The document describes a study that induced type 1 diabetes in rats using streptozotocin injections to study the progression of diabetic nephropathy. Rats were divided into normal, diabetic, and diabetic fed a high-fat diet groups. Over 8 weeks, the diabetic and diabetic high-fat diet rats displayed increased blood glucose, water intake, urine output and glomerular mesangial expansion compared to normal rats, indicating the onset and progression of diabetic nephropathy. Streptozotocin successfully induced diabetes in rats and the addition of a high-fat diet exacerbated nephropathy severity.

1. “to study the severity of nephropathy in high fat diet
fed streptozotocin induced Diabetic rats”
Dissertation Report
Submitted to
Department of Biochemistry
Dr. R.M.L. Avadh university Faizabad
Supervisor Submitted by
Dr.Swasti tivari senior scientist Firoz Ahmad
Division of molecular medicine M.sc.Biochemistry 4th sem.
SGPGIMS LKO. Dr.R.M.L.Avadh university
Faizabad (UP)

2. • The Objective of this study is to study the severity of nephropathy in
high fat diet fed Streptozotocin induced diabetic rats.
• The type-I DM was induced by intraperitoneal injection of 50mg/kg
dose of Streptozotocin in adult wistar rats weighted (200-300) grams,
causes degeneration in beta cells and induces diabetes mellitus within
(2-4) days.
• The diabetic and normal animals were weight and kept in the
metabolic cages separately for the measurement of consumption of
food and water and urine volume.
• Then these quantities were compared. After Induction of diabetes,
consumption of food and water, volume of urine, urinary albumin and
blood glucose level was increased in the diabetic as well as diabetic
rats fed with high fat diet in comparison to the normal rats, but body
weight, blood glucose level, water intake, urine output, albumin and
creatinine clearance decreased in diabetic animals fed with high fat
diet in compared to diabetic rats.

3. Based on biochemical parameter and histopathology the
present study show that young male Wistar rats treated by
a single intraperitoneal STZ dose of 50 mg/kg developed a
persistent disease state characterized by severe
hyperglycaemia with major clinical signs of diabetes
mellitus
.
1. Onset of DN in rats begins subtly after one month of
Diabetes. It has been shown that addition of cholesterol to
the diet exacerbate the severity of Diabetic Nephropathy.

4. Introduction
 Diabetes mellitus (DM) is a group of diseases characterized by high
levels of blood glucose resulting from defects action of insulin
production, insulin action, or both.
 The term diabetes mellitus describes a metabolic disorder of multiple
aetiology characterized by chronic hyperglycaemia with disturbances
of carbohydrate, fat and protein metabolism resulting from defects in
insulin secretion, insulin action, or both.
 The effects of diabetes mellitus include long–term damage,
dysfunction and failure of various organs of our body.

5. Diabetes
 Diabetes mellitus may present with characteristic symptoms such
as polydipsia, polyuria, blurring of vision, and weight loss.
 In its most severe forms, ketoacidosis or a non–ketotic
hyperosmolar state may develop and lead to stupor, coma and, in
absence of effective treatment, death.
 Often symptoms are not severe, or may be absent, and
consequently hyperglycaemia sufficient to cause pathological
and functional changes may be present for a long time before the
diagnosis is made.

6. Burden of Diabetes
 The development of diabetes is projected to reach pandemic
proportions over the next 10-20 years.
 International Diabetes Federation (IDF) data indicate that by the year
2025, the number of people affected will reach 333 million – 90% of
these people will have Type 2 diabetes.
 In most Western societies, the overall prevalence has reached 4-6%,
and is as high as 10-12% among 60-70-year-old people.
 The annual health costs caused by diabetes and its complications
account for around 6-12% of all health-care expenditure.

7. Types of Diabetes
There are mainly three types of diabetes:
 Type -1 Diabetes Mellitus ( IDDM )
 Type -2 Diabetes Mellitus ( NIDDM )
 Gestational Diabetes

8. Type -1 Diabetes Mellitus
 Type -1 DM is called insulin-dependent diabetes mellitus
(IDDM) or juvenile-onset diabetes.
 Type 1 diabetes develops when the body’s immune system
destroys the pancreatic beta cells, these cells produced insulin
hormone it regulates transport & metabolism of blood glucose.
 This form of diabetes usually strikes children and young
adults, although disease onset can occur at any age.
 Type -1 diabetes may account for 5% to 10% of all diagnosed
cases of diabetes.
 Risk factors for type -1 diabetes may include autoimmune,
genetic, and environmental factors.

9. Type - 2 Diabetes Mellitus
 Non-insulin-dependent diabetes mellitus (NIDDM) or
adult-onset diabetes.
 It account for about 90% to 95% of all diagnosed cases of
diabetes.
 It usually begins as insulin resistance, a disorder in which
the cells do not use insulin properly. As the need for
insulin rises, the pancreas gradually loses its ability to
produce insulin.
 Type -2 diabetes is associated with older age, obesity,
family history of diabetes, history of gestational diabetes,
impaired glucose metabolism, physical inactivity, and
race/ethnicity.
 Type -2 diabetes is increasingly being diagnosed in
children and adolescents.

10. Comparison between two types of
Diabetes

11. Mechanism of Insulin Action

12. Streptozotocin
• Streptozotocin (STZ) is a Monofunctional Nitrosourea Derivative.
Produced by the bacterium (Streptomyces achromogenes) that
exhibits broad spectrum Antibacterial properties (Vavra JJ et al.,
1959). Streptozotocin is a mixture of α - and β-stereoisomers that
appear as a pale yellow or off-white crystalline powder. STZ is very
soluble in water, ketones, and lower alcohols and only slightly soluble
in polar organic solvents.

13. Properties of Streptozotocin
Chemical Name 2-Deoxy-2-[(methylnitrosoamino)carbonyl]amino)-D-
Glucopyranose
Chemical
Structure
Cytotoxic Methylnitrosourea Moiety (N-methyl-N-
Nitrosourea) attached to the glucose (2-deoxyglucose)
molecule; glucosamine derivative
Chemical
Properties
 Hydrophilic, Beta cell-toxic glucose analogue
 Relatively stable at pH 7.4 and 37o C (at least for up
to 1 h)
Chemical
Reactivity’s
 DNA alkylating agent
 Protein alkylating agent
 NO donor
Mode of Toxicity DNA alkylation

14. Mechanism Action of Streptozotocin
Streptozotocin
Selective β-cell
uptake by the
GLUT 2 glucose
transporter
β-cell toxicity
through alkylation
Insulin-dependent Diabetes
mellitus
β-cell death
through necrosis

15. High Fat Diet
• The interrelation between diabetes,hypercholesterolemia,
atherosclerosis, and renal disease are well recognized.
• Approximately 30% of new cases of end stage renal
disease are attributed to diabetic nephropathy.
• Diabetes mellitus markedly increases the risk of coronary
artery disease.
• It has been proposed that a persistent subclinical increase
in urine albumin excretion in diabetics is associated with
the later occurrence of nephropathy..
• These complications include hyperglycemia hyper
cholesterolemia , hypertension , and obesity . Each of these
conditions can affect renal function independently, or in
combination, and lead to end stage renal disease.

16. • In a number of experimental animal models, it has been shown
that addition of cholesterol to the diet favors the development
of glomerulosclerosis.
• On the other hand, a reduction in serum cholesterol
ameliorates the development of Glomerulosclerosis in several
experimental animal models.
• Hyper Cholesterolemia and hypertriglyceridemia, recognized as
contributing to Atherosclerosis, are also emerging as risk factors
for progression of renal disease
• In a number of models of experimental renal disease obtained
from patients with a variety of renal diseases, lipid and
apolipoprotein deposition, particularly in mesangial cells and
macrophages, has been documented.

17. Diabetic Nephropathy
 It is progressive rise in urine albumin excretion, coupled with
increasing Blood Pressure, leading to declining glomerular filtration
and elevated arterial blood pressure.
 Diabetic nephropathy is now the single commonest cause of end-stage
kidney failure worldwide and is acknowledged as an independent risk
factor for cardiovascular disease.

 The earliest clinical manifestation is microalbuminuria.
 Diabetic Nephropathy is characterized by:
 Structural canges especially with the degree of mesangial expansion.
 Thickening of Glomerular basement membrane.
 Changes in the structure and mumber of podocytes.

18. STUDY DESIGN
(Preparation and Validation of animal model of DN)
50 mg/kg STZ induced Diabetic Rat (n=6) Normal Rat (n=3)
Diabetic Rats (n=3) Diabetic Rats fed High cholesterol Diet (n=3)
After 8th week rat are sacrificed for study the severity of Diabetic nephropathy

19. • Animal Handling
• Injection Techniques
• Intermusculer injection
• Interparitonial injection
• Oral gawage
• Glucose Tolerance Test
• Organ seperation
• Histopatology
• Tissue preparation for histology
• Prepration of paraffin blocks of kidney
• Sectioning by microtome
• Staning
• PAS staning (periodic acid schiff,base staning)

20. Results
Figure 1. Body weight (g) at weekly intervals. Data are mean ± SE. Type 1 diabetes
mellitus (DM) rats lost weight by the end of the study period

21. Biochemical Parameters
Figure 2. Blood glucose levels (mg/dL) obtained by tail vein puncture during the study
period at weekly intervals. Data are mean ± SE. STZ induced Diabetic rats had elevated
blood glucose levels throughout the study compared to control groups

22. Biochemical Parameters
Figure 3. 24 hours urine output (ml). Data are mean ± SE. Type 1 diabetes mellitus (DM)
had significantly higher 24 hrs urine output compared to control groups by the end of the
study period

23. Biochemical parameters
Figure 4. 24 hours water intake (ml). Data are mean ± SE. Type 1 diabetes mellitus (DM)
had significantly higher 24 hrs water intake compared to control groups by the end of the
study period

24. Biochemical Parameters
Figure 5. Glucose Tolerance Tests (GTT): Area under the curve (AUC) for glucose tolerance was compared among the groups.
Type 1 DM resulted in higher AUC ** (p<0.01) compared to control rats

25. PAS STAINING
Figure 7. Histology [Jones periodic acid-Schiff stain (PAS)] of representative glomeruli at 40X magnification.
Jones PAS-stained glomeruli showed expanded mesangium in diabetic groups relative to control.
Type I diabetic animals were most affected, showing substantially more widening of matrix than control groups

26. Conclusion
 50 mg/kg body weight of Streptozotocin
successfully induce diabetes in rats
 Onset of DN in rats begins subtly after one
month of diabetes
 Most of the parameter showed correlation to
glycemic levels. Hence persistent glycemic
levels will results in diabetic complications
such as Diabetic nephropathy