ZDSD Overview a model of obesity, metabolic syndrome and diabetes, 24 May-2011

INDEX
1. Introduction Page 2-
2. Spontaneous or Synchronous Diabetes Page 7-
3. Metabolic Syndrome Elements Page 10-
A. Visceral Obesity Page 13-
B. Insulin Resistance Page 19-
C. Clamp Study Page 28-
D. Dyslipidemia Page 32-
E. Hypertension Page 35-
4. Eating Behavior Page 37- →
5. Beta cell Failure Page 38-
6. Renal Injury Page 41-
A. Urinary biomarkers, Exp. 1, 2 Page 44-
B. RBM Biomarkers – Renal, Exp 3 Page 55-
C. Glomerular Pathology, EM, Exp 4 Page 64-
D. Synchronized Nephropathy, Exp 5 Page 76-
7. Osteoporosis Page 86-
8. Wound Healing Page 92-
9. RBM Biomarkers – Pro-Thrombotic Page 96-
10. RBM Biomarkers - Inflammation Page 102-
11. Therapeutic Efficacy
A. Common Anti-diabetic Compounds Page 110-
B. Rimonabant Page 115-
C. Niacin Page 123-
12. Summary Page 125-
1

← →

The ZDSD Rat as a Translational Model for the
Development of Drugs for Obesity, Metabolic
Syndrome and Diabetes that Demonstrates
Many of the Serious Complications of Diabetes.

PreClinOmics, Inc.
PDF Return to Index, For
Web Use Refresh Above

2

• Most rodent models of type 2 diabetes
Background have a monogenetic mutation that is
responsible for the initiation of obesity
and subsequent insulin resistance.
• The two most common obesity-
causing mutations are
← – the leptin receptor →
• Zucker Fatty; ZF rat
• Zucker Diabetic Fatty; ZDF rat
• db/db mouse
– the leptin molecule
• ob/ob mouse
• Both leptin and leptin receptor
mutations are rare in humans.
• The ZDSD rat does not have these
mutations but still has obesity PDF Return to Index, For

metabolic syndrome and diabetes. 3

Development Scheme
Zucker Diabetic-Sprague Dawley Rat (ZDSD)
• Produced by crossing diet induced obese (DIO) rat derived
from the Crl:CD(SD) strain (exhibiting polygenetic obesity and
← insulin resistance) with homozygous lean ZDF/Crl rat (which →
will express beta cell failure with the Leprfa/Leprfa genotype).
• Selectively bred for obesity and diabetes.
• Selected for genetically matched breeders to develop
phenotypic homogeneity.
• Studied male rats at different ages.


4

Characteristics of The ZDSD Rat
• Unique translational model for obesity, metabolic
syndrome/type II diabetes - 35 generations inbred
• Polygenic obesity and phenotype can be modulated by diet.
• Phenotype is expressed in the presence of a functional leptin
← pathway. →
• Insulin resistance development starts at an early age.
• Early onset of hyperglycemia and slower progression to frank
diabetes when compared to the ZDF rat.
– Slower deterioration of beta cell function.
• Manifests diabetic complications:
Diabetic nephropathy Hypertension
Cardiovascular markers Inflammation
Osteoporosis Delayed Wound Healing
• In production PDF Return to Index, For

5

The ZDSD Rat:
One rodent – Many Models ZDSD
Obesity
Metabolic Syndrome
Diabetes

Obesity Model
before
Metabolic Syndrome Diabetes Model
diabetes develops,

← →
5-16 weeks of age

Natural/Spontaneous Diet Synchronized
Insulin Development (LabDiet 5008) (RD D12468 or Purina Test
Resistance Slower & more random Diet 5SCA)

Diabetic Diabetic
Hyperlipidemia Nephropathy Nephropathy

Obesity Osteoporosis Osteoporosis

Cardiovascular/ Cardiovascular/
Inflammatory Inflammatory
Hypertension Biomarkers Biomarkers

Delayed Wound Delayed Wound
Delayed Wound Healing Healing
Healing
6

Spontaneous 700

development of 600

diabetes 500

Body weight (g)
400

The ZDSD Rat when 300

maintained on Lab Diet 5008
200
chow will spontaneously
← develop diabetes as it ages 100
→
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
beyond 16 wks. As fed serum Age (weeks)

glucose levels begin to 450

increase above ~350 mg/dl, 400

body weight begins to Serum glucose (mg/dL)
350

decrease. 300

250

200

150

100
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Age (weeks) PDF Return to Index, For

7

Synchronization 600

of diabetic onset

Glucose (mg/dl)
400 SD Male Rats

The ZDSD Rat can be placed ZDSD Males

on either D12468 (Research 200
ZDSD Females

Diets) or 5SCA (LabDiet) to
synchronize the onset of 0
diabetes.
← 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Age (wks) →
When the ZDSD rat was 600

placed on either diet at 17
wks of age, the plasma 400

Weight (g)
glucose levels of the animals
averaged over 450 mg/dl 200
SD Male Rats

ZDSD Males
within 1 week. Following a
ZDSD Females
return to LabDiet 5008 at 19 0
wks of age, the animals 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Age (wks)
maintained the diabetic state.
PCO now recommends a 3 Area shaded in grey indicates
week synchronization time frame of diabetogenic diet PDF Return to Index, For

protocol
8

Synchronization
of diabetic onset
% Body Fat by QNMR
Body composition 30

changes in response 25

← to diabetogenic diet 20 SD Male Rats
→

% Body Fat
ZDSD Males
(5SCA or D12468). 15
ZDSD Females
10

5

0
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Age (wks)

Area shaded in grey indicates
time frame of diabetogenic diet


9

ZDSD as a preclinical model of Metabolic Syndrome
Metabolic syndrome affects a large proportion of the population and is becoming
increasingly important in adolescents. The syndrome has many components
including central obesity, insulin resistance, dyslipidemia and hypertension. In
addition, the syndrome features a chronic low grade inflammatory state, vascular
endothelial dysfunction, and a prothrombotic environment. Long standing
← →
metabolic syndrome can thus pre-dispose to atherosclerosis, microvasculature
disease (retina), stroke, renal injury and diabetes. Due to the complicated
mechanisms involved in the syndrome and its sequelae, current standard of care
reflects poly-pharmacy and is aimed at controlling atherogenic dyslipidemia,
hyperglycemia and hypertension as well as intervening in secondary diseases such
as renal dysfunction, stroke, and micro-vascular disease related to retinopathy.
Development of new chemical entities with the potential to control more than one
risk factor is hampered by currently available animal models. To that end, the ZDSD
rat was designed to spontaneously develop a phenotype that mimics many aspects
of the human metabolic syndrome, including hypertension and the progression to
frank diabetes with long-standing disease.

10

Metabolic Syndrome
• Metabolic syndrome is most frequently
defined by a presence of certain traits,
← →
including:
– abdominal obesity
– insulin resistance
– Dyslipidemia
– elevated blood pressure and
– pro-thrombotic and pro-inflammatory states

11

Characteristics of Metabolic
Syndrome Seen in the ZDSD Rat
• Increased body weight with increased
abdominal fat
← →
• Insulin resistance / Glucose intolerance
• Hyper-lipidemia
• Increased blood pressure / Hypertension
• Increased Serum BioMarkers of Coagulation
inflamation and Vascular Disease
• Increased fed and fasting glucose and

HbA1c levels Web Use Refresh Above

12

Visceral Obesity

A prominent component of metabolic syndrome is insulin resistance which is
thought to be mediated by an increase in metabolically active visceral fat. Visceral
fat accumulation occurs in human patients in the presence of a functional leptin
← pathway as leptin deficiencies and receptor defects are rarely reported. According →
to published growth charts for male leptin resistant ZDF rats, the new ZDSD rats are
heavier when fed a normal diet (PMI 5008) and exhibit a body composition
(increased % fat) comparable to age matched DIO-LE model which is a mainstay for
anti-obesity research. In addition, the ZDSD responds to a common reference anti-
obesity agent (rimonabant) with significant loss of body fat. Interestingly, ZDSD rats
are not typically nocturnal in that they exhibit significant feed intake during the
daylight hours. Exogenously administered leptin results in an acute anorexic effect
quite similar to normal SD rats and indicates the presence of a functioning leptin
pathway


13

Comparative Growth Curves
in SD and ZDSD Rat Fed 5008 chow

ZDSD rats were significantly
← (15%) heavier than their SD →
counterparts at 8 weeks of
age. In addition, the rate of
body weight gain was
increased in ZDSD animals as
evidenced by an 82% vs 62%
weight gain in SD animals
during the 24 weeks.

All time points statistically different
14
Study # 09-550-170

Spontaneous Development of Obesity in ZDSD
Rats Fed 5008 Chow

Body composition was
assessed using QNMR . The
percentage of body weight
← identified as fat was 50 %
→
higher in ZDSD compared to
SD controls as early as 8 weeks
of age. Body fat percentage
continued to increase
throughout the study and
remained significantly higher
than control rats at each time-
point.

15
Study # 09-550-170

Visceral Obesity in the ZDSD Rat
CT Scan

Sub-cutaneous fat
← →

Retroperitoneal fat

Visceral fat


16

Terminal Body Weight Comparison
ZDF
CRL-SD, CD
700
+/fa
ZDSD, Diabetic 7-11 weeks

600

← →
500
Weight (g)

400

300

200

100

Terminal animal weights in diabetic and control animals. Web Use Refresh Above

17

Terminal Comparison
Liver Weight Food & Water Consumption
30 ZDF
350
CRL-SD, CD
+/fa
ZDSD, Diabetic 7-11 weeks ZDF
25 300
ZDSD, Diabetic 12-21 weeks CRL-SD, CD
+/fa

← 250
→

Amount/rat (gram)
20
Weight (gram)

200
15
150

10
100

5 50

0 0

Terminal liver weights, water intake and food consumption are highest in the diabetic groups.


18

Development of Insulin Resistance in the
ZDSD Rat on 5008 Purina chow
• Rats tested started at 8 weeks of age (SD & ZDSD)
• Weight, glucose and insulin measured weekly
← • Animals fasted every two weeks for OGTT →
• Data analyzed
– Weight
– Body composition
– Glucose levels
– OGTT glucose and insulin
– Glucose disposal
– HOMA-IR

19

Comparative Growth Curves
in SD and ZDSD Rat Fed 5008 chow

ZDSD rats were significantly
← (15%) heavier than their SD →
counterparts at 8 weeks of
age. In addition, the rate of
body weight gain was
increased in ZDSD animals as
evidenced by an 82% vs 62%
weight gain in SD animals
during the 24 weeks.

20
Study # 09-550-170

Spontaneous Development of Obesity in ZDSD
Rats Fed 5008 Chow

Body composition was
assessed using QNMR . The
percentage of body weight
← identified as fat was 50 %
→
higher in ZDSD compared to
SD controls as early as 8 weeks
of age. Body fat percentage
continued to increase
throughout the study and
remained significantly higher
than control rats at each time-
point.

21
Study # 09-550-170

Spontaneous Development of Hyperglycemia in
ZDSD Rats Fed 5008 Chow

← →

22
Study # 09-550-170

Spontaneous Development of Glucose Intolerance
Shown by OGTT in ZDSD Rats Fed 5008 Chow

← →


23

Spontaneous Development of Insulin Resistance
Shown by OGTT in ZDSD Rats Fed 5008 Chow

← →


24

Spontaneous Development of Impairment in Glucose
Disposal in ZDSD Rats as Demonstrated by OGTT, AUC

Impairment in glucose
disposal as represented
by the area under the
← glucose curve during an
→
oral glucose tolerance
test developed
spontaneously in ZDSD
rats and was evident as
early as 8 weeks of age
(fed Purina 5008 chow).

25
Study # 09-550-170

Progressive Development of Insulin Resistance
(HOMA-IR) in ZDSD Rats

ZDSD rats become
increasingly more insulin
← resistant with age as →
evidenced by the calculated
HOMA-IR. The insulin
resistance is evident
compared to SD rats as early
as 8 weeks of age (fed Purina
5008 chow).

26

Glucose and Glycated Hemoglobin Levels in CD
and Prediabetic ZDSD Rats

Glucose in CD vs ZDSD Glycated Hb in CD vs ZDSD
16 weeks of age 16 weeks of age
150 4.0
CD CD
Blood Glucose mg/dL

← 140 ZDSD 3.8 ZDSD
→

Glycated Hb
130 3.6

120 3.4

110 3.2

100 3.0

D

SD
D

SD

C
C

ZD
ZD


27

Pre-diabetic Insulin Design
Sensitivity, – Rosiglitazone treatment: 3 mg/kg
hyperinsulinemic- PO, QD for 2 weeks
euglycemic glucose – Comparison of insulin sensitivity
clamp at 9 wks of age in
• The ZDSD Rat,
← →
• Zucker Fatty (ZF), and
• Sprague Dawley (SD) rats
– Assessed by exogenous glucose
infusion rate (GIR) during
hyperinsulinemic (25
mU/kg/min)-euglycemic glucose
clamp


28

Body Weight and Basal Glucose of Rats Before
Undergoing Glucose Clamp

Basal Blood Glucose (mg/dl)
500 200
Vehicle (n=6-8)
Rosiglitazone (3 mg/kg PO, n=6-8)
400 # 160
Body Weight (g)


 

300 120
← →
200 80

100 40

0 0
SD (age) SD (wt) ZDSD ZF SD (age) SD (wt) ZDSD ZF
Rat Strain (SD rats are age or weight matched) Rat Strain (SD rats are age or weight matched)

* P<0.05 compared to SD rat (age matched) group
# P<0.05 compared to vehicle treated group


29

ZDSD and ZF Rats are Insulin Resistant which
Improved with Rosiglitazone Treatment
60 Vehicle (n=6-8)
Rosiglitazone (3 mg/kg PO, n=6-8)
Glucose Infusion Rate

50
(mg/kg/min)

#
← 40 # →

30 

20

10

0
SD (age) SD (wt) ZDSD ZF
Rat Strain (SD rats are age or weight matched)

* P<0.05 compared to SD rat (age matched) group PDF Return to Index, For

30

ZDSD Rats Have Modest Visceral and
Whole Body Obesity Unlike ZF Rats
12 # 40  #
Vehicle (n=6-8)
Epididymal Fat Pad (g)

Rosiglitazone (3 mg/kg PO, n=6-8)  Vehicle (n=6-8)
10
30

Body Fat (%)
8

← 6 20 →
4   
10
2

0 0
SD (age) SD (wt) ZDSD ZF SD (age) SD (wt) ZDSD ZF
Rat Strain (SD rats are age or weight matched) Rat Strain (SD rats are age or weight matched)

* P<0.05 compared to SD rat (age matched) group


31

Dyslipidemia

Patients with Type II diabetes and metabolic syndrome often present with
dyslipidemia including elevated cholesterol and triglycerides and decreased HDL-
← C. These lipids have been shown to impact cardiovascular and renal co- →
morbidities. Hypertriglyceridemia expresses as early as 12 weeks of age in ZDSD
rats when maintained on a normal diet and levels progress up to 500 mg/dL by 15
weeks. Similar to the fructose fed rat, a model commonly used for the study of
dyslipidemia, the spontaneous nature of the ZDSD lipid abnormality may provide a
relevant model for the examination of compounds affecting the up-regulated
lipogenic pathway seen in metabolic syndrome. The dyslipidemia in this model
responds to classic reference agents including rosiglitazone. Increases in
cholesterol are not as dramatic and may be induced by feeding a high fat diet


32

Terminal Comparison of Models
1300
1200
1100
1000 ZDF
CRL-SD, CD
700 +/fa
900 ZDSD, Diabetic 7-11 weeks
(mg/dL) ZDSD, Diabetic 12-21 weeks

800
600
← 700 →
Analytes

500
600
Weight (g)

500
400
400
300
300
200
100
200
0
100
TG
e

L
s

O
co

H
C
lu
G

Glucose, triglyceride and cholesterol levels. Glucose, triglyceride and cholesterol
levels are elevated in all of the diabetic groups (ZDF and ZDSD). The model and Web Use Refresh Above
duration of diabetes did not have a significant effect on these measurements.
33

Dyslipidemia in ZDSD
Treatment ofEffect of 7 Days of Niacin Treatment with Niacin
Weight Glucose
Vehicle 250 Vehicle
600
Niacin Niacin
200

Glucose (mg/dL)
Weight (grams)

400 150

100 *
200
50

← 0 0
→
Fed Fasted Fed Fasted Fed Fasted Fed Fasted
Before Treatment After Treatment Before Treatment After Treatment

Triglyceride Free Fatty Acids
600 Vehicle 1.5 Vehicle
Niacin Niacin
Triglyceride (mg/dL)

FFA (mEq/L)
400 1.0

*
200 0.5
* * *
0 0.0
Fed Fasted Fed Fasted Fed Fasted Fed Fasted
Before Treatment After Treatment Before Treatment After Treatment PDF Return to Index, For

7 days of treatment 34

Hypertension

← →
High blood pressure is a key symptom of metabolic syndrome and is a major
contributor to the increased risk of cardiovascular disease, kidney disease and
ischemic stroke seen in these patients. Examination of the interactions of all the
components of the syndrome in rats is complicated by the absence of high blood
pressure in current models (i.e., Zucker fatty rat). Indirect evidence of probable
elevated pressure in the form of elevated biomarkers for an activated RAAS
,endothelial dysfunction and aberrant vasoconstriction is noted in ZDSD rats.
Direct evidence of Hypertension has been confirmed in the pre-diabetic state via
the tail-cuff method.


35

Blood pressure in ZDSD vs CD Rats
8-16 weeks of age
160
ZDSD
CD
140
Systolic BP

← →
120

100

80
60 70 80 90 100
Age in Days
Blood pressure data produced in collaboration with
Dr. Subah Packer’s Laboratory, IU School of Medicine PDF Return to Index, For

36

30
ANOVA/pooled t

Leptin Physiology
dark cycle (p<0.05) compared
light cycle to SD animals
25 daily total

Food intake (g/period)
Food intake of ZDSD Rats is 20

more evenly divided between 15

day and night
10

← 5
→
0
SD ZDSD

10
ANOVA/pooled t (p<0.05) compared

Food Intake of ZDSD Rats is
9 to corresponding saline control

Food intake 4 hrs after treatment
8

Reduced in Response to 7

Leptin Indicating a 6

Functioning Leptin Pathway. 5

Leptin was given just before 4

3
the start of the dark cycle and 2
food intake was measured for 1

the first 4 dark hours. 0
SD-saline SD-leptin ZDSD-saline ZDSD-leptin
Assessment of leptin pathway function as determined by Web Use Refresh Above
feeding response to leptin injection (1 mg/kg, IP)
37

Insulin Levels Decline as Diabetes Progresses

Glucose Insulin
8

600 7

11-13 WEEKS

← 500 6
15 WEEKS
17 WEEKS →

Insulin (ng/ml)
5
Glucose (mg/dl)

400

4
300
11-17 WEEKS
3

200
2

100 1

0 0

5 7 9 11 13 15 17 19 21 23 25 27 29 5 7 9 11 13 15 17 19 21 23 25 27 29
Age (wks) Age (wks)

Insulin levels of the group that become diabetic between 11-17 weeks of age. The animals that
become diabetic earlier have higher insulin levels than those who become diabetic later.


38

Average Glucose
Pancreatic Insulin Content % Diabetic
500

The glucose levels for ZDSD rats 400

Glucose (mg/dL)
were followed from 16 to 28 300

weeks of age (upper figure). At 200
28 weeks of age approximately 100 3.6% 13.1% 18.0% 32.8% 52.5% 62.3% 70.5% 75.8%

75% of the animals were overtly
← diabetic. The average glucose
0
15 20 25 30 →
levels for each animal (16 to 28 Age (weeks)

weeks) were correlated with
Correlation Between
insulin content of the pancreas Pancreatic Insulin and
when the animals were Blood Glucose Level
terminated at about 28 weeks of 600

age (lower figure). Higher
Glucose (mg/dL)

400
average glucose levels were
associated with lower insulin
200
content in the pancreas.
0 PDF Return to Index, For
0 200 400 600 800
Insulin ng/g 39

Representative Islets from ZDSD Rats
Pre-diabetic Diabetic

← →


40

Renal Injury

Obesity and metabolic syndrome are clear predictors
← of chronic kidney disease largely due to the →
potentiation of chronic inflammation by insulin
resistance. In addition, the lipoprotein abnormalities,
increased hemodynamics, hypercoagulability and
vascular dysfunction associated with metabolic
syndrome have all been implicated as causative for
renal disease. Biomarkers for renal dysfunction (i.e.,
IL6, TNF-α,NGAL,KIM-1, VEGF etc.) as well as significant
albuminuria , elevated free fatty acids with oxidative
stress, and histological analysis have shown the ZDSD
rat to exhibit nephropathy that closely mimics that
observed in obese insulin resistant patients.

41

Diabetic Nephropathy
in the ZDSD Rat
• Increased kidney weight
• Increased urinary markers for kidney disease
← →
• Increased serum markers for kidney disease
• Glomerular sclerosis
• Nodular sclerosis, KW nodules
• Thickening basement membrane of
glomerular capillaries
• Podocyte effacement on capillaries PDF Return to Index, For

42

Terminal Comparison
Kidney Weight Urine Analysis
300
7
ZDF
CRL-SD, CD
+/fa 250
6 ZDSD, Diabetic 7-11 weeks
ZDF
CRL-SD, CD
+/fa
5 200 ZDSD, Diabetic 7-11 weeks

← →
Weight (gram)

4 150

3 100

2
50

1
0

0

Terminal kidney weights are highest in the ZDSD Rat groups. These increased kidney weights and high
urinary volume along with increased micro-albumin concentration and the total amount of micro-
albumin indicate that there may be significant diabetic nephropathy in the ZDSD Rat model.

43

Experiment 1
← ZDSD Diabetic Nephropathy →
Spontaneous Diabetes
ELISA Analysis of Markers


44

WEIGHT GLUCOSE
600 600
SD SD
ZDSD ZDSD
Weight (grams)

Glucose (mg/dL)
500 400

400 200

← 300
10 12 14 16 18 20 22 24 26 28 30
0
10 12 14 16 18 20 22 24 26 28 30
→
Age (weeks) Age (weeks)

Urine volume
200
Urinary volume (mls/24hr)

SD
ZDSD
150

100

50

0
10 20 22 24 26 30
Age (weeks)

45

Urinary albumin beta-2 microglobulin

Urinary  -2 microglobulin (g/day)
150 2000
Urinary albumin (mg/day)

SD SD
125 ZDSD ZDSD
1500
100

75 1000

50
500
25

← 0
10 20 22 24 26 30
0
10 20 22 24 26 30 →

Cystatin C KIM-1
30 15.0
Urinary cystatin C ( g/day)

SD SD

Urinary KIM-1 (ng/day)
ZDSD 12.5 ZDSD
20 10.0

7.5

10 5.0

2.5

0 0.0
10 20 22 24 26 30 10 20 22 24 26 30

46

Experiment 2
Urine BioMarkers of Renal Disease
Study Details
← • Male ZDSD Rats were allowed to become diabetic →
spontaineously on Purina 5008 and aged to 33 weeks.
Two groups of animals were selected for further study: animals that
were diabetic for longer than 16 weeks and animals that were
diabetic for less than 8 weeks.
• Mesoscale (MSD) urine panels were run on urine (Argutus
AKI test, Kidney Injury Panel 1 and Rat Clusterin)

• Pathological evaluation of the kidneys was done.

47

Data From Urinary Excretion Study

← →


48

Urinary Excretion of Kidney Markers

← →


49

Urinary Excretion of Kidney Markers

← →


50

Pathological Evaluation of Kidney
• Glomerulopathy: Changes in the renal glomeruli consisted of one or more of the following: increased
cellularity in the mesangium; increased in mesangial connective tissue; thickening of Bowman’s capsule;
hypertrophy of capsular epithelium; dilation of the capsular space. Individual glomeruli appeared
moderately enlarged. The lesions were highly variable within individual glomeruli and between glomeruli
within a kidney. The changes were most usually segmental, although a rare glomeruli was fibrotic
(condensed). Expanded mesangial material stained positively with the PAS stain and to a lesser extent
with the Trichrome stain.

←
• Tubular dilation/degeneration: This change was mainly in the cortex and consisted of irregularly dilated, →
empty tubules, that sometimes were lined by cuboidal epithelium that stained basophilic compared to the
expected normal eosinophilic tubular epithelium. In some individual tubules the epithelium were
flattened. These dilated/degenerate tubules were randomly scattered throughout the cortex, and
sometimes were associated with protein casts and/or non-suppurative inflammation (see below). Focal
mild increases in fibrous connective tissue within the interstitial space was present, frequently in
association with the interstitial inflammatory response, but not restrictively so.

• Protein casts: Individual tubules contained acellular, uniformly staining eosinophilic material consistent
with protein. These protein casts were present in the cortex and in the medulla, as well as at the cortico-
medullary junction in various sections. Often, several such dilated tubules containing protein casts were
clustered together, usually in the cortex.

• Inflammation: The inflammatory process consisted of focal collections of lymphocytes and
macrophages, which were seen in the cortical interstitial space, adjacent to individual glomeruli PDF Return to Index, For
and individual blood vessels, and in association with the renal pelvic epithelium.
51

← →


52

← →


53

Kidney Histopathology of the ZDSD Rat
4.0
A Novel Animal Model of Diabetes
non-diabetic
compared to Non-diabetic animals (t-test)
diabetic
3.5
Histopathology Score (0-5)

3.0

← 2.5 →

2.0

1.5

1.0

0.5

0.0
Glomerulopathy Tubular dilation Protein casts Inflammation PDF Return to Index, For
/degeneration
54

Experiment 3
← →
Serum BioMarkers of Renal Disease
RBM Collaboration


55
Rules Based Medicine

Renal Injury

Obesity and metabolic syndrome are clear predictors of
chronic kidney disease largely due to the potentiation of
chronic inflammation by insulin resistance. In addition, the
← →
lipoprotein abnormalities, increased hemodynamics,
hypercoagulability and vascular dysfunction associated with
metabolic syndrome have all been implicated as causative
for renal disease. Biomarkers for renal dysfunction (i.e., IL6,
TNF-α, NGAL, KIM-1, VEGF etc.) as well as significant
albuminuria , elevated free fatty acids with oxidative stress,
and histological analysis have shown the ZDSD rat to exhibit
nephropathy that closely mimics that observed in obese
insulin resistant patients. PDF Return to Index, For

56

Experimental details
• Male ZDSD rats were placed on a high-fat diet
(RD12468) between 17 and 19 weeks of age. 15 out
← of 21 animals in this experiment developed diabetes →
during this period (this is usually 90%+).
• Rules Based Medicine panels (Rat Metabolic MAP,
Rat Kidney MAP and RodentMAP™) were run on
serum samples that were collected:
– before diabetes developed (14 weeks)
– while diabetic on the high fat diet (18 weeks) and
– one week after they were taken off the high fat diet PDF Return to Index, For

(20 weeks). Web Use Refresh Above

57


Neutrophil Gelatinous Associated Protein
•also called lipocalin2
compared to SD
•levels up-regulated during 1400 Sprague-Dawley (5)
inflammation ZDSD (6) 
Diabetic ZDSD (15)
•protective protein can trigger 1200
nephrogenesis

Serum NGAL (ng/ml)
← •associated with obesity, insulin 1000 →
resistance and hyperglycemia
800


600


400

200
14 18 20

Age (weeks)

14 weeks = non-diabetic
18 weeks = on diabetogenic diet one week
20 weeks = off diabetogenic diet one week

58


Beta-2-microglobulin
80
•a protein present in all nucleated cells
Sprague-Dawley (5)
which is normally reabsorbed in renal ZDSD (6) 

Serum beta-2-microglobulin (ug/ml)
tubules 75 Diabetic ZDSD (15) 

•increased circulating levels indicate
glomerular membrane disease and 70

← inflammation

 →
65

#
60

55 
# Diabetic vs. non-diabetic
50
14 18 20

Age (weeks)


59


Kidney-injury molecule-1
0.30
•a membrane protein, not normally compared to SD
Sprague-Dawley (5)
# diabetic vs. non-diabetic
present but appears in urine in ZDSD (6)
0.25 Diabetic ZDSD (15) 
response to acute kidney tubular
injury 

Serum Kim-1 (ng/ml)
0.20
•highly sensitive predictor of renal #

← injury when elevated in urine
0.15
→
0.10

0.05

0.00
14 18 20

Age (weeks)


60


Glutathione-S-transferase-alpha 60
•enzyme that reduces toxin levels by Sprague-Dawley (5)
ZDSD (6)
50
conjugation with glutathione Diabetic ZDSD (15)


Serum GST-alpha (ng/ml)
•localized in proximal convoluted 40
tubules, medullary tubules and loop of
Henle 30

← •in diabetes, hyperglycemia triggers  →
oxidative stress which increases the 20 #

renal excretion of this enzyme and

therefore removes this protective 10

function and increases blood levels.
0
•Biomarker for tubular kidney disease
14 18 20

Age (weeks)



61


Clusterin
500
•(apolipoprotein J) is a protein highly Sprague-Dawley (5) comapred to SD
correlated with apoptosis and the ZDSD (6) # Diabetic vs. non-diabetic
clearance of cellular debri Diabetic ZDSD (15)
400
•It is elevated in glomeruli and tubules

Serum Clusterin (g/ml)
of diabetic kidneys
← 300
→

200

#

100
14 18 20

Age (weeks)


62


von Willebrand factor 300
•required for platelet adhesion, makes 

Serum von Willebrand Factor (ng/ml)
platelets "sticky“ 250 #

•vWF binds inactive Factor VIII, 
#
protecting it from degradation 200
•defiency leads to bleeding disorders 

•increased levels predispose to stroke 
← •increases precede microalbuminuria
150 →
in diabetic nephropathy
100

Sprague-Dawley (5)
50 ZDSD (6)
Diabetic ZDSD (15)

0
14 18 20

Age (weeks)


63

Experiment 4
← →
Diabetic Nephropathy,
EM of Glomerular Pathology


64

Experimental details
• Male ZDSD rats allowed to become spontaneously
diabetic.
← • Animals were terminated by perfusion at about 35 →
weeks of age. We evaluated the following groups:
– Control CD rats
– ZDSD rats that had been diabetic from 12-13 weeks
– ZDSD rats that had been diabetic from 16-17 weeks
• Took pictures of glomerular capillaries and BM
– Measured GBM thickness
– Evaluated podocyte morphology PDF Return to Index, For

65

Glomerular Capillary
Control, Age Matched Diabetic, 12 Weeks

← →

66

66

Glomerular Capillary, Basement Membrane

← →


67


← →


68

Glomerular Capillary
Control, Age Matched Diabetic, 16.5 Weeks

← →

69

69

Control, Age Matched Diabetic, 16.5 Weeks

← →


70

Glomerular Basement
Membrane Thickness
500

Thickness in nm 400
← →
300

200

100

0
ks

ks
ol
tr

ee

ee
on

W

W
C

12

.5
D

16
C

Time of diabetes in the ZDSD Rat 09-550-187


71

Scanning Microscopy
Glomerular Capillaries
Control Diabetic

← →


72

Scanning Microscopy
Control Glomerular Capillary with Normal Podocyte Foot Processes

← →


73

Scanning Microscopy
Diabetic Glomerular Capillaries Demonstrating Effacement

← →


74

ZDSD Overview a model of obesity, metabolic syndrome and diabetes, 24 May-2011

ZDSD Overview a model of obesity, metabolic syndrome and diabetes, 24 May-2011

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ZDSD Overview a model of obesity, metabolic syndrome and diabetes, 24 May-2011