Islamic University-Gaza

Deanery of Higher Education

Faculty of Science

Master of Biological Sciences

Medical Technology�Department

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Early Markers for Diabetic Nephropathy in Urine

of Type 2 Diabetics�in Southern Gaza Strip

ƍƄƂƃŒ�¾ƚřŵƚƃ�ŖŧƂŕƆ�ŘƙƙťƐ�ƇƆ�ƑƈœśƃŒ�ųƍƈƃœŕ�ƇƒŕœŮƆƃŒ�Ǝťƃ���ƐŧƂŪƃŒ�ůŧƆ�ŖŨŹ�ųœűſ�ŔƍƈŞ�Ƒż��

Prepared By

Abed EL-Rahman Abu Hilal

Supervisor

Prof. Dr. Mohammad Shubair

A thesis submitted in partial fulfillment of the requirements for

the master degree of Biological Sciences/Medical Technology

June, 2009

id3671015 pdfMachine by Broadgun Software - a great PDF writer! - a great PDF creator! - http://www.pdfmachine.com http://www.broadgun.com

- I -

Declaration

I hereby declare that this submission is my own work and that, to the best of my

knowledge and belief, it contains material neither previously published or written

by another person nor material which to a substantial extent has been accepted

for the award of any other degree of the university of other institute, except where

due a acknowledgment has been made in the next.

Name

ABED R. HILAL

Date

June, 2009

Signature

ABED R. HILAL

Copy Right

All rights reserved 2009: No part of this work can be copied, translated or stored in

any retrieval system, without prior permission of the author.

Correspondence to: abed67hil@yahoo.com

mailto:abed67hil@yahoo.com

- II -

Dedication

To my late father who put me on the right way ..

To my Lovely mother and wife who supported me on the

way of success ..

To my daughter Mona who change my life ..

To my Palestinian people who are still suffering..

..

To all of them I dedicate this work

Abed R. Hilal

June, 2009

- III -

Acknowledgment

I would like to express my deepest gratitude and appreciation to my supervisor

Prof. Dr. Mohammad Shubair Professor of Medical Technology, Faculty of

Science, for his initiation and planning of this study, keen supervision and the

great valuable scientific help that leads to achieve this work.

I would like to express my deepest thanks to Mr Kamal Bakeer, senior laboratory

of Abu Yousef El Najar hospital and to the technical staff for their helping in the

biochemical analyses.

My special thanks to the UNRWA laboratory team of Rafah clinic and to the team

of non-communication disease department there for their kind co-operation in the

data collection processes.

My thanks and respects also extended to everyone who encourages or gives me

a help in this research.

Abed R. Hilal

June, 2009

- IV -

Early Markers for Diabetic Nephropathy in Urine of Type 2

Diabetics�in Southern Gaza Strip

Abstract

Diabetic nephropathy represents one of the major problems developed in

Diabetics that eventually lead to renal failure. By time the kidneys performs

dysfunctional progression from hyperfiltration to micro-to macroalbuminuria to

renal failure, hence the onset of diabetic nephropathy can be predicted by

detection of alterations in release of enzymes or albumin in the urine. The aim of

this study is to detect and asses some urinary enzymes and microalbuminuria in

urine of diabetic patients and healthy subjects. It also aims to find relationship

between these bio-markers and the progression of diabetic nephropathy as

suitable noninvasive assay for early detection of DNP in type-2 DM patients� in

Gaza Strip. Random 61 urine samples of diabetics (33 females and 28 males)

and 53 healthy subjects (23 females and 30 males) that matched the diabetic

case in sex and age were collected. Samples were tested for microalbuminuria

(MAU), alkaline phosphatase (ALP), alanine aminopeptidase (AAP), acid

phosphatase (ACP) and creatinine (Cr) using Hitachi 902 (Roche) auto analyzer

and Biosystems BTS-310 spectrophotometer. Turbidimetric bio-systems kits for

MAU were used while the other biomarkers measured spectrophotometry using

Far-diagnostic kit. Data were collected and analyzed using SPSS (version15.0).

The means of the markers in patients group were: MAU 28.6 mg/L, ALP 15.8 U/L,

AAP 13.0 U/L, ACP 418.2 U/L and Cr 82.5 mg/dl, and in control group they were:

10.8 mg/L, 7.4 U/L, 6.2 U/L, 178.3 U/L and 158.6 mg/dl, respectively.

Comparison of the markers means among males in patient and control groups

showed significant difference (P= 0.000 for each marker), and among females in

both groups showed P-values of 0.000, �����, �����, ����� and ����� for MAU,

ALP, AAP, ACP and Cr, respectively. Comparison between the means of the

markers ratios in patient and control showed significant difference (P= 0.000) for

both males and females. The results of the study also showed that 49% of the

DM population (30 subjects; 15 males and 15 females) had microalbuminuria and

- V -

60.6% had elevated level of tested enzymes (37 subjects; 19 males and18

females) while the control group showed normal results.

The results also showed positive correlation, and a significant relationship

between markers and markers ratios and each of body mass index (BMI),

duration of DM and blood pressure p-values: 0.016, 0.000 and 0.001,

respectively. Furthermore this study shows that more attention should be taken

by diabetics concerning lifestyle and food quality.

In conclusion, these markers and their ratios may be used as noninvasive early

indicators for renal deterioration in DM patients.

Key words: Diabetic nephropathy, Gaza Strip, microalbuminuria, urinary

enzymes

- VI -

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- VII -

Table of Contents

page

Contents

I Declaration

II Dedication

III Acknowledgement

IV Abstract (English)

VI Abstract (Arabic)

VII Table of Contents

X List of tables

XI List of figures

XII Abbreviations

2 3 5 6

Chapter 1: Introduction 1.1 Overview 1.2 Objectives 1.3 Significant of the study

7 8 9

12 12 13 17 18 19 20 20 21 23 23 23 26 29 33 34 35

Chapter 2: Literature review 2.1 Definition and history of DM 2.2 Epidemiological view 2.3 Causes and types of DM

2.3.1 Type-1 diabetes mellitus 2.3.2 Type-2 diabetes mellitus 2.3.3 Gestational diabetes mellitus 2.3.4 Other specific types of DM

2.4 DM diagnosis and criteria 2.5 Diabetic complications

2.5.1 Short-term DM complications 2.5.2 Long-term DM complications

2.6 Diabetic Nephropathy DNP 2.6.1 Definition of DNP 2.6.2 Stages and risk factors 2.6.3 Pathology and patho-physiology of DNP 2.6.4 Molecular and cellular mechanisms 2.6.5 Markers for Diabetic Nephropathy 2.6.5.1 Microalbuminuria

2.6.5.2 Urinary Enzyme (AAP, ALP and AP)

- VIII -

41 42 42 42 42 42 43 43 43 43 44 44 46 47 49 50 51 52 53

Chapter 3: Materials and methods 3.1 Study design 3.2 Study population 3.3 Sample size 3.4 Inclusion criteria 3.5 Exclusion criteria 3.6 Ethical consideration 3.7 Data collection 3.7.1 Questionnaire 3.7.2 Sampling and sample collection 3.7.3 Biochemical analysis

3.7.3.1 Determination of routine urine analysis 3.7.3.2 Determination of AAP 3.7.3.3 Determination of Microalbuminuria 3.7.3.4 Determination of Alkaline Phosphatase 3.7.3.5 Determination of Acid Phosphatase 3.7.3.6 Determination of creatinine 3.7.3.7 Determination of glucose

3.8 Data analysis

54 55

55 55

56

57

58 59

60

62 62 63

64

67 67 68 68

69

Chapter 4: Results 4.1 Overview 4.2 Personal, clinical and Socio-demographic characteristics of the

study population 4.2.1 Distribution of the study population by gender 4.2.2 Distribution of the study population by personal and clinical

characteristics 4.2.3 History of diabetic patients: Systolic, Diastolic, duration and

BMI of DM group 4.2.4 Comparison between the means for age and BMI between

males and females for patient group 4.2.5 Relationship between Gender and BMI of patient group

4.3 Comparison of the means of the urinary markers between patients and control

4.4 Comparison between the means of the markers by gender in patient and healthy groups

4.4.1 Comparisons among males in patient and control 4.4.2 Comparisons among females in patient and control

4.5 Comparison of means for the four marker ratios between patient and control group

4.6 Comparison between the means of the markers ratios by gender in patient and control group

4.6.1 Comparisons among males in patient and control 4.6.2 Comparisons among females in patient and control

4.7 Distribution of MAU ratios in patient and control groups 4.8 Relationships between some variables and marker ratios of

DM study group

- IX -

71 Chapter 5: Discussion

79 80 81

Chapter 6: Conclusion and recommendation 6.1 Conclusion 6.2 Recommendations

82 Chapter 7: Bibliography

Appendices A. approval from Helsinki committee- Gaza Strip B. permission to conduct the study in MOH clinics and laboratories C. permission to conduct the study in UNRWA clinics D. The questionnaire

- X -

List of tables

Table 2-1 Values for diagnosis of DM and other form of hyperglycaemia.

20

Table 4-1 Some of personal and clinical characteristics of patients. 57

Table 4-2 Systolic, Diastolic, Duration and BMI of DM patients. 58

Table 4-3 Comparison between age, DM duration and BMI for both sexes of patient.

58

Table 4-4 Relationship between Gender and BMI of patient group. 59

Table 4-5 Comparison between the urinary markers among patient and control groups

60

Table 4-6 Comparison of means of the markers among males in both groups.

63

Table 4-7 Comparison of means for the markers among females in both groups.

64

Table 4-8 Comparison of ratios between patient and control groups. 65

Table 4-9 Comparisons between the means of markers ratios among males in the patients and controls.

67

Table 4 -10 Comparisons between the means of markers ratios among Females in the patients and controls.

68

Table 4-11 Duration of DM, ages of healthy and DM, Systolic and Diastolic blood pressure of the patient group

70

- XI -

List of figures

Figure 2.1 Global projections for the diabetes epidemic. 10

Figure 2-2 Stages of DNP. 24

Figure 2-3 Lumen of glomerular capillary. 28

Figure 2-4 Intracellular mechanisms of high glucose and stretch induced effects. 32

Figure 4-1 Distribution of study population by gender. 56

Figure 4-2 Multiple bar chart of the means of the MAU, ALP and AAP in patient and control groups.

61

Figure 4-3 Multiple bar chart of the means of the ACP and Cr in patient and control groups.

62

Figure 4-4 Multiple bar charts of the means of the markers ratios in patient and control groups. 66

Figure 4-5 Distribution of MAU subjects by patient and control groups. 69

- XII -

Abbreviations

Alanine Amino Peptidase AAP

Acid Phosphatase ACP

Advanced glycation end products AGE

Alkaline Phosphatase ALP

American Diabetes Association ADA

Angiotensin receptor blockers ARBs

Blood Glucose Level BGL

Body mass index BMI

Cardio vascular disease CVD

Chronic kidney disease CKD

Coronary Artery Disease

Creatinine Cr

Diabetic Ketoacidosis DKA

Diabetes Mellitus DM

Diabetic Nephropathy DNP

End-Stage Renal Disease ESRD

Fasting plasma glucose FPG

Gestational Diabetes Mellitus GDM

ã-glutamyl transpeptidase GGT

Glomerular basement membrane GBM

Glomerular filtration rate GFR

Hyperosmolar nonketotic states HNS

Impaired fasting glucose IFG

Impaired glucose tolerance IGT

Insulin-dependent diabetes mellitus IDDM

International Diabetes Federation IDF

Maturity onset diabetes of the young MODY

- XIII -

Microalbuminuria MAU

Ministry of Health MOH

Mitogen-activated protein kinases MAPK

N-acetyl-â-D-glucosaminidase NAG

National Institutes of Health NIH

Non-insulin-dependent diabetes mellitus NIDDM

Oral glucose tolerance test OGTT

Protein kinase C PK-C

Reactive Oxygen species ROS

Signal Transducers and Activators of Transcription STAT

Statistical Package of Social Sciences SPSS

Transforming growth factor-â TGF-â

urinary albumin excretion UAE

United Kingdom Prospective Diabetes Study UKPDS

United Nation Refugee Work Agency UNRWA

Vascular endothelial growth factors VEGF

World Health Organization WHO

Chapter 1

Introduction

�

1. Introduction

1.1 Overview Diabetes mellitus (DM) prevalence increases continually around the world; it

became one of the major global problems for the developing as well as the

developed countries. It is affecting millions of peoples, about 6-7% of the

worlds population (1, 2, 3).

DM is a metabolic disorder characterized by chronic hyperglycemia with

disturbances of carbohydrate, fat and protein metabolism resulting from defects

in insulin secretion, insulin action, or both (2, 4, 5).

The most prevalent diabetes form in human is type-1 and type-2 D M, the

latest accounts for more than 90% of patients (1, 2, 6). Many patients with type-

2 diabetes are asymptomatic; there are no sharp clinical manifestations; and

hence patients may remain undiagnosed for many years.� Type-2 DM is

characterized by two major defects, impaired insulin secretion or a decrease in

its peripheral action (2, 7, 8). The cause of Type-2 DM is multifactorial. Genetic

susceptibility plays a crucial role in the etiology and manifestations of type-2 DM,

with roots in the interaction of environmental factors; physical inactivity, obesity,

ethnic, drugs and toxic agents, viral infection, and location; individual with a

susceptible gene may become diabetic if environmental factors modify the

expression of these genes (1, 5, 9). It is evident that environmental factors are

playing a more increasing role in the cause of DM (1, 2, 8, 10).

The complications of diabetes Iinclude microvascular diseases due to

damage to small blood vessels that affect eyes, nerves and kidney and

macrovascular diseases due to damage to the arteries that affect brain, heart

and extremities (11, 12). Strict controling of BGL and blood pressure play an

important role in delaying diabetic complications (13, 14, 15, 16). On the other

hand, less glycemic control, smoking, high blood pressure, elevated cholesterol

levels, obesity, and lack of regular exercise are considered to be risk factors

that accelerate the deleterious effects of diabetes complications (2, 17, 18).

One third or more of the DM patients develop DNP with progressive

�

deterioration of renal function and structure in their life time (6, 19, 20).

DNP is defined as urinary albumin excretion equals to or more than 300

mg/24hr and more commonly represented by persistent albuminuria which is

detected by various dipsticks (21). This condition is irreversible and also known

as overt nephropathy, clinical nephropathy, proteinuria, or macro-albuminuria

(20, 22). Earliest clinical manifestations of DNP are the appearance of

microalbuminuria (MAU) and excretion of other tubular enzymes in the patients

urine. MAU is defined as an elevation of urinary albumin excretion rate below

the level of clinical albuminuria (between 20-300 mg/24h); this indicates a high

probability of damage of the glomerular filtration capacity of the kidney and is of

great diagnostic relevance in diabetic patients for early diagnosis of DNP (23,

24).

Once MAU is detected in diabetic patients, this is called renal

involvement or termed as incipient nephropathy (22). MAU affects 20 to 40

percent of patients 10 to 15 years after the onset of diabetes (20). In both type-1

and type-2 DM patients, the progression from MAU to macro-proteinuria is

irreversible and once proteinuria developed a relentless deterioration in renal

functions is inevitable and renal failure occurs; despite cardiovascular death

which may interrupt this progression (25).

Several studies demonstrated that many abnormal urinary excretions of

smaller molecular weight proteins and enzymes can be detected in the

preclinical stage of DNP along with the increased of MAU or even before. About

50 enzymes and protein activity in urine was studied and tested as early

biomarkers for DNP (26, 27): alanine aminopeptidase enzyme (AAP), N-acetyl-

â-D-glucosaminidase (NAG), alkaline phosphatase (ALP), glutathione-S-

transferase (GST), acid phosphates (ACP), â2-microglobulin, ã-glutamyl

transpeptidase (GGT), transferrin and other markers which indicate a good

evaluation for proximal tubular deterioration and kidney dysfunction, it may

identify diabetic patients at risk of developing diabetic nephropathy (28, 29, 30,

31). Consequently, as these urinary biomarkers assess different areas of the

renal architecture, estimation of renal tubular function and integrity may provide

a site-specific and early indication of renal insult in subjects with diabetes (28,

31).

�

DNP is considered the most common single cause of end-stage renal disease

(ESRD) in the United States and Europe. Most of DM patients have increased

risk for developing nephropathy disease by time; they have about five times

greater risk than normal subjects (32). Kidney failure typically occurs after 20-23

years of diabetes (2, 8, 18). Then patients must undergo either dialysis or a

kidney transplant (22).

There are limited available data that discuss diabetic nephropathy in

Palestine. Recently two theses were submitted to the Master of Biological

Sciences in the Islamic University. The first study looked at MAU and

macroalbuminuria in DM patients (33), and the second study looked at some

enzymes� N-acetyl-â-D-glucosaminidase, ã-glutamyl transpeptidase and

transferring in the urine among type-2 diabetics in Gaza strip (34).

1.2 Objectives

The main purpose of this study is to assess some of early markers for DNP in

type-2 diabetic patients in southern Gaza Strip.

The Specific objectives:

To determine the urinary level of, MAU, AAP, ALP, ACP and creatinine in

patients and healthy controls.

To ascertain whether an association exist between the duration of DM

and these markers.

To investigate the association between the urinary enzyme activities and

the progressions of MAU in DM type-2.

To define risk factor associated with the development and progression of

MAU among type-2 D M patients.

To assess the interrelation of Cr with the other biomarkers: determination

of MAU, AAP, ALP and ACP /creatinine ratios in a urine sample to

predict future complications.

�

1.3 Significance

Diabetic nephropathy represents one of the major problems developed in type-2

DM patients as they have five times greater risk for developing nephropathy

than healthy persons, which is a common cause for renal failure and ESRD.

Early detection of renal involvement in diabetic nephropathy, through testing of

MAU and other markers such as urinary Alanine aminopeptidase, Alkaline and

Acid Phosphatase enzymes can draw the attention of both physicians and

patients for continuous monitoring of blood glucose level in order to prevent

disease progression before onset of clinical symptoms, thereby leading to

increased survival and lower treatment costs.

Special efforts for early detection of albumin / creatinine ratio in urine,

treatments and lifestyle adjustments should be taken to halt the progression

from micro- to macroalbuminuria and eventually ESRD.

The use of a sensitive and specific noninvasive method with simple steps for

the detection of such markers, in the patients urine, as early as possible, is very

important.

�

Chapter 2

Literature Review

�

2. Literature Review

Diabetes is one of the most common non communicable diseases affecting

almost 7% of the world's population (2, 18); there is currently a global epidemic

of type-2 diabetes as it forms more than 95% of all DM (32, 35). DNP is

considered as one of the major chronic complications in DM patients that lead

to serious medical condition of ESRD. It is reported that about one third of all

people with diabetes develop DNP by time (18, 20, 36). Early detection of

specific markers and markers/ creatinine ratios in urine, treatments and lifestyle

adjustments could be beneficial and one of preventive actions in follow-up and

controlling the disease.

2.1 Definition and history of DM

The most accurate description of DM disease and its complications appeared in

a book, The Law in Medicine, by President Ibn Sina in the 10th century.

Diabetes is derived from the Greek words means "passing through" or "siphon",

that referred to the major symptoms in excessive urine production and In 1675

Thomas Willis, London Great Britain, added the word mellitus which is Latin

word meaning "honey" referred to the sweet taste of the urine. Islets of

Langerhans are pancreatic cells, discovered in 1869 by Paul Langerhans the

German pathological anatomist and took his name (10, 37).

In 1921 after the first discovery of insulin and its metabolic role by Sir Frederick

Grant Banting and Charles Herbert Best, the distinction between diabetes as

type-1 and type-2 was first published in January 1936 by Sir Harold Percival

Himsworth. In 1942 Sir Frederick Sanger discovered the amino acid sequence

of the insulin, where in 1980 the first human insulin was produced by genetic

engineering (37, 38). In 1991, The IDF and the WHO have launched World

Diabetes Day in response to the alarming rise in diabetes around the world. (3)

DM is one of the most common endocrine disorders that affect the world's

population (1, 3, 6). It is found in almost every population in the world, and

�

epidemiological evidence suggests that DM is steady increases in huge number

that will cause globally big burden for the healthcare systems (10, 32, 35).

DM is a syndrome characterized by disorder metabolism of the blood sugar,

causing a chronic state of high BGL (hyperglycemia); it arise when the pancreas

fails to secret enough insulin or when the body cannot effectively use the insulin

produced via insulin receptors (insulin resistance) or both (6, 8).

Insulin hormone, secreted by â-cells of Islets of Langerhans in the pancreas, is

responsible for the regulation of glucose uptake into most human being cells for

use as primary source of energy in the muscles and adipose tissues or for

storage in liver. If the insulin is deficient, defective or there is insensitivity of its

receptors, a state of high BGL, poor protein synthesis, and acidosis will occure

(11).

Usually the most filtered glucose by the kidneys is reabsorbed by proximal

tubules into the blood, when BGL reaches 8 mmol/l (renal threshold value)

tubular re-absorptive capacity will be reduced and glucosuria occurs (11, 12,

39).

This chronic metabolic disorder affects the metabolism of carbohydrates,

protein, fat, water, and electrolytes. Over time hyperglycemia damages the

basement cell membrane of the blood vessels causing dysfunction and failure

of various organ systems in the body, especially the eyes, kidneys, nerves,

heart and blood vessels that called multi system failure (1, 40). The most

common types of human diabetes mellitus are type-2 referred to as non-insulin

dependent diabetes mellitus (NIDDM) and type-1 which is referred to as insulin-

dependent diabetes mellitus (IDDM) (6, 8).

2.2 Epidemiological view

According to the World Health Organization (WHO) global report of 2003, there

are approximately 171 million diabetic patients around the world, and this trend

is likely to continue as WHO projects an increase in the number of people with

diabetes to 366 million patients worldwide by the year 2030 (2, 6, 41).

In 2007 the lDF estimated that Diabetes affects 246 million people worldwide

(an increase of 55%), and is predicted to affect 380 million patients by 2025.

��

IDF also estimated that 7.3% of adults aged 20-79 in all IDF member countries

have diabetes (3).� The greatest increases will take place in developing

countries in Asia and Africa where diabetes rates are projected to rise to two or

three times those experienced today (40), due to a complex interplay of genetic,

environmental and social factors. Figure 2-1 shows world map, the number of

DM patients in 2007 and the expected number by the year 2025 with increased

percent.

Figure 2-1: Global projections for the diabetes epidemic in 2007-2025 (3, 42)

The prevalence of type-2 DM ranged from 4.6 to 40% in the Middle East, 0.3 to

17.9% in Africa, 1.2 to 14.6% in Asia, 0.7 to 11.6% in Europe, 6.69 to 28.2% in

North America, and 2.01 to 17.4% in South America (1, 9, 10). In the United

States there are 20.8 million DM patients, this means 7% of the population, (2,

9) while recent studies demonstrated that the total prevalence of diabetes

increased 13.5% from 2005 - 2007 (3) with a prevalence of approximately 9.3%

(diagnosed and undiagnosed) (43).

The prevalence of diagnosed diabetes amongst adults in England aged 16 and

above is about 3 % (40). A high-prevalence in Saudi Arabia is 12-23.7% (44).

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High prevalence was also seen in Palestine 9.8% (1). In the United Arab

Emirates a study by El Mugamer and his colleagues in 1995 found that diabetes

prevalence was 6% (45). A cross-sectional study of 2,128 Bahrainis in �����

reported that prevalence of diabetes was 25% in Jaafari Arabs, 48% in Sunni

Arabs, and 23% in Iranians (46).

A cross sectional study by Jamil and his colleagues in 2008 estimated the

prevalence of diabetes among Whites (n = 212), Arabs (n = 1,303), Chaldeans

(n = 828), and Blacks (n = 789) in southeast Michigan. The overall prevalence

of diabetes was 7.0%, and the highest was for Blacks 8.0% followed by Arabs

and Whites 7.0% for each group and for Chaldeans it was 6.0% (P 0.005) (43).

There are about 110 millions patients in the developing countries suffering from

DM (6, 10, 47). As indicated by WHO report, there are more than 2.6 millions

DM patients in Egypt, It is predicted that this number will jump to more than 6

millions in 2030, In Libya, it is estimated that there are about 88,000 diabetics,

in Iran 2 million patients, 0.5 million patients in each of Syria and Morocco, and

about 1.6 million patients in Arabian Gulf. This trend is likely to continue

increase and duplicated in the number of people with diabetes worldwide by the

year 2030 as WHO projects (18, 19, 47); and type-2 diabetes has reached the

epidemic proportions in many of such countries (22, 48).

In Palestine, a statistics held in 2003 on MOH website showed that there are

about 134,560 DM patients registered in the governmental diabetic clinics in the

West Bank and 15,844 diabetic Palestinian refugee patients in Gaza Strip,

including DM patients having hypertension, are under supervision of UNRWA in

Gaza. The prevalence rate of diabetes mellitus in patients over 40 years was

4.3% in 2000 and 4.7% in 2001 (49, 50). In a cross-sectional survey of rural

population of Ramallah, Palestinian, Husseini and his colleagues investigated

the prevalence of diabetes. They found that the prevalence was 9.6% and

10.0% in females and males respectively (51). Another cross-sectional survey

of urban Palestinian population of 492 men and women aged 3065 years were

studied by Abdul-Rahim and his colleagues. They found DM in 12.0% of the

surveyed population, including 9.4% previously diagnosed (52).

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2.3 Causes and types of DM

The cause of DM is multi-factorial. Both genetic and environmental factors play

a contributing role. In general the environmental include physical inactivity,

drugs and toxic agents, obesity, viral infection, and location (1). Various global

sources WHO, IDF, DAD and International Statistical and Classification of

Diseases (ICD) have recognized three major types of DM: type-1 insulin-

dependant diabetes, type-2 non insulin- dependant diabetes and gestational

diabetes, and a rare specific types of diabetes due to other causes do not

match to type-1, type-2, or gestational diabetes. Impaired glucose tolerance is a

condition closely related to Type-2 diabetes (3, 18).

2.3.1 Type-1 diabetes mellitus

Type-1 DM, also known as insulin-dependent diabetes IDDM, juvenile diabetes

or childhood diabetes, is characterized by autoimmune destruction of the beta

cells of the islets of Langerhans, the pancreas unable to secrete insulin and

usually leading to absolute insulin deficiency. Viral infection, environmental or

toxins are thought to act as a trigger that stimulates immunological mediated

destruction of the beta cells in the pancreas; this mainly caused by T-cell

mediated autoimmune attack (1, 2, 11, 53). The rate of â-cells destruction is

quite variable being rapid in some individuals, infants and children and slow in

others (adults) (2).

Type-1 is strongly associated with certain HLA haplotypes with linkage to

the DQA and DQB genes, and patients with HLA tissue types and DR3 DR4 are

most at risk (2, 9, 11, 12, 39). The heterozygous state DR3/DR4 and the genes

which encode them at the populations and from HLA-DQ are loci on

chromosome 6 (9).

Symptoms of diabetes are present when endogenous insulin production

is unable to meet the bodys requirements. Typical symptoms is polyuria

polydepsia, polyphagia, rapid weight loss, nausea, fatigue, and abdominal pain

and mental confusion and possible loss of consciousness due to increased

glucose to brain(2, 54) in newly diagnosed patients. Sensitivity and

responsiveness to insulin therapy are usually normal especially in the early

��

stages. Unlike type-2 exercises and diet cannot prevent type-1 diabetes

progresion. patients are prone to diabetic ketoacidosis, can be treated only with

insulin, with careful monitoring of BGL. Without insulin, diabetic ketoacidosis

can lead to coma or death will result (11, 37).

Type-1 accounts form 5% to 10% of DM total diagnosed cases in the

world and the highest prevalence and incidence cases of type-1 diabetes are in

the Nordic countries (1, 2). The incidence in the Middle East ranges from 2.62

to 20.18/100,000/yr (6, 10); a highest incidence was reported in 1999 in Kuwait

20.18/100,000/yr (1, 55).

2.3.2 Type-2 diabetes mellitus

Over the past decade it has been obvious that the prevalence of DM is

increasing rapidly, this projected increase in the number of diabetic patients will

cause a big burden for the healthcare systems (1). The majority of patients have

Type-2 diabetes, which accounts for more than 90% of all diagnosed cases of

diabetes (1, 6, 7, 22).

Previously Type-2 was named: noninsulin-dependent diabetes mellitus

(NIDDM), adult-onset diabetes, adult-onset diabetes mellitus, and maturity-

onset diabetes mellitus. (6, 10, 56)

Type-2 is characterised by impaired insulin secretion or defective its

action at responsive tissues (insulin resistance: an attenuated effect of insulin in

target body tissues, mainly muscle, fat and liver), a combination of resistance to

insulin action and an inadequate compensatory insulin may occur. Insulin

resistance is manifested as impaired glucose utilisations which lead to

increased BGL and decreased intracellular glucose. This will enhance the

pancreas to produce more insulin to reduce BGL. Muscle tissue can take up

glucose, and any excess glucose is stored at three sites: muscle, fat and liver,

over time the production of insulin will be reduced (1, 6, 7, 10, 54).

There is currently a global epidemic of type-2 diabetes (32, 35). It is considered

a lifestyle-dependent disease that associated with a strong genetic factor and

requires long-term of medical control and attention both to limit and to manage

the development of the complications when they do occur.

��

Type-2 diabetes tends to develop slowly, most patients of this condition

are asymptomatic, and therefore the diagnosis is often missed, so

complications may present at the time of diagnosis as a degree of

hyperglycaemia sufficient to cause pathologic and functional changes in various

target tissues for a long period of time before diabetes is detected (2, 42). The

same disease process of Type-2 can cause impaired fasting glucose (IFG)

and/or impaired glucose tolerance (IGT) without fulfilling the criteria for the

diagnosis of diabetes (2). The condition of IGT also constitutes a major public

health problem, sometimes referred to as pre-diabetes since individuals with

IGT are at high risk of progressing to type-2 diabetes. Impaired glucose

tolerance is now recognized as being a stage in the transition from normality to

diabetes, although such progression is not inevitable (3, 42).

The rise in the prevalence of type-2 in the world and the variability

among ethnic groups is due to many reasons including: The silent nature of

DM-2, genetic factors, environmental factors, population ageing unhealthy diet

(amount and origin of dietary protein), overweight or obesity, history of

gestational diabetes and impaired glucose metabolism, physical inactivity,

and sedentary lifestyle, all seems to play a role as risk factors (1, 3, 4). Type-2

begins as improper insulin use by body cells (insulin resistance); gradually the

pancreas loses its ability to produce insulin as its need rises (5).

There is dramatic changing in the lifestyles of people all around the world

because of rapid socioeconomic development (51). The global basis of rise in

type-2 diabetes rates is clearly linked to the growth in urbanization, economic

development and mal-adaptation to a rapidly changing environment, which is in

turn associated with changing of dietary and lifestyle patterns. The outcome of

this change results in obesity and inactivity, it is most prevalent in urban areas

than rural ones (42).

Over the past 20 years, obesity rates have tripled in developing countries

that have been adopting a Western lifestyle involving decreased physical

activity and over consumption of cheap, energy-dense food. Obesity is strongly

associated with type-2 in adult, children and adolescents. About 80-85% of

type-2 DM are obese, body mass index is directly associated with increased risk

��

of type-2 in many ethnic groups (1, 9, 57, 58, 59). Excess weight, abdominal fat

in particular the visceral rather than subcutaneous depots, increases insulin

requirements and compounds the problem of insensitivity to insulin (2, 58).

Many investigators have reported a strong correlation between obesity and

type-2 diabetes. In a study of the pattern of diabetes in Kuwait on 3229 patients,

it was shown that 57.7% of the diabetic women were obese and 30.2% were

overweight (60).

In a study in Germany, men and women waist measurements were found to be

associated with a substantially increased risk of DM complications: more than

102 cm in men or more than 88 cm in women (58).

In the United Arab Emirates it was found that diabetes prevalence was

6% (11% in male and 7% in female subjects aged 30-64 years). Urban

residence was associated with higher BGL and with higher body mass index

(BMI), 27% of all urban residents were obese (45). A cross-sectional study in

Bahrain in 1998 showed prevalence of Obesity (28% of participants had BMI 30

kg/m2) among diabetes (46). In Libya, according to local epidemiological

studies, the prevalence for known diabetic patients aged over 20 years was

3.8%, most of them were obese (61).

In Palestine, Husseini and his colleagues found that DM was associated

with increased of BMI and waist-hip ratio (OR = 2.13, 95%CI =1.313.45). They

added that DM may also be associated with the urbanization of rural areas and

the socioeconomic changes that go along with it (62).

In another study, it was found that DM prevalence in an urban Palestinian West

Bank population was high; 12.0% of the surveyed population (52).

In many cases obesity alone may be sufficient to lead to DM, two of

interrelated risk factors associated with type-2 DM are generalized and central

obesity (58).

Why most of type-2 diabetes patients are obese:

First, in obesity increased lipolysis associated with visceral adipose tissue result

in release of free fatty acids, this have negative effects on insulin sensitivity in

muscle and liver; decreased insulin binding and its action; result in metabolic

products (acetyl-CoA and citrate) which inhibit glucose mobilization, moreover

��

lead to deregulated production and secretion of adipose derived bio-molecules

(signalling proteins) like vasoprine, leptin, resistin, and adiponectin that act as

antagonizes (especially leptin) to insulin. After eating, Insulin signals the satiety

(appetite-control) center of the brain to stop dietary glucose intake, while leptin

and adiponectin act to antagonizes insulin signaling (by inhibiting insulin binding

to adipocyte insulin receptors). Net result; high BGL occurs (58, 59).

Second, increase food uptake and excess weight of the obese lead to a state of

â-cells, insulin exhaustion and insensitivity to insulin.

A study performed in Saudi Arabia showed that age adjusted (15 years and

older) prevalence of DM was significantly higher in the urban population (males

12%, females 14%) compared to rural (males 7%, females 7.7%) population,

and the highest prevalence of obesity, BMI>30, was among urban female

subjects (57). Another study in Saudi Arabia Kingdom showed that the

prevalence of DM in males and females were 26.2% and 21.5%. DM was more

prevalent among Saudis living in urban areas (25.5%) compared to rural Saudis

(19.5%) (63).

Al-Moosa and his colleagues in Oman found that the prevalence of

diabetes in the capital region was 17.7% compared to 10.5% in rural areas, with

high prevalence of obesity among urban individuals (64). The difference

between the prevalence of diabetes in the urban and rural setting may be due to

two reasons, people in the urban setting may be more affluent and eat more

junk food in contrast to people in the rural setting. Second, people in the rural

setting are more likely to be involved in more physical activity compared to their

urban counterparts, thereby reducing the developing type-2 diabetes (1, 65).

The diabetes epidemic is accelerating in the developing countries, with

an increasing proportion of affected people in younger age groups (2, 5, 42).

It has been considered as a public health problem in children and adolescents,

lifestyle changes are also affecting children. Over the past years, the age of

onset of Type-2 DM has been steadily decreasing in many population groups (5,

10, 14, 65). Recent reports in the Arabian Gulf described that Type-2 DM is not

only affecting older people but also spreading to children and adolescents who

��

are overweight or obese and with family history. being overweight and having

high blood pressure or high cholesterol and physical inactivity increase the risk

(85%) of getting Type-2 DM (63- 65).

It has been shown that regular physical activity increases insulin sensitivity and

glucose tolerance, it was also shown that physical activity reduces the risk of

type-2 DM (1, 9, 59, 66). In a study performed on an African American

population in the United States, it was observed that the prevalence of diabetes

increases with the degree of inactivity and obesity (1, 9, 66).

Type-2 diabetes is usually first treated by attention to change physical

activity (exercise), the diet to decrease carbohydrate intake, sedentary life and

weight loss. The modifiable lifestyle factors (life style and/or intensive

behavioural actions) are appearing to be the most important in terms of

treatments. Type-2 can be prevented or post pended in many cases by making

changes in diet and increasing physical activity (2, 58, 59, 67).

Type-2 DM is a progressive and the insulin deficiency will worsen over

time, and around 50% of patients will require; in addition to oral BGL-lowering

drugs; insulin therapy to maintain normal BGL (18, 3). This means an increase

in the effective cost if incurring DM patients. It is not surprising that, direct and

indirect medical expenditures attributable to diabetes in 2002 were estimated at

$132 billion in the USA (68).

2.3.3 Gestational diabetes mellitus GDM

Gestational diabetes is defined as any degree of glucose intolerance in some

women during pregnancy; associated with increased perinatal complications; it

is more common among obese women and women with a family history of

diabetes. It may be transient, resembling type-2 diabetes in several respects

and about 20%50% of women with GDM develop type-2 diabetes later in life

(2, 6, 68, 69). It is believed that the high BGL resulting of the hormones that

produced during pregnancy and other factors reduce the tissues sensitivity to

insulin by interfering with the action of insulin as it binds to the insulin receptor

(37, 69). GDM affects approximately 4 - 7% of all pregnancies in the United

States. It is temporary but treatable, if untreated it can lead to fetal macrosomia

(high birth weight), malformation, congenital heart disease, and hyper-

��

bilirubinemia in severe cases, perinatal death may occur. In addition GDM

mothers have increased rates of caesarean delivery and chronic hypertension

(56, 69). To avoid GDM complications, screening test should be done at 24-28

weeks of gestation, and careful medical supervision during the pregnancy is

required (69).

2.3.4 Other specific types of DM

There are other types of DM secondary to rare conditions do not match with

type-1, type-2, or GDM like:

a) Genetic defects in beta cells. Maturity onset diabetes of the young

(MODY) refers to a number of rare hereditary mutations lead to forms of

diabetes (2, 3, 7) due to: defects of insulin secretion, inability to convert

proinsulin to insulin and production of mutant insulin (impaired receptor

binding) (2).

b) Genetically related defects in insulin action: mutations of the insulin

receptor

c) Diseases of the pancreas: chronic pancreatitis, cystic fibrosis, Neoplasia;

haemochromatosis.

d) Endocrino-pathies or hormonal defects: growth hormones, cortisol,

glucagons and epinephrine antagonize insulin action. (Acromegaly,

Hyperthyroidism and Cushings syndrome)

e) Drug or chemical induced diabetes: might arise from the use of steroids,

diazoxide, thiazides or pentamadine

f) Infections associated with the development of diabetes including

congenital rubella, coxsackie, cytomegalovirus (CMV), and mumps.

g) Uncommon forms of immune-mediated diabetes: Anti-insulin receptor

antibodies can cause diabetes by binding to insulin receptor, blocking the

action of insulin in target tissues (insulin agonist), and can thereby cause

hypoglycaemia, previously known as type-B insulin resistance (2, 3).

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2.4 Diabetes mellitus diagnosis

Diabetes mellitus is diagnosed by laboratory tests, the two most common

screening tests are the fasting blood sugar test and glucose tolerance test.

The term diagnosis refers to confirmation of suspected people who have

symptoms or who have a positive screening test, the symptoms may include:

polydipsia, polyphagia, polyuria, glucosuria, rapid weight loss and mental

confusion and possible loss of consciousness due to increased levels of

glucose in brain (2, 54).

The screening test may be considered a diagnostic test, in someone who has

symptoms ADA recommended more than 126 mg/dl in fasting plasma glucose

(FPG) concentration test on two different days or a casual plasma glucose

concentration more 200 mg/dl at random time of the day (1, 2, 16). The WHO

continues to recommend oral glucose tolerance test OGTT to confirm the

diagnosis in asymptomatic individuals (5, 6).

Fasting blood sugar measures the BGL after a 12-hour fast (11, 12, 39).

OGTT described by the WHO consists of a 75 g glucose load taken in the

morning after an 8 -10 hour fast patient, venous blood samples are taken

in the fasting state and again at half, one, one-half and 2 hour to

compare BGL. Curve is plotted to watch their pattern in response to

sugar drink. Normally the blood sugar level is lower before the drink,

rises quickly during the first few hours, and slowly drops again. In insulin

resistance, the BGL rises but stays abnormally high because it is

resistant to being removed from blood into tissues by insulin.

Urinary glucose testing by routine dipstick glucosuria occurs when BGL

exceeds roughly 180 mg\dl in individual with normal kidney function (54).

2.4.1 Criteria for the diagnosis of DM

Normal level should be below 100 mg/dl. A value in range 100 to 125 mg/dl is

considered evidence of insulin resistance, IFG or pre-diabetic. A value of 126

mg/dl or more is considered diabetic (2, 3, 5).

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Table 2-1: Values for diagnosis of DM and other form of hyperglycaemia

Normal Impaired fasting glucose*

Impaired glucose tolerance** Diabetes

FPG < 6.1 mmol/L

< 110 mg/dL

6.1 - 6.9 mmol/L*

110 - 126 mg/dL*

≥ 7.0 mmol/L

≥ 126 mg/dL

2hr PG < 7.8 mmol/L

< 126 mg/dL

7.8 - 11 mmol/L**

126 - 200 mg/dL**

≥ 11.1 mmol/L

≥ 200 mg/dL

To convert mg/dl to mmol, divide by18. example: 200 mg/dl / 18 = 11.1 mmol

Evidence tests between type-1/2 DM:

ketones in the urine indicate insulin deficiency and point towards the

diagnosis of type-1

Fasting C-peptide and insulin levels are usually elevated in type-2

diabetes.

The presence of islet-cell antibodies/GAD antibodies in type-1 diabetes.

2.5 Diabetic complications

Diabetes as a chronic condition requires careful control. Without proper control,

management and follow-up it can lead to various complications. These

complications may be divided to short and long term complications.

2.5.1 Short-term DM complications

It is acute metabolic complications manifested by increased lipolysis with fatty

acid release and accumulation of fat in parenchymal organs further aggravates

the metabolic disturbance.

Ketoacidosis (DKA): Insulin is principal signal in converting many of

metabolisms from a catabolic to anabolic direction and vice versa. Lack

of insulin with excess of glucagons permits to gluconeogenesis and

lipolysis. acetoacetate, -hydroxybutyrate and acetone are produced

��

leading to decreases the blood's pH. Diabetic Ketoacidosis can cause

sever dehydration, electrolyte disturbances, hypotension, shock, and

death may occur. Individuals with type-1 have tendency to produce

ketones while it is rare in type-2, as still producing insulin, who have

greater tendency to develop hyperosmolar nonketotic states (HNS)

resulting in concomitant loss of water and if it is prolonged, it will result in

electrolyte imbalances and may progress to coma (11, 37, 39, 54).

Hyperosmolar nonketotic states (HNS) insulin deficiency leads to a high

BGL (usually considered to be above 300mg/dl) and resulting in serum

osmolarity which leads to osmotic diuresis (resulting in concomitant loss

of water; polyuria), volume depletion and hemoconcentration that cause

a further increase in BGL. Unlike DKA, Ketosis is absent because the

presence of some insulin inhibits lipolysis. If HNS is prolonged, it will

result in electrolyte imbalances, Hyperviscosity and increased risk of

thrombosis, disordered mental functioning or focal seizures or motor

abnormalities. if untreated, it will progress to coma or to death. This

condition is more frequent in elderly DM (39).

Hypoglycemia: involves decreased plasma glucose levels. Excess insulin

(if the glucose intake does not match the treatment) results in BGL below

normal fasting level leading to mental confusion and possible loss of

consciousness, coma and/or seizures, or even brain damage and death.

Infections: bacterial/fungal: diabetics have increased risk of cystitis and,

more important, of serious upper urinary tract infection as well as ear,

nose, and throat infections, necrotizing otitis externa principally occurs.

Skin and soft tissue infections are common in DM and may spread to

adjacent bone causing osteomyelitis infection (56, 70).

2.5.2 Long-term DM complications

Many patients with type-2 diabetes are asymptomatic, and their disease is

undiagnosed for many years. The kidney plays a pivotal role in myocardial

failure. Therefore, interfering with the cardio-renal axis is an important

��

therapeutic objective. The duration and intensity of high BGL play important role

in glycosylation of proteins and leads to changes in the shape of the endothelial

cells lining the blood vessels; glycoprotein formation; basement membrane

become thickening and weak (37, 59). The complications of DM can be divided

into macro-vascular damage (the arteries) and micro-vascular (damage to small

blood vessels) diseases:

Micro-vascular diseases include:

o Nephropathy

o neuroparthy

o retinopathy

Macro-vascular diseases (cardiovascular disease) are more common

among patients with type-2 DM (74), they include:

o Cerebro-vascular disease

o Coronary artery disease

o Peripheral vascular disease (diabetic foot)

Amputation: Peripheral vascular disease, are often seen in in patients who

have foot infections. Poorly controlled DM lead to impaired circulation and

slowly heal from small cuts. The untreated damage, or failing to heal or

unnoticed minor trauma may result in an infection especially in the lower-

extremity, where the blood flow delays, and micro-angiopathic lesions lead to

cellulitis, osteomyelitis, or nonclostridial gangrene that end in amputation. In

2004 about 60% of non-traumatic lower limb amputations occur in people with

diabetes in USA. The complications of diabetes are far less common and less

severe in people who have well-controlled blood sugar levels (2, 3).

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2.6 Diabetic Nephropathy

DNP is a state of a progressive rise in urine albumin excretion (UAE), coupled

with increasing blood pressure. It develops approximately in about 40% of

patients with type-1 and 2 DM even when high BGL are maintained for long

periods of time (22). Those affected will have progressive deterioration of renal

function, declining of glomerular filtration and eventually ESRD (20, 36, 72, 73)

as well as increased cardiovascular mortality (71). Because of its increasing in

prevalence, particularly type-2, and having patients living more, DM is

considered the main cause of increasing incidence of DNP (3, 36, 37). As most

of type-2 DM patients are some how asymptomatic and they maybe

undiagnosed for many years (10), a higher proportion of the patients are found

to having MAU and overt nephropathy shortly after the diagnosis is made (36).

About 80% of ESRD that is caused by DNP occurs in patients with type-2 DM

(17). DNP is considered the most common single disorder leading to ESRD in

the USA and Europe. In 2005, it was responsible for causing more than 43% of

ESRD new cases in USA, with treatment cost excess of $32 billions (74).

Statistics show that there is a racial and ethnicity difference in the prevalence of

DNP and ESRD (36), and the risk of nephropathy is strongly determined by

genetic factors (16).

2.6.1 Definition of DNP

It is a chronic progressive complication result from long term of DM. It has been

classically defined by proteinuria, the presence of protein in urine more than or

equals to 300 mg/24hr in DM patients with absence of other renal diseases.

This condition can be obviously detected by various common Albustix, and also

known as overt nephropathy, proteinuria, clinical nephropathy, or

macroalbuminuria (20, 22, 32, 36, 75).

2.6.2 Stages and risk factors of DNP

DNP is multi stage conditions that take several years to develop ESRD. The

earliest detectable change is in the thickening of the glomerular basement

membrane (GBM) that filters the blood, the damage to the membrane and the

cells next to it in the capillary walls causes albumin to leak from the blood into

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the urine; this is called albuminuria and proteinuria. Depending on the values of

MAU and based on the assumption the rate of urinary albumin excretion (UAE),

DNP is divided primary into 5 stages: the two stages hyperfiltration and silent

phase are normo-albuminuria, MAU is stage 3 and proteinuria stage 4 and

ESRD is the last one. Figure 2-2 shows the various stages of DNP.

Figure 2-2: stages of DNP. (116)

Normoalbuminuria is a primary step includes both Hyperfiltration and

Silent phase stages, in this step there maybe a functional (glomerular

hypertension and hyperfiltration) and structural changes (detectable GBM

thickening on biopsy but no clinical manifestations). Patients are usually with

normal renal function and albumin excretion rate concentration < 20 µg per

minute {less than 30 mg/24hr, or < 20 mg/l} (76).

MAU stage; an early manifestation of DNP is the presence of MAU; which is

defined as an elevation of urinary albumin excretion rate from 20 to 200 µg per

minute in an overnight urine sample (albumin-creatinine ratio of 30 to 300 mg/g

in a random urine specimen or ��- 200 mg/l), it is considered as the first stage

of renal involvement in type-2 diabetes (20, 24, 36, 76). In this stage there is

normal renal function, blood pressure may be increased and MAU is undetected

by dipsticks which show negative result for albuminuria. It was found that after

10 years of follow-up for a group of DM patients, the risk of DNP was 29 times

greater in patients with type-2 diabetes with UAE values >10µg/min (22). MAU

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affects a proportion of type-2 DM patients (about 7%) shortly after the diagnosis

is made; some of them already have MAU at that time; and about 40% of them

after 10 years of having the disease (20, 22, 74). American Diabetes

Association (ADA) recommends screening for MAU 5 years after the onset of

type-1 and at the time of diagnosis in type-2 DM. As the disease progresses,

more albumin leaks into the urine, the kidneys filtering function usually begins

to drop, and development to the later stage which is the presence of macro-

albuminuria or proteinuria.

In the overt nephropathy stage, hypertension is found, serum creatinine

is normal or raised, urinary albumin excretion rate >200 µg per min (more than

300 mg/24 h or urine albumin concentration > 200 mg/l), and MAU can be

detected by commercial dipsticks (22, 74, 76). This stage is irreversible, which

leads to increase in blood pressure that develops more and more damage to

the kidneys with leakage of more protein leading to ESRD; last stage kidney

failure occurs, serum creatinine is > 500 mol/L, that requires dialysis or

transplant to maintain life (76).

Current evidence suggests that both genetic and environmental factors

determine the risk for and susceptibility to develop DNP. Many studies have

identified factors associated with a high risk of diabetes nephropathy:

hyperglycaemia, progressive MAU, increase serum lipids or lipid disorders,

blood pressure levels, glycosylated haemoglobin, smoking, physical inactivity,

older ages, high level of insulin resistance and origin (the amount and source)

of dietary protein also seem to play a role as DNP risk factors (20, 22, 74).

Some medications may be harmful to the kidneys, especially non-steroidal anti-

inflammatory drugs and some antibiotics. X-ray tests such as angiograms,

intravenous pyelography, and some CT scans requires IV contrast material (IV

dye) can cause further kidney damage (76).

The risk of developing cardiovascular disease increases as the MAU

excretion progresses to albuminuria. Data from the United Kingdom Prospective

Diabetes Study UKPDS showed that once patients with type-2 diabetes develop

albuminuria, their annual risk of dying of CVD is greater than their risk of

progressing to a stage of ESRED (36).

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MAU excretion is reversible state and can be resolved by glycemic control and

specific medications like angiotensin-converting enzyme inhibitors (ACE) and

angiotensin receptor blockers (ARBs), these drugs help to reduce MAU, and a

combination of these two types of drugs may be the best to avoid going to

proteinuria stage (76).

2.6.3 Pathology and pathophysiology of DNP

DNP is a major micro-vascular complication caused by DM by time; it differs

from other causes of chronic kidney disease (CKD) in its predictability of

functional progression from hyperfiltration, to MAU, macro-albuminuria and

renal failure (14, 36). As mentioned above, glomerular hypertrophy and

hyperfiltration are early renal abnormalities (first step) in DNP patients.

Functional abnormalities take place in the glomerulus filter apparatus include

glomerular hypertrophy, thickening of GBM, and expansion of mesangial extra

cellular matrix with development of proteinuria and subsequent fall in glomerular

filtration rate (GFR) (77, 78, 79).

Many studies focused on the cellular and molecular mechanisms

abnormalities of glomerulus and tubular-interstitial injury that lead to DNP as

predictor of renal dysfunction. In the first step of DNP (hyperfiltration), the

hyperglycaemia state will cause injury to lining cells of glomerulus; each

glomerulus will receive more blood at a higher pressure and therefore will filter

more fluid per minute into tubules after injury to maintain homeostasis. Studies

have shown that hyperfiltration, are associated with the degree of

hyperglycaemia and present in early phases of both type-1 and type-2 for

several years (80). The hyperglycaemic state seems to sensitize the

endothelium to injury from elevated blood pressure, causing hyperfiltration and

lead to secretion of intra-renal vasoactive hormones, such as angiotensin and

catecholamines that constrict efferent arterioles (77).

Morphological and structural changes take place in the glomerulus of

DNP patients; Glomerular hypertrophy is one of the histological changes seen

in DNP (35). Earliest changes take place in the capillary of GBM and become

more thick; a loss of GBM charge-selective properties (due to decrements in the

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anionic components of the glomerular capillary wall); mesangial expansion with

extra cellular matrix (ECM) accumulation, and subsequent increase in pores

size leading to loss of filtration surface area podocytes (14, 36, 77, 79). By time

a glomerulus with dilated afferent and constricted efferent arterioles and

abnormal basement membrane permeability will cause damage to the

glomerular capillary, proceeds to mesangial and GBM injury, and in-turn it will

stimulate the release of different cytokines. These effects can produce further

relentless injury to the cells and cause further nephron loss (80).

Ultimately, there is irreversible changes such as loss of podocytes and

development of arteriolar hyalinosis, glomerulosclerosis, and tubulointerstitial

fibrosis occur (14, 36), this means progression to the latest step of DNP. The

increased thickening of GBM is associated with increased MAU, and eventually

the glomerular filtration rate (GFR) begins to fall (36, 77).

The pathological development of CKD due to DM in the human was discussed

by Erwin Bo¨ttinger. He termed the early finding affected cells (morphological

substrates) through DNP by podocyte depletion, these cells found to be crucial

during the disease pathogenesis (Figure 2-3). Podocytes, elaborately shaped

visceral epithelial cell, are highly differentiated glomerular epithelial cells and

appear to be incapable to divide or replicate in adult animals (81). These cells

are essential for glomerular structure and function; they are surrounding the

glomerular capillaries and appeared to form foot like processes contributing to

the filtration barrier and responsible for maintaining and supporting the

glomerular basement membrane so as to facilitate efficient filtration (36).

Studies demonstrated that loss of podocytes is an early feature of DNP; a clear

loss was found after onset of hyperglycaemia and contributes to the progression

of albuminuria in Pima Indians with type-2 DM (14, 77, 81). Hence the onset of

DNP can be predicted, and can be significantly ameliorated, at an early stage

when MAU is detected (72).

Susztak and his colleagues discussed genomic strategies to find new diagnostic

and therapeutic targets in DNP of streptozotocin (STZ) induced mouse models

of type-1 and type-2 diabetes. They demonstrate that podocyte apoptosis

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increased sharply with onset of hyperglycemia. In an induction of a 30-mmol/l

glucose solution in both STZ model and cell cultured experiment, the podocytes

showed increased apoptosis with exposure to the elevated glucose levels (13).

Figure 2-3 shows high power electron micrograph of the glomerular filtration

barrier and podocyte foot process (PFC). The podocyte, via the foot processes,

provides structural support for the glomerular capillaries (82).

Figure 2-3: Lumen of normal glomerular capillary (82)

Pagtalunan and his colleagues assessed podocyte number and histology in

diabetic patients and concluded that the same pathologic processes that cause

glomerular injury are present in type-1 as well as in type-2 diabetic patients;

subjects with clinical nephropathy exhibit a marked reduction in the podocytes

numbers due to mesangial expansion and structural changes in subjects with

MAU are modest (81). This was agreed by Vestra and his colleagues in a study

that evaluated podocytes density, number, and structure in 67 white patients

with type-2 DM. They demonstrated that changes in podocyte structure and

numerical density are founded at early stages of DNP, with mesangial

expansion and GBM thickening. They concluded that abnormal albuminuria in

type-2 diabetic without glomerulo-pathy is related to podocyte structural

changes occurring in the patients (83).

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Recent studies demonstrate that loss of podocytes is an early feature of

DNP that predicts its progressive course. Among various glomerular

morphological characteristics, the decreased number of podocytes per

glomerulus was the strongest predictor of progression of diabetic nephropathy

(77).

Other material that has important role in the pathogenesis of DNP disease is â-

1 integrin; inhibition of â-1 integrin expression plays a role in cell adhesion to

extra cellular matrix substrates and act as modulators of podocyte detachment

(14, 36). To study the podocyte abnormalities in diabetes, Dessapt and his

colleagues assessed modulators of podocyte detachment via inhibition of â-1

integrin expression. They found that the exposure to 25 mmol/l glucose and

cyclic exposure to 20% mechanical stress both decreased â-1 integrin

expression by about 15% (14).

2.6.4 Molecular and cellular mechanisms of DNP

DNP is characterised by increased glomerular permeability to proteins, the

GBM thickening (plays key role in UAE), and excessive ECM accumulation in

the mesangium. Both podocytes and mesangial cells play a pivotal role in the

pathogenesis (79). Glomerulo-sclerosis is advanced phase in DNP

characterized by increased ECM deposition lead to GBM thickening and

mesangial expansion. Mesangial expansions result from imbalance between

mesangial matrix protein production and degradation, favouring of the

accumulation matrix protein leading to the thickening. The production of

mesangial matrix proteins is accelerated by hyperglycaemia-driven synthesis of

cytokines such as transforming growth factor-B (TGF-â) family, angiotensin-2,

and other growth factors (75, 77).

Wolf and Ritz 2003 suggested a mechanism involved in the development of

DNP, the chronic high BGL will increase super oxide formation due to increased

mitochondrial oxidation of glucose that generates reactive oxygen species

(ROS). Extracellular glucose to about 30 mmol/l will rapidly stimulate generation

of these molecules that play a pivotal role in proteins modified {Amadori

products, and advanced glycation end products (AGE) that cause GBM

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thickening and kidneys deterioration}. Oxidation of glucose and ROS also

activates protein kinase C (PK-C) and mitogen-activated protein kinases

(MAPK), which induces TGF-â1 and fibronectin production. All are involved in

the development of diabetic complications particularly accumulation of ECM and

leading to renal hypertrophy (16). Hermann Haller found that hyperglycaemia

increased PK-C with a subsequent increase in endothelial cell permeability in

DNP patients (14).

Longstanding of hyperglycaemia and glucose intermediaries interact with

various metabolic pathways in different cell types of the kidney which lead to

generate AGEs, free amino groups of proteins modified by glucose and its

metabolites to form Amadori-glycated albumin and AGEs that crosslink to the

GBM and other vascular membranes through AGE binding proteins to produce

functional changes causing harmful deteriorations (81). Binding of AGEs to its

receptors activates cell signalling mechanisms to increased expression of PK-C,

TGF-â and vascular endothelial growth factors (VEGF). These cytokines

appears to play a crucial role in the development of renal hypertrophy and

mediator of accumulation of ECM (16, 75). TGF-â1, a potent pro-sclerotic

cytokine, is produced by mesangial cells upon its degredation and binds to the

TGF-â receptors exposed on it (it was found that high BGL induces TGF-â

receptor expression). TGF-â1 induces excessive ECM accumulation by

enhancing synthesis of collagen, fibronectin, and laminin as well as by inhibiting

the expression of metalloproteinases mediated extra cellular matrix degradation.

Figure 2-4 (79). These molecules amplifies the deleterious effects of BP within

the glomerulus by inducing impairment in the auto-regulation of the glomerular

microcirculation (both metabolic and hemodynamic) which act as a stimuli

leading to the activation and/or increased of expression of TGF-â, vascular

endothelial growth factor (VEGF) and GTP-binding proteins (78, 79).

Susztak and his colleagues demonstrated for the first time that high

extracellular glucose induces ROS production, activation of proapoptotic p38

MAPK, and apoptosis of cultured podocytes. They suggested that podocyte

apoptosis/depletion represents a novel early patho-mechanism leading to

diabetic nephropathy, and concluded that NADPH oxidase is a key mediator of

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podocyte apoptosis and subsequent DNP in vivo, thus mechanistically linking:

hyperglycemia, ROS, podocyte apoptosis, podocyte depletion, and diabetic

nephropathy. Systemic inhibition of NADPH oxidase prevents podocyte

apoptosis, reduces loss of podocytes, and ameliorates UAE and mesangial

matrix expansion in vivo of db/db mice (77).

Cellular and molecular mechanisms of multiple pathways of hyperglycaemia

and glomerular hypertension induced damage that converge at the cellular level

(both MC and podocyte) were discussed (79). Many in-vitro investigations have

explored the intracellular mechanisms which can induce, directly or by induction

of both cytokines and mesangial cells dysfunction or damage. A brief summary

for the multiple pathways in gluco-toxicity /DNP of the study is shown in Figure

2-4. They concluded that evidences indicating that both PKC and TGF-â1 are

important mediators of the glomerular injury in diabetes. They declared that a lot

of clinical trials are currently held to test safety and efficacy of PKC andTGF-â1

inhibition in DM complications in humans.

High glucose in the milieu increases intracellular glucose levels. Both high

glucose and stretch induce over-expression of the glucose transporter GLUT-1

further enhancing glucose entry into mesangial cells. Within the cell high

glucose induces ROS formation. This reduces glucose catabolism through the

glycolytic pathway and enhances the glucose flux through alternative signalling

and metabolic pathways. Stretch- and high glucose-induced activation of the

PKC-MAP kinase pathway results in activation of transcription factors

increasing the transcription of genes encoding for ECM components and

prosclerotic cytokines (79).

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Figure 2-4: Intracellular mechanisms of high glucose and stretch induced

effects. GLUT-1: glucose transporter, ROS: reactive oxygen species, PKC: protein kinase-C,

MAPK: mitogen-activated protein kinases, AP-1: Protein-1 Transcription Factor, NFKB: Nuclear Factor kB, STAT: Signal Transducers and Activators of Transcription, TGF: Transforming

Growth Factor â1, CTGF: Connective Tissue Growth Factor, IGF-I: Insulin growth factor-1,

Ang II: angiotensin II enzyme, VEGF: vascular endothelial growth factor, MCP-1: Monocyte

Chemoattractant Protein-1. Adapted (79)

Haemodynamic Insult Stretch

Metabolic Insult High glucose

Signaling pathways PKC-MAPK

GLUT-1

Intracellular Glucose ROS

Glycolysis

Alternative Metabolic Pathways Poly, HSBP, AGEs

Transcription Factor AP-1, NFKB, STAT

Enhanced Extra cellular Matrix

Cytokines TGF, CTGF, IGF-I

Ang II, VEGF, MCP-1

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2.6.5 Markers for diabetic nephropathy

DNP and its complication (glomerular lesions and changes to the tubular-

interstitial) have been extensively studied worldwide. In the early stages of DNP,

there are no clinical signs or symptoms of renal disease. Several recent studies

demonstrated that the cells of the kidneys (proximal tubules) are very sensitive

to any toxic or immunologic alterations and release enzymes or proteins upon

exposure to such alterations. These urinary albumin and/or lysosomal

glycosidases investigated and found to be of particular diagnostic value in the

early detection of diabetic nephropathy (28, 36, 84). The preclinical stage of

DNP is characterised by decreased renal tubular reabsorption capacity,

elevated low molecular weight protein and increased proximal tubular

enzymuria. Investigation of serum creatinine is of limited value in the early

detection of renal insult (30). The assay of preclinical stage of diabetic

nephropathy, include the activities of the specific tubular enzymes, such as N-

acetyl-â-Dglucosaminidase (NAG; â-hexosaminidase), â-glucuronidase, â-

galactosidase, acid phosphatase, alanine- (leu-gly) aminopeptidase,

imunoglogulin G, micro- albumin and other markers in the patients urine can be

used as an early non-invasive indicator for kidney involvement of diabetic

nephropathy (28, 85).

The significance of these markers

First: they provide a sensitive non-invasive test for renal damage.

Second: careful selection of enzymes, they can be used to determine the

primary site of damage in the nephron (71, 85). For example, â-hexosaminidase,

the most studied urinary lysosomal enzyme, ALP, AAP and â-glucuronidase are

all located in the proximal tubule of the nephron (31, 86, 87), while the specific

activity of acid phosphatase is enriched in the glomeruli.

Third: because the excretion of urinary enzymes varies with the activity of renal

disease, they can be used to monitor the rate of recovery (88).

The increased urinary excretion of these markers in the preclinical stage of

diabetic patients without the clinical signs of diabetic nephropathy (proteinuria)

manifested is a significant indicators of diabetic nephropathy.

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2.6.5.1 Microalbuminuria

MAU affects 20 to 40 % of patients over 10 years after the onset of diabetes.

Progression to MAU or overt nephropathy occurs in 20 to 40 % of patients over

a period of 15 years after the onset of diabetes (22, 74). MAU has been used

for many years as a predictor of incipient DNP, reflecting the loss of glomerular

selectivity, estimation of renal tubular function and integrity (30). The first sign of

renal involvement in patients with type-2 DM is most often MAU (UAE, 20 to

200 µg per minute in an overnight) (20), and patients are classified to as having

incipient nephropathy (36). The screening for MAU in diabetic patients is at

present the earliest clinical marker identifying patients who are at risk for

developing diabetic nephropathy (89). MAU occurs when urinary albumin

excretion increases but remains below detectable level by routine laboratory

methods (28, 90, 91).

Extensive studies have demonstrated that diabetic patients diagnosed

with MAU have increased risk of progression to macroalbuminuria, which lead

to renal failure. The progression of diabetic nephropathy from the detection of

proteinuria to ESRD is usually irreversible. In Type 2 diabetic patients about

20% develop ESRD. (92, 93) Early medical treatment and lifestyle adjustments

have been shown to halt the progression from micro- to macroalbuminuria,

detection of MAU as early as possible in the course of the disease is very

important (89, 93, 94).

Various epidemiological and cross-sectional studies have reported

marked variation in the prevalence of MAU among diabetes patients, and

besides being a predictor for incipient nephropathy; it also represent a good

marker for greatly increased cardiovascular morbidity and mortality in patients

with type 1 or type-2 DM (95, 96). Measuring albumin/ creatinine ratio to detect

early manifestations of renal impairment is the most widely used method in the

United Kingdom. It is proved to be a more accurate indicator than albumin

concentration (96). Many studies focused on the detection of MAU in DM

patient and to find the correlation with creatinine, enzymes and the duration of

DM in order to predict and treat DNP.

Cross sectional study on 91 Type-1 and 153 Type-2 diabetic patients by

Lutale et al., 2007 in Dar Es Salaam Tanzania, estimated MAU among Type-2

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patients, was 9.8% , and 7.2% had macroalbuminuria. The duration of diabetes

was the strongest predictor in Type-2 patients with abnormal albumin excretion

rate had longer diabetes duration of 7.5 years (93).

In china, urine albumin and some urinary enzymes excretion in random urine

samples of 157 DM Chinese patients and 54 healthy subjects were studied (97).

Alb/Cr ratio was greater than 26.8 mg/mmol in 14.6% of subjects, and between

2.5 - 26.8 mg /mmol in 54.1% of subjects. Another cross sectional study in

Hong Kong aimed to examine the prevalence of MAU among patients with type-

2 DM in four primary care clinics, showed significant association of MAU in

patients with DM with advanced age, female sex, poor glycaemic control,

HbA1c level, long duration of DM, diabetic retinopathy, and coexisting

hypertension in correlation analysis (95). The effective screening of MAU is

important to optimize the renal outcome of patients with DM (95).

In a study conducted in Japan including 29 medical clinics from different areas,

14919 patients with type-2 diabetes, diagnosed according to the Japan Diabetes

Society Criteria, the prevalence of MAU in Japanese type-2 diabetic patients

was 32%. This indicates that the Japanese type-2 diabetic population may be

susceptible to developing MAU (98). Screening of MAU is recommended to be

performed every 6 months for patients, who have had diabetes starting 5 years

after diagnosis in type-1 DM, and at time for patients with type-2, screening

should be performed at diagnosis and every year thereafter (22, 90, 91).

In 1996 DM was found to be responsible for 14.5% of the ESRD new cases in

Egypt. (99)

In Gaza strip, Altibi assessed the MAU among type-2 diabetic patients

(44 males and 55 females). He found that 22.2% were microalbuminuric, 22.2%

were macroalbuminuric and 55.5% were normoalbuminuric (33).

2.6.5.2 Urinary Enzymes (AAP, ALP and ACP)

The use of enzymatic determinations as routine methods of clinical chemistry

has markedly improved the diagnostic possibilities in clinical medicine. Attention

has been focused on the diagnostic applicability of urinary enzyme

determinations as a tool for the diagnosis and follow-up of many common

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diseases since the last decade (29). Renal function assessment is important in

DM patients and indicators are needed to identify the early structural and

functional changes in DNP. The kidney is the principal source of urinary

lysosomal enzymes, as several enzymes localized in the renal parenchyma

which can be measured in urine to detect early renal tubular damage (71).

Under pathologic conditions the evaluation of urinary enzymes for diagnostic

purposes is an important tool in clinical medicine. Alkaline phosphatase, ALP

(EC 3.1.3.1), Acid phosphatase, ACP and Alanine aminopeptidase, [AAP;

microsomal alpha aminoacyl-peptide hydrolase, EC 3.4.11.2] are proximal-

tubule brush-border enzymes have proved to be a suitable urinary e