Diabetic Nephropathy in the ZDSD Rat

PreClinOmics, Inc.Contact Richard G. Peterson for more information.

rpeterson@preclinomics.com317-872-6001 x 13

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.

Renal Injury

2

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

3

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.

Terminal Comparison

Kidney Weight Urine Analysis

0

1

2

3

4

5

6

7CRL-SD, CD+/fa

ZDF

ZDSD, Diabetic 12-21 weeks

ZDSD, Diabetic 7-11 weeks

We

igh

t (g

)

4

0

50

100

150

200

250

300

CRL-SD, CD

+/fa

ZDF

ZDSD, Diabetic 12-21 weeks

ZDSD, Diabetic 7-11 weeks

Experiment 1

ZDSD Diabetic Nephropathy

Spontaneous Diabetes

ELISA Analysis of Markers

5

6

Weight

10 12 14 16 18 20 22 24 26 28 30300

400

500

600

SD

ZDSD

Age (weeks)

We

igh

t (g

)Glucose

10 12 14 16 18 20 22 24 26 28 300

200

400

600SD

ZDSD

Age (weeks)

Glu

co

se

(m

g/d

L)

Urine Volume

10 20 22 24 26 300

50

100

150

200SD

ZDSD

Age (weeks)

Uri

na

ry V

olu

me

(m

l/2

4h

r)

7

Urinary albumin

10 20 22 24 26 300

25

50

75

100

125

150SD

ZDSD

Age (weeks)

Uri

nary

alb

um

in (

mg

/day)

beta-2 microglobulin

10 20 22 24 26 300

500

1000

1500

2000SD

ZDSD

Age (weeks)

Uri

naryb

-2 m

icro

glo

bu

lin

( mg

/da

y)

Cystatin C

10 20 22 24 26 300

10

20

30SD

ZDSD

Age (weeks)

Uri

nary

cys

tati

nC

( mg

/da

y)

KIM-1

10 20 22 24 26 300.0

2.5

5.0

7.5

10.0

12.5

15.0SD

ZDSD

Age (weeks)

Uri

nary

KIM

-1 (

ng

/day)

Experiment 2Urine 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.

8

Data From Urinary Excretion Study

9

Urinary Excretion of Kidney Markers

10

Urinary Excretion of Kidney Markers

11

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

and individual blood vessels, and in association with the renal pelvic epithelium.

12

13

Kidney Histopathology of the ZDSD Rat

A Novel Animal Model of Diabetes

14

Glomerulopathy Tubular dilation Protein casts Inflammation

His

topath

olo

gy S

core

(0-5

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0Non-diabetic

Diabetic

* * **

* compared to Non-diabetic animals (t-test)

/degeneration

Experiment 3

Diabetic Nephropathy,

EM of Glomerular Pathology

15

Experimental details

• Male ZDSD rats allowed to become spontaneously

diabetic.

• Animals were terminated and perfused fixed at about

35 weeks of age.

– 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

16

Glomerular Capillary, Basement Membrane

Control, Age Matched Diabetic, 12 Weeks

17

Glomerular Capillary, Basement Membrane

Control, Age Matched Diabetic, 16.5 Weeks

18

19

Glomerular Basement Membrane Thickness

Time of Diabetes in the ZDSD Rat

Th

ickn

ess

(mm

)

CD Control 12 Weeks 16.5 Weeks

0

100

200

300

400

500

20

Scanning Microscopy

Control Glomerular Capillary with Normal Podocyte Foot Processes

21

Scanning Microscopy

Diabetic Glomerular Capillaries Demonstrating Effacement

Experiment 4

Diabetic Nephropathy,

Synchronized Diabetes:

Clinical Data and

LM of Glomerular Pathology

22

Experimental details

• Male ZDSD rats synchronized to become diabetic

by feeding them Purina 5SCA.

• Animals were put on 5SCA at 19 weeks of age

and were diabetic by 20 weeks of age. They were

monitored until they were 47 weeks old. We

evaluated the following groups:

– ZDSD rats that had been diabetic for 27 weeks

(14)

– ZDSD rats that failed to become diabetic (4)

• Graphed terminal data and evaluated pictures of

glomeruli and other kidney pathology

23

24

Body weight

0 7

14

21

28

35

42

70

10

5

12

6

15

5

17

2

18

5

19

6

350

400

450

500

550

600 Diabetic

Non-diabetic

Day of study

Bo

dy w

eig

ht

(g)

Glucose

0 7

14

21

28

35

42

70

10

5

12

6

15

5

17

2

0

200

400

600

800Diabetic

Non-diabetic

Day of study

Glu

co

se (

mg

/dL

)

Diabetic

Non-diabetic

43 47

0

5

10

15* t-test

**

Age (weeks)

Hb

A1c (

%)

HbA1c

43

0.0

0.2

0.4

0.6

0.8

1.0Diabetic

Non-diabetic

Age (weeks)

NE

FA

(m

Eq

/L)

NEFA

25

Diabetic Non-diabetic0

5

10

15

20

25 Diabetic

Non-diabetic

*

* t-testL

iver

weig

ht

(g)

47 w

eeks o

f ag

e

Liver Weight

Diabetic Non-diabetic0

2

4

6Diabetic

Non-diabetic

Kid

ney w

eig

ht

(g)

47 w

eeks o

f ag

e

* t-test

*

Kidney Weight

43 470

100

200

300Diabetic

Non-diabetic

Age (weeks)

Uri

ne v

olu

me (

mls

/24 h

r)

Urine Volume

430

20

40

60

80Diabetic

Non-diabetic

*

* t-test

Age (weeks)

Uri

ne a

lbu

min

(m

g/d

ay)

Urinary Albumin

Blood Chemistry

26

43 45 470

10

20

30Diabetic

Non-diabetic

* t-test

***

Age (weeks)

Seru

m B

UN

(m

g/d

L)

BUN

45 470.0

0.1

0.2

0.3

0.4

0.5Diabetic

Non-diabetic

Age (weeks)

Seru

m c

reati

nin

e(m

g/d

L)

Creatinine

43 470

50

100

150

200Diabetic

Non-diabetic* t-test

**

Age (weeks)

Seru

m c

ho

leste

rol (m

g/d

L)

Cholesterol

43 470

500

1000

1500Diabetic

Non-diabetic* t-test

**

Age (weeks)

Seru

m t

rig

lyceri

des (

mg

/dL

)

Triglyceride

47 Week-old, 27 Weeks Diabetes

Non-Diabetic Diabetic

Diabetic Diabetic27

28

47 Week-old, 27 Weeks Diabetes

28

Non-Diabetic Diabetic

47 Week-old, 27 Weeks Diabetes

29

Non-Diabetic Diabetic

47 Week-old, 27 Weeks Diabetes

30

Follow-up study, Synchronized

• Diabetes was induced by putting ZDSD rats on

5SCA diet when they were 21 weeks of age.

This can be done anytime after 16 weeks of age.

• Weight and glucose were followed periodically.

• 24 hour urine was collected and urinary albumin

and creatinine were measured so that total 24

hour albumin and albumin/creatinine ratios could

be determined.

• Terminal data were collected when the rats had

been diabetic 13-14 weeks.

31

32

Progression of Diabetes and Urinary Albumin in ZDSD Rats

Diabetes was synchronized with 5SCA diet (3 weeks) when ZDSD rats were about 20 weeks old.

Glucose rose rapidly while animals were on 5SCA diet and they remained hyperglycemic when they

were taken off diet; overtly diabetic animals lost weigh. Urinary volume increased steadily while the

urinary albumin levels had a rapid increase between 11 and 15 weeks after 5SCA was started.

Glu

co

se (

mg

/dL

)

0 4 8 12 160

200

400

600

Weig

ht

(g)

0 4 8 12 160

200

400

600

Weeks After 5SCA

Uri

nary

vo

lum

e m

L

0 4 8 12 160

100

200

300

Weeks After 5SCA

Uri

nary

alb

um

in (

mg

/day)

0 4 8 12 160

100

200

300

400

Glomerular Proteomics

• Nondiabetic, prediabetic and diabetic

glomeruli were collected and analyzed for

protein expression.

• Data were analyzed using volcano plotting

to show the differences in expression of

proteins in the three groups.

• The different groups were compared in the

next three slides (33-35).

• Slide 36 demonstrates down-regulation of

glyoxalase 1 with diabetes.

33

Protein Expression in the ZDSD

Glomerulus

34

Protein Expression in the ZDSD

Glomerulus

35

Protein Expression in the ZDSD

Glomerulus

36

Glyoxalase 1 (GLO-1) down regulated in Diabetic Nephropathy

observed in the ZDSD Rat Glomerulus

37

Protocol-Lisinopril treatment of diabetic

nephropathy in ZDSD rats• 18 week-old ZDSD rats were fed a diabetogenic diet (Purina 5SCA)

for 2 weeks. Following this induction phase, ZDSD rats were maintained on regular rodent chow (Purina 5008) for the remainder of study.

• The diabetic ZDSDs were sorted into matched untreated (N=12) and treated groups (N=13).

• Treatments were administered by diet admixture; 5008 or 5008 with Lisinopril, 250 ppm. Based on feed intake and average body weight, Lisinopril was delivered at approximately 29 mg/kg/day for 4 weeks.

• Blood and urine data were collected before and after 4 weeks of treatment.

• Treatment was started at 5-13 weeks after ZDSD rats became diabetic.

• Glucose, HbA1c, creatinine and BUN were measured in blood; albumin, and creatinine were measured in urine.

• Differences were determined using Student’s t-tests. Significance was determined with p-values <0.05, data was graphed as Mean ±SEM.

38

Data following 4 weeks of treatment. There were no differences in weight or glucose at baseline. Weight loss is a consequence of overt diabetes in the ZDSD rat. Asterisks* represent significant effects of Lisinopril compared to vehicle.

Weight and glucose levels after treatment,The effect of Lisinopril on nephropathy in the ZDSD rat

-15

-10

-5

0

Bo

dy w

eig

ht

(% c

ha

ng

e f

rom

base

lin

e)

Vehicle

Lisinopril *

0

200

400

600

800

Se

rum

glu

co

se(m

g/d

L)

4 w

eek

s o

f tr

eatm

en

t

Vehicle

Lisinopril

*

49

Data following 4 weeks of treatment. There were no differences in creatinine or BUN at baseline. Asterisks* represent significant effects of Lisinopril compared to vehicle.

0.0

0.2

0.4

0.6

Seru

m C

rea

tin

ine (

mg

/dL

)

Vehicle

Lisinopril*

0

10

20

30

40

50

Se

rum

BU

N (

mg

/dL

) Vehicle

Lisinopril *

Creatinine and BUN levels after treatment,The effect of Lisinopril on nephropathy in the ZDSD rat

40

Data following 4 weeks of treatment. There was no difference in albumin excretion at baseline. Asterisks* represent significant effects of Lisinopril compared to vehicle.

0

25

50

75

100

125

150

175

200

Uri

nary

alb

um

in

( m

g/d

ay)

Vehicle

Lisinopril

* -100

0

100

200

300

400

500

Uri

nary

alb

um

in

(% c

ha

ng

e f

rom

base

lin

e)

Vehicle

Lisinopril

*

Absolute and % change of urinary albumin levels after treatment,

The effect of Lisinopril on nephropathy in the ZDSD rat

41

Data following 4 weeks of treatment. The change in urinary albumin was greater in vehicle animals when they had a shorter duration of diabetes since they had lower urinary albumin levels before treatment started. Asterisks* represent significant effects of Lisinopril compared to vehicle.

Percent change of urinary albumin level is dependent on duration of diabetes,The effect of Lisinopril on nephropathy in the ZDSD rat

13 9 5-200

0

200

400

600

800

% Change in UA

Weeks of Diabetes Before Treatment

Uri

nary

Alb

um

in

(% c

ha

ng

e f

rom

base

lin

e)

Vehicle

Lisinopril

N=3 3 4 5 5 5

* * *

42

Summary-The effect of Lisinopril on nephropathy in the

ZDSD rat

• ZDSD weight decreased in both untreated and treated diabetic groups with greater decreases in the treated group.

• Blood levels of glucose, creatinine and BUN increased with Lisinopril treatment.

• Urinary albumin decreased significantly (~87%) in the Lisinopril treated group while it increased significantly (~400%) in the untreated diabetic ZDSD rats.

• Irrespective of the time the ZDSD rats had diabetes and their initial level of urinary albumin, the age subgroups of animals had 82-91% decreases in the amount of urinary albumin excreted after 4 weeks of Lisinopril treatment.

43

Summary: Diabetic Nephropathy (DN) in the ZDSD Rat.

The following are supported by data in this slide set!

• Diabetic nephropathy (DN) develops with spontaneous

or synchronized diabetes.

• Increased urinary albumin and other urinary biomarkers

increase with duration of diabetes.

• Increased BUN and blood creatinine levels.

• LM and EM morphological changes consistent with DN.

• Glomerular BM thickening and podocyte effacement.

• Changes in glomerular protein expression similar to

human DN.

• Urinary albumin secretion significantly improved with

ACE inhibitor (Lisinopril) treatment, similar to human DN.

• ACE-I treatment effective at all stages of DN examined.44