard diagnostic techniques.

Selection of subjects
The subjects for this project were selected according to the following criteria:

Blood sample
About 10mL of venous blood was collected from each subject into lithium heparin bottle.

Preparation of blood plasma
The blood samples were separated as plasma into plain labeled bottles after spinning in a centrifuge at 4,000 rpm for 20 min.

24 hr urine sample
A preservative of 2M HCl (20mL) was added into a sterile 5 litre container for voided 24 hour timed urine by all subjects [28].

Preparation of 24 hr urine sample
Timed urine of 24 hour was collected from each of the subjects in each group and was measured using cylinder. About 10 mL of the measured urine was aliquoted into a plain labelled bottle before spinning in a centrifuge at 4,000 rpm for 20 min. The urine sample was used to confirm presence of protein in urine as well as clearance rates (of creatinine and urea).

Estimation of creatinine (in plasma and urine) and creatinine clearance
Plasma and urine concentration of creatinine was estimated spectrophotometrically according to Jaffe's reaction method [22].

Procedure
Distilled water (0.5 mL) was pipetted into test and standard tubes. 1 mL of plasma/urine and standard (1 mg/mL) was added into their tubes respectively. Thereafter, 0.5 mL of both sodium tungstate (5%) and sulphuric acid (0.33 mol/L) were added to all the tubes, and the content mixed thoroughly. The suspension was centrifuged for 10 min. at 5000 rpm. In the second stage, 1 mL of each tube supernatant was taken into other arranged tubes for test and standard. 1 ml of picric acid (0.04 mol/L) and sodium hydroxide (0.75 mol/L) were then added to all tubes, with the content mixed appropriately. It was incubate at room temperature for 15 min. before the absorbance was read at 520 nm against reagent blank.

Calculation:
Concentration of Plasma / urine (Creatinine (umol/L)) = Absorbance of test X Concentration of standard / Absorbance of standard

Estimation of urea (in plasma and urine) and urea clearance
The concentration of plasma and urine urea was estimated spectrophotometrically using diacetylmonoxime method [22].

Procedure
Distilled water (1 mL) was pipetted into tubes of test and standard. 0.01 mL equal volume of plasma/urine and standard (200 mg / 100 mL) was added to their tubes respectively and the suspension mixed. The same volume of 1 mL of urea colour reagent [containing diacetylmonoxime (20 g/L) and thiosemicarbazide (5 g / L)] and urea acid reagent [containing FeCl 3 .6H 2 O (20 g/L) and sulphuric acid (2.0 mol/L)] was added to all tubes. The mixture was boiled in water bath at 100°C for 20 min., then removed and cooled in cold water. The absorbance of test and standard was read at 520 nm against reagent blank.

Calculation:
Concentration of Plasma / urine (Urea (mmol/L)) =Absorbance of test X Concentration of standard / Absorbance of standard

Determination of plasma and urine sodium and potassium concentrations
Plasma and urine concentrations of sodium and potassium were determined using the flame photometry method [23].

Procedure
Plasma/urine and standard [containing 58.5 g/L of NaCl and 7.46 g/L of KCl] of equal volume of 0.1 mL was taken with pipette into respective electrolyte bottle (of test and standard) and diluted with distilled water (10 mL). The flame photometer was turned on and blanked with distilled water. Required electrolyte filter was selected, after which the nebulizer was introduced into the diluted standard and test. The concentration of sodium and potassium in the plasma/urine samples and standard in mmol/L was visualised through the read-out device of the flame photometer.

Determination of urine glucose
Qualitative urine detection of glucose was determined using the clinistix strip method [23].

Procedure
The clinistix band area was completely immersed in urine specimen collected into a sterile container. The strip was removed and tapped against the side of urine container to remove excess urine. The strip was held close to colour chart on the clinistix bottle, and the investigation was carefully recorded by comparing glucose band colour area with corresponding colour chart on the bottle after 2 min.

Estimation of 24 hour urine protein concentration
Concentration of 24 hour urine protein was estimated spectrophotometrically according to turbidimetric method [24].

Procedure
24 hour urine sample and standard (100 mg/dL) of equal volume of 0.5 mL were pipetted to their test tubes and 3% trichloroacetic acid (1.5 mL) was added to each tubes. The reaction mixture were mixed and incubated for 5 min. at room temperature, after which the absorbance of the urine and standard were read against reagent blank at 470 nm.

Statistical Analysis
Results are expressed as mean ± SEM. Statistical difference was determined by one-way analysis of variance (ANOVA) followed by a post hoc test (Student Newman-Keuls Test (SNK)). Difference was considered statistically significant with p < 0.05. Computer software Graph pad PRISM ® version 3.00 was used for the analysis.

RESULTS
The results of this study showed that in women with metabolic syndrome plasma concentrations of creatinine and potassium were significantly raised at (P<0.05) while the levels of urea and sodium were significantly raised at (P<0.01) in the investigated renal indices when compared with normotensive women (see Table 1).
Women with metabolic syndrome however revealed non-significant increase at (P>0.05) in urine volume and urine protein, as well as a nonsignificant decrease (P>0.05) in urea clearance rates and urine sodium in women with metabolic syndrome when compared to normotensive women. Moreover, in the same comparison, significant decreases were observed in urine creatinine, creatinine clearance rates and urine potassium respectively at (P<0.001) but at (P<0.05) in urine urea (see Table 2).

DISCUSSION
Results obtained from this study showed that plasma creatinine and urea concentrations were significantly raised in women with metabolic syndrome than the normotensive groups. This observation could be due to endogenous degradation/destruction of muscle and tissues from raised blood pressure vasoconstriction (kidney hypertension) in metabolic syndrome individual as well as vascular dysregulation/ endotheliosis of arteries to facilitate kidney necrosis that elevates synthesis of creatinine and urea. This result is similar to previous reports [4,25].  However, the urine creatinine and urea concentrations, as well as creatinine clearance rates were decreased significantly while urea clearance rates were non-significantly decreased in women with metabolic syndrome than the normotensive groups. The observations are similar to the previous findings [4, 25,26]. This result could be attributed to reflections of endogenous degradation / destruction of muscle and tissues as well as vascular dysregulation/ endotheliosis of arteries to facilitate kidney necrosis that elevates synthesis of plasma creatinine and urea with reduced clearance rates of creatinine and urea in urine.
Plasma concentrations of sodium and potassium were observed to be significantly raised in women with metabolic syndrome than the normotensive groups, while in urine, sodium was non-significantly decreased and potassium concentration was significantly decreased in same comparison. The observed hypernataemia and hyperkalaemia might be due to increased kidney vasoconstriction in metabolic syndrome with resultant changes in the kidneys' ability to remove salt for increased retention of sodium and potassium in blood with simultaneous reduction of sodium and potassium in urine, thus enhancing high blood pressure, heart disease and stroke. This result is as well similar to previous reports [27,28].
The 24 hour urine volume and urine protein concentration were non-significantly raised in women with metabolic syndrome when compared with the normotensive groups. Similar previous reports have been made [29,28,[30][31][32]. The result might be due to increased blood pressure (kidney hypertension) vasoconstriction in metabolic syndrome that initiates resultant changes in the kidneys' ability for increased thirst and increased urination as well as hyperproteinuria due to increase kidney intratubular pressure with ureteral obstruction, and efferent glomerular arteriole constriction to filter out protein with failure of tubular reabsorption.

CONCLUSION
Metabolic syndrome as expressed in this study has negative effects on the renal indices investigated which were attributed to the characterizing high blood pressure and vessel dysregulation to possibly result in kidney dysfunction in its sufferer. This work thus showed that renal indices can be employed with other investigations to diagnose metabolic syndrome, prevents its medical complications and monitor treatment progress.

LIMITATIONS OF THE STUDY
The limitation attached to this study is based on the rare occurrence rate of metabolic syndrome in ilesa metropolis that has warranted using smaller subjects number for the study.

CONSENT
Author declares that written informed consent was obtained from all the subjects studied in this work.

ETHICAL APPROVAL
Author hereby declare that all experiments have been examined and approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.