Stages Of Progression Of Diabetic Nephropathy Biology Essay


Diabetic nephropathy is histopathologically characterized by several changes in the kidney, such as nodular glomerulosclerosis, mesangial expansion, basement membrane thickening and interstitial fibrosis. Clinically, diabetic nephropathy is usually a constellation of persistent albuminuria, elevated arterial blood pressure and decline in kidney function [13].

Stage I: Hypertrophic hyper filtration. In this stage, GFR is either normal or increased. Stage I lasts approximately five years from the onset of the disease. The size of the kidneys is increased by approximately 20% and renal plasma flow is increased by 10%-15%, while albuminuria and blood pressure remain within the normal range.

Stage II: The quiet stage. This stage starts approximately two years after the onset of the disease and is characterized by kidney damage with basement membrane thickening and mesangial proliferation. There are still no clinical signs of the disease. GFR returns to normal values. Many patients remain in this stage until the end of their life.

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Stage III: The microalbuminuria stage (albumin 30-300 mg/dU) or initial nephropathy. This is the first clinically detectable sign of glomerular damage. It usually occurs five to ten years after the onset of the disease. Blood pressure may be increased or normal. Approximately 40% of patients reach this stage.

Stage IV: Chronic kidney failure (CKF) is the irreversible stage. Proteinuria develops (albumin > 300 mg/dU), GFR decreases below 60 mL/min/1.73 m2, and blood pressure increases above normal values.

Stage V: Terminal kidney failure (TKF) (GFR < 15 mL/min/1.73 m2). Approximately 50% of the patients with TKF require kidney replacement therapy (peritoneal dialysis, hemodialysis, kidney transplantation) [5].

In the initial stages of diabetic nephropathy, increased kidney size and changed Doppler indicators may be the early morphological signs of renal damage, while proteinuria and GFR are the best indicators of the degree of the damage [6].Changes in albuminuria are considered a hallmark of onset or progression of nephropathy. .

Study Setting: The subjects will be required at outpatient's clinic of nephropathy, University Diabetes Center, King Abdul Aziz University Hospital, King Saud University, Riyadh. All laboratory work will be performed at Strategic Centre for Diabetic Research, King Saud University.

Study Design:

The case-control studies will require 586 subjects for this study. The 600 samples will be recruited according to the Raosoft, the error margin of the group will give 5% and 95 % (CI) confidence level. The group will be dived in different stages of disease progression.

Study subjects will be recruited from the out-patient department at University Diabetes Center, King Saud University. The randomly selected subjects will be recruited for this study and will be classified as "known diabetic subjects" according to American Diabetes Association criteria (ADA 2012), if they stated that they had diabetes and were on the treatment. After first screening they will be invited to the center for detailed testing, including an oral glucose tolerance test in those without self-reported diabetes. Those who confirmed by oral glucose tolerance test to have 2-hour plasma glucose value of 11.1 mmol/L or more (200 mg/dL) based on World Health Organization consulting group criteria were labelled as "newly detected diabetic subjects (NDD)," those with 2-hour post glucose value of at least 7.8 mmol/L (140 mg/dL) and less than 11.1 mmol/L (200 mg/dL) as subjects with IGT, and those with 2-hour post glucose value of less than 7.8 mmol/L (140 mg/dL) as subjects with normal glucose tolerance (NGT).

For Screening of diabetic Nephropathy following criteria will be adopted.

According to the ADA criteria


Albuminuria cutoff values

Clinical characteristics


20-199 µg/min

Abnormal nocturnal decrease of blood pressure levels.

30-299 mg/24 h

Increase triglycerides, total and LDL cholesterol, and saturated fatty acids.

30-299 mg/g*

Increased frequency of metabolic syndrome components.

Endothelial dysfunction.

Association with diabetic retinopathy, amputation and cardiovascular disease.

Increased cardiovascular mortality.

Stable GFR


≥200 µg/min

≥300 mg/24 h

≥300 mg/g*


Increased triglycerides and total and HDL cholesterol.

Asymptomatic myocardial ischemia.

Progressive GFR decline

Exclusion Criteria: Patients with a diagnosis of reduced renal function, proteinuria (≥300 mg/dl) and creatinine levels (≥2mg/dl), history of alcohol intake or smoking.

Anthropometric measurements

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It's included weight, height, and waist and will be obtained using standardized techniques. Height will be measured with a tape measure to the nearest centimeter. Weight will be measured with traditional spring balance that was kept on a firm horizontal surface. Waist will be measured using a non stretchable fiber measuring tape. The body mass index (BMI) will be calculated as the weight in kilograms divided by the square of height in meters. Blood pressure will be recorded from the right arm in a sitting position to the nearest 2 mm Hg with a mercury sphygmomanometer.

Biochemical parameters

Fasting plasma glucose (glucose oxidase-peroxidase method), serum cholesterol (cholesterol oxidase-peroxidase- amidopyrine method), serum triglycerides (glycerol phosphate oxidase-peroxidase-amidopyrine method), and high-density lipoprotein (HDL) cholesterol (direct methodpolyethylene glycol-pretreated enzymes) will be measured using BS200 Auto-analyzer (Mindray, China). Glycated hemoglobin (HbA1c) will be estimated by NycoCard - HbA1c Test.

Biomarker Analysis:

Biochip Assay Methodology is based on standard immunoassay techniques. In most test panels, antibodies are attached to the surface of the biochip and analyses in the patient's samples binds to them. Competitive and sandwich immunoassay techniques are used for biochip assays. The methodology adopted is panel-specific and dependent on molecular weight of the analytes of interest. The competitive assays utilize an enzyme-labelled analyte for signal production whereas the sandwich assay utilizes an enzyme-labelled antibody.

One biochip is used per sample to produce multiple test results simultaneously, Each biochips contain internal control sites, which are always on the same position on every biochips. The control sites have sets target levels in order to identify problems.The detection system is based on chemiluminescent signal. An enzyme is used to catalyse the chemical reaction on the biochip which generates the chemiluminescent signal. The light emitted from each DTRs is simultaneously detected and Quantified using Charge-Coupled Device (CCD) camera. Analyse a complete profile of biomarkers from as little as 7 µl of samples.

ELISA Based Method;

Adiponectin, insulin, leptin, resistin and other markers that not available in biochip array will be measured by enzyme-linked immunosorbent assay (ELISA). In brief, monoclonal antibody specific for marker were pre-coated onto a micro plate. Standards and samples will be pipette into the wells, and specific marker present will be bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked monoclonal antibody specific for marker will be added to the wells. After a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells; and colour developed in proportion to the amount of marker bound in the initial step. Absorbance will be read at 450 nm. The values would be expressed in pictograms per milliliter units.

Statistical Analysis: Student t test or 1-way analysis of variance will be used to compare groups for continuous variables, whereas χ2 test or Fisher exact test as appropriate used to compare proportions. Pearson correlation analysis will be carried out to determine the relation of different biomarker with other risk variables. Multiple linear regression analysis was used to determine the association of significant biomarker with different biochemical parameters. All analyses will be done using Windows-based SPSS statistical package and P values of < .05 will be taken as significant.

4.3 Management Plan:

The research team was established based on recommendations that "multidisciplinary approaches to research" is a major drive for successful research projects. The research team for this project includes: Dr. Khalid Al-Rubeaan (PI), Consultant Endocrinologist, Director of the University Diabetes Center Fellow the Royal College of physician of Canada, F.R.C.P(C), Dr. Khalid Siddiqui, PhD. in Biochemistry and Dr. Dhekra Al-Naqeeb, Bachelor Degree in Medicine and Surgery (MBBS).

Dr. Khalid Al-Rubeaan (PI) is the Director of the University Diabetes Center, King Saud University. He is working as a Consultant Endocrinologist and Assistant Professor, College of Medicine at King Saud University. He is also the Executive Director of Strategic Center for Diabetes Research, Editor-in-Chief of International Journal of Diabetes Mellitus Scientific Journal and Al-Sukari Magazine for Patient Education in the Middle East, the Head of the Saudi National Diabetes Registry and a former president of the Pan Arab Group for the Study of Endocrinology and Diabetes. His major interest is Endocrine Disease with special interest on Diabetes Mellitus covering all aspects i.e. basic research, education, prevention and epidemiology. Dr. Al-Rubeaan is a member of many Diabetes Associations and Federations and had numerous publications in the form of chapters and books or scientific papers covering many fields of diabetes. Dr. Al-Rubeaan chaired many training courses in the field of Diabetology for physicians, educators, foot care specialist and nutritionists. He had been an organizer of undergraduate courses and former head of endocrine division, Medical College at King Saud University. He was awarded the outstanding postgraduate achievement by the Saudi Arabian Education Mission in 1988 and the Arabian Award for Diabetes Education by the Pan-Arab Society for Diabetes and Endocrinology in 2001. He was also awarded the Novo Nordisk Research Merit Award in diabetes in 2002. He has obtained patent in the field of nano and IT technology in the area of diabetes management.

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Dr. Khalid Siddiqui, is currently the Head, Basic Science (Genetics & Biochemistry) Strategic Centre for Diabetic Research, King Saud University. He pursued his doctoral degree in the field of Molecular Genetics and biochemistry from International Max Planck Research School, Germany and degree was awarded by Goethe University Frankfurt Germany. Following his PhD he worked as Postdoctoral research associate in Faculty of Life Science at The University of Manchester, England. He will be responsible for supervising the laboratory part of the project. In addition He will also responsible for the statistical analysis, writing results and manuscripts.

Dr. Dhekra Al-Naqeeb is a Research Physician in University Diabetes Center (UDC), King Saud University. She will be responsible for recruitment of patients and clinical diagnosis following STRICT criteria in addition to the writing of the reports and the manuscript.







Dr. Khalid Al-Rubeaan

Dr. Khalid Siddiqui

Dr. Dhekra Al-Naqeeb