Study of urinary protein and leucocytes in normotensive and hypertensive children and adults.
An important indicator of hypertensive vascular disease in adults is the target organ damage (TOD) caused by elevated blood pressure.  Target organ damages include changes in heart (left ventricular hypertrophy), blood vessels (increased carotid artery intimal thickness), eyes (hypertensive retinopathy), kidneys (hypertensive nephropathy) and other organs.[i-6l However, studies by different workers have suggested that TODs are less prominent in hypertensive children. [2.7]
The definition of hypertension in adults is based on the relationship between blood pressure levels and incidence of adverse cardiovascular events like stroke, myocardial infarction, hypertensive nephropathy and hypertensive retinopathy.  Similar data to define paediatric hypertension that are based on target organ damage are not available.  Children often do not develop the target organ damages to such an extent to warrant clinical manifestations. [2.7]
Chronic hypertension leads to hypertensive nephropathy, a common cause of kidney disease.  Proteinuria is a known indicator of early stages of hypertensive nephropathy.  Excretion of albumin in the range of 30-300 mg in 24 hours or an Albumin: Creatinine Ratio (ACR) of 30 to 300 [micro]g albumin/mg creatinine is an indicator of microalbuminuria.  Aggressive blood pressure control to 130/80 mmHg or lower slows the progression of all forms of chronic kidney disease, especially when proteinuria is present. [10,11]
Microalbuminuria predicts progressive deterioration of renal function and an increased risk of cardiovascular (CV) events/death in adults.  Recently, Assadi demonstrated that even in children with essential hypertension, the treatment of microalbuminuria by renoprotective therapy (Angiotensin converting enzyme inhibitors or angiotensin-receptor blockers) was associated with regression of left ventricular hypertrophy. 
The purpose of this study is to investigate the prevalence of proteinuria and pyuria in children and adults with hypertension, and compare it with the prevalence in normotensive population in both children and adults.
MATERIALS AND METHODS
The subjects were chosen from school-going children of different schools in Kolkata (for < 18 years of age). Subjects of more than 18 years of age were selected from the students and staff of our Institution. Informed consent of the individuals was taken. The study was approved by the Institutional Ethical Committee of KPC Medical College and Hospital, Jadavpur, Kolkata.
All consenting individuals between 10 years to 60 years of age.
* Presence of known primary renal disease.
* Presence of known non-hypertensive causes of nephropathy (e.g. Diabetes mellitus).
* Individuals taking drugs that can alter urinary protein output (e.g. ACE inhibitors).
* Individuals with clinical features of active urinary tract infection.
Stratified random sampling method was used for the selection of study subjects. 1000 respondents who were part of the study were divided into two strata. The first stratum included individuals < 18 years of age and the second stratum included individuals > 18 years of age. In each stratum 100 hypertensive and 100 normotensive subjects were selected by random sampling. Total number of subjects was 400.
The Following Formula was used to calculate the Sample Size-
n = [Z.sup.2]pq/[e.sup.2], where, n = required sample size.
Z = normal standard deviation which is equal to 1.96% at 5% significance level.
p = prevalence of proteinuria among hypertensive subjects in India, which is 37.5%. 
q = (100 - p), i.e. (100 - 37.5) % = 62.5%.
The allowable error (e) is 5%.
Therefore, the minimum sample size (n) = [Z.sup.2]pq/[e.sup.2]
= [(1.96).sup.2] x 37.5 x 62.5/[(5).sup.2]
Considering 10% non-response rate, the sample size (n) would be- n = [360 + (10% of 360)] = 360 + 36 = 396.
We studied 400 subjects after stratified random sampling.
Urine from 200 hypertensive and 200 normotensive individuals was collected. Out of 200 individuals in each group, 100 were below 18 years of age, and 100 were above 18 years of age. The urine samples were tested for presence of protein and leucocytes or pus cells.
A strip test with multiple parameters (Multistix) was used for urinalysis. A standard urine test strip comprises of 10 different chemical pads or reagents which react (Change colour) when immersed in, and then removed from, a urine sample. The test can often be read in as little as 60 to 120 seconds after dipping. 
Multistix contains tetrabromophenol blue indicator for protein detection with a buffer to maintain the pH at a constant level. As the protein concentration increases, the colour of the indicator progresses through various shades of green and finally to blue. The interpretation of the result was done according to the guidelines provided with the kit  [Figure 1].
Presence of leucocytes or pus cells was further confirmed by microscopic examination of urine under high power objective (400 x magnification) and reported in units of numbers of cells per high power field (n/HPF).
A sample of well-mixed urine measuring 10 mL is centrifuged in a centrifuge tube at 3,000 rpm for 10 minutes until a deposit is produced at the bottom of the tube. The supernate is decanted until a volume of 0.5 mL is left inside the tube. The sediment is dissolved in the residual supernate by tapping the bottom of the tube. A drop of resuspended sediment is placed on a glass slide, secured with coverslip and examined under 40x objective (high power) of microscope. Usually there are about 0 - 5 leucocytes per high power field in a normal sample of urine, with females having slightly higher in numbers due to vaginal contamination.
The urine strip test for white blood cells detects leucocyte esterase, which is present in azurophilic granules of leucocytes. The urine strip test reaction for leucocytes is based on the action of leucocyte esterase in catalysing the hydrolysis of an ester of indolecarboxylic acid. The indoxyl that is liberated combines with a diazonium salt in order to produce a violet coloured azole dye. [14,15]
Reactions Catalysed by Leucocyte Esterase [14,15] are-
i. Indolecarboxylic acid ester [right arrow] Indoxyl + Acid.
ii. Indoxyl + Diazonium salt [right arrow] Violet azole dye.
The esterase reaction needs about 5 minutes to take place. The presence of strong oxidising agents or formaldehyde can cause false positives. False negative results are associated with elevated concentrations of protein (Greater than 500 mg/dL), glucose (Greater than 3 g/dL), oxalic acid and ascorbic acid. Urine with a high specific gravity can also cause leucocyte crenation, which can impede the liberation of the esterases. 
The data were analysed using Microsoft Excel 2007. Piecharts and bar-diagrams were constructed representing the distribution of the urinary findings in the two age groups. Accordingly, the prevalence of proteinuria and pyuria in normotensive and hypertensive children and adults were calculated and compared.
Urine was tested for protein and leucocytes in 200 hypertensive individuals and 200 normotensive individuals (with each group comprising of 100 individuals under 18 years of age, 100 individuals at or above 18 years of age).
The findings are presented in Charts 1 - 4.
The prevalence of proteinuria was markedly higher in hypertensives, compared to normotensive subjects in both adults and children.
The severity of proteinuria among hypertensive and normotensive subjects is presented below.
Many authors have implicated chronic pyelonephritis as an important secondary cause of paediatric hypertension.  Chronic pyelonephritis is associated with increase in pus cells in urine. In our study; however, the prevalence of pyuria was not found to be remarkably higher in hypertensive children, compared to the normotensive children.
Proteinuria, particularly microalbuminuria, is a known early marker of hypertensive nephropathy in adults.  In our study, prevalence of proteinuria (Estimated by dipstick method) was markedly higher in hypertensive individuals, compared to normotensive individuals. An interesting finding was that the prevalence of proteinuria was also higher in hypertensive children, as compared to their age-matched counterparts. This is important, as we often think that hypertension in children may not produce target organ changes.
Previous studies have shown that children with hypertension develop subtle target organ damages like increased carotid artery intimal medial thickness,  left ventricular enlargement, structural changes in forearm vessels,  narrowing of retinal arterioles,  among other changes. Proteinuria; however, is easier to estimate and far less expensive compared to the above parameters. Studies conducted by Assadi  has demonstrated that controlling microalbuminuria in children by ACE inhibitors or Angiotensin receptor blockers causes regression of left ventricular hypertrophy. Thus, detection of proteinuria in hypertensive children may have both diagnostic and prognostic implications.
In our study, 17% of the hypertensive subjects in the younger age group (<18 years of age) were diagnosed with proteinuria; in contrast to only 2% of the normotensive subjects in the same age group who had proteinuria.
The prevalence of proteinuria in the hypertensive adults was even higher. Sixty-two percent of the hypertensive adults were diagnosed to have proteinuria in comparison with only 4% of normotensive adults who had a positive test for protein in their urine.
The significance of the findings may be an indication that children with raised blood pressure are prone to target organ changes as seen in adults. It may be therefore useful to screen children with proteinuria for hypertension, and hypertensive children may be advised for baseline urinary protein estimation.
Our study indicates that children who suffer from hypertension can also develop proteinuria indicating renal damage. It may be useful to screen hypertensive children for proteinuria and vice versa, to improve monitoring of hypertensive target organ damage and initiate appropriate therapeutic measures. Our findings suggest that paediatric hypertension can also be associated with proteinuria which can be used as a marker for target organ injury in children.
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 Falkner B. Hypertension in children and adolescents: epidemiology and natural history. Pediatr Nephrol 2010;25(7):1219-24.
 Daniels SR, Loggie JMH, Khoury P, et al. Left ventricular geometry and severe left ventricular hypertrophy in children and adolescents with essential hypertension. Circulation 1998;97(19):1907-11.
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 Larson EW, Edwards WD. Risk factors for aortic dissection: a necropsy study of 161 cases. American Journal of Cardiology 1984;53(6):849-55.
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 Jalal S, Sofi FA, Sidiqqi MA, et al. Prevalence of Microalbuminuria in essential hypertension: a study of patients with mild to moderate hypertension. Indian J Nephrol 2001;11:6-11.
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Tirthankar Guha Thakurta (1), Pulak Panda (2)
(1) Postgraduate Trainee, Department of Physiology, KPC Medical College and Hospital, Jadavpur, Kolkata.
(2) Professor and HOD, Department of Physiology, KPC Medical College and Hospital, Jadavpur, Kolkata.
Financial or Other, Competing Interest: None.
Submission 12-02-2017, Peer Review 15-03-2017, Acceptance 21-03-2017, Published 27-03-2017.
Dr. Tirthankar Guha Thakurta, 4A East Road, Jadavpur, Kolkata-700032.
Caption: Figure 1. Grading of leucocyte count and proteinuria by urine dipstick test (Multistix)
Chart 1. Prevalence of proteinuria revealed by dipstick examination of random sample of urine in hypertensive and normotensive subjects of different age groups Normontensive Hypertensive Age < 18 years 2% 4% Age > 18 years 17% 62% Note: Table made from bar graph. Chart 2. Findings of urine examination for protein in normotensive and hypertensive individuals Normotensive subjects Hypertensive subjects No Proteinuria 97% 60% 1+ Proteinuria 1% 5% 2+ proteinuria 3% Trace Proteinuria 2% 32% Note: Table made from pie chart. Chart 3. Prevalence of pyuria revealed by dipstick examination of urine in hypertensive and normotensive subjects of different age groups. Normotensive Hypertensive Age < 18 years 5% 7% Age > 18 years 6% 7% Note: Table made from bar graph.
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|Title Annotation:||Original Research Article|
|Author:||Thakurta, Tirthankar Guha; Panda, Pulak|
|Publication:||Journal of Evolution of Medical and Dental Sciences|
|Date:||Mar 27, 2017|
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