mechanisms of hypertension and diabetic...

Mechanisms of hypertension and diabetic nephropathy

Richard J Roman Ph DRichard J. Roman, Ph.D.

Department of Pharmacology

The economic burden of kidney disease

• Hypertension and Diabetes are the leading cause of end stage renal disease. 66% of new cases.  

• Number of patients with chronic renal disease is exploding currently estimated at 30 million or 13% of the population. 

• Currently, approximately 500,000 Americans have been diagnosed with kidney failure. The number of Americans with this advanced stage of the disease is expected to grow to 785,000 by 2020. p g y

• The annual cost of treating kidney disease is $42 billion, 25% of Medicare spending. 

• There is a critical need for development of new therapies to slow or reverse the progression of chronic kidney disease. 

http://www.asn‐online.org/facts_and_statistics/kd‐health‐threat

Impaired autoregulation and 20Impaired autoregulation and 20--HETE, MMP2,TGFHETE, MMP2,TGF--ββ InteractionInteractionin the Pathogenesis of Renal Diseasein the Pathogenesis of Renal Disease

Hypertension Diabetes

20-HETE SS

Autoregulation Myogenic tone FHH-

TGF

P

TGF

gc

TGF MMP2 TGF

Palb

TGF -glomerulus

Myoepithelialtransformation

TGF --

Palb

TGF -glomerulus

Myoepithelialtransformation

TGF - Palb

TGF -glomerulusMMP2 , TGF

glomerulusMyoepithelial

transformation TGF -

Myoepithelialtransformation

TGF --

Proteinuria GlomerulosclerosisInterstitial

Fibrosis Tubulonecrosis

Proteinuria GlomerulosclerosisInterstitial

Fibrosis Tubulonecrosis

ProteinuriaProteinuria GlomerulosclerosisGlomerulosclerosisInterstitial

Fibrosis Tubulonecrosis

Interstitial Fibrosis

Tubulonecrosis

RENAL FAILURERENAL FAILURERENAL FAILURE

TGF Responses in Dahl S and R RatsTGF Responses in Dahl S and R Rats

50

55 H

g50

55 Dahl R (n=13, 5 rats)Dahl S (n=15, 5 rats) H

g

45

ure

mm

45

ure

mm

35

40

Pres

su

35

40

Pres

su

25

30

op F

low

25

30

op F

low

20

25

Sto

0 10 20 30 40 5020

25

Sto

0 10 20 30 40 50Perfusion Rate (nl/min)

0 10 20 30 40 50Perfusion Rate (nl/min)

0 10 20 30 40 50

Efferent arteriole

Macula Densa

Efferent arterioleAfferent ArtrioleDistal Tubule

Afferent arterioleDistal tubule

GlumerulusMacula Densa

Effect of 20‐HETE on TGF

18

21

riole 

m) 15

18

ent Arte

iam

eter

(um

12

of Affere

(µm)

Di

9

ameter o

20‐HETE in tubule (10 µm)

NaClNaCl

NaClETE

0

Dia 20‐HETE in tubule (10 µm)

10mMNa

80mMNa

10mMNa

80mMNaCl+20HET

Control 

60mmHG  120mmHG

Effect of 20‐HETE on Myogenic Response

16

e 

12

14

Arteriole

amet

er(u

m)

10

12

ferent A

Dia

8

20-HETEter of Af

0

6

60 120 60 120 60 120

20 HETE

HET-0016

Diamet

(µm)

Pressure(mmHg)

60 120 60 120 60 120

Pgc during the During the Development of Hypertension in Dahl S rats yp

55

g)

50SS4A+ * *

(mm

Hg

45 *

†

Pgc

40†

†

30

35

30LS HS HS+HET0016

MMP‐2 and TGF‐β1 levels in Dahl S rats fed i h l l hi h l dieither a low salt or high salt diet

mg)

1600

2000*

/mg)

16

20

*

vels

(pg/

1200

vels

(pg/

12

MM

P-2

lev

400

800

GF-1

lev

4

8

M

0LS(3)

HS(3)

TG0

LS(3)

HS(3)(3) (3) (3) (3)

Hypertension

XL784 reversibility study in Dahl S rats

Vehicle (12)Lisinopril + Losartan (12)XL784 (12)

g) 180

220

g/da

y)

250

300

XL784 (12)Lisinopril + Losartan + XL784 (12)

MA

P (m

mH

g

140

180

**

cret

ion

(m

100

150

200

**

100Vehicle XL784 Lisinopril+

Losartan+XL784

(12)(12)

(12)Lisinopril+Losartan

Prot

ein

exc

0

50

100 ***** **

(12)(12)

Weeks on High Salt Diet

P

Control 5 6 7 8 9 10

Vehicle Lisinopril

XL784XL784

Effect of Hypertension on the Expression yp pof TGF-β in Dahl S Rats

Effect of TGF-β Ab on Proteinuria and Glomerular Injury in Dahl S Rats fed a High Salt Diet for 3

Weeks. Weeks.

100 *Females (n=10)Males ( 10)

80

g/da

y)* *

(n=10)

60

retio

n (m

g

* †

40

umin

Excr

* †

20

Mic

roal

bu

Low Salt High Salt TGF -b Ab0

Figure 1 B

6% BSA 4% BSA 4% BSA4% BSA 4% BSA

H20H20

6% BSA

FITC-Dx250

4% BSA

FITC-Dx250

6% BSA

FITC-Dx250

H20 H20

FITC‐Dextran 250 kD

Labeling and Isolation of Glomeruli and Imaging

SD glomerulus albumin and dextran labeled

Live Image of glomeruli

Figure 3A 

110

100

110

cenc

e

80

90

Fluo

resc

70

80

% F

50

60 6-5% (12, 59)6-4% (16 108)

0 10 20 30 40 50 6040

50 6-4% (16, 108) 6-3% (17, 98) 6-2% (26, 100)

Time(seconds)0 10 20 30 40 50 60

Figure 5B

110nc

e100

uore

sce

80

90

% F

lu

70

80

60

70 Vehicle ( 6, 37 )TGF- ng /ml(6, 44)

Time (seconds)0 10 20 30 40 50

60

Hypertensive model

A B

ce

100

110FHH (8,82)SD (12,206)

* * * * * * * e

100

110 SD (5,41)Dahl s low salt (5,43) Dahl s high salt (5,64)

***

% F

luor

esce

nc

80

90* * * * * * * * *

Fluo

resc

ence

80

90*

* * * *

%

60

70

% F

60

70Expected Δ Expected Δ

Time (seconds)0 20 40 60 80 100 120 140 160

Time (seconds)0 20 40 60 80

60

Effect of Hypertension on Glomerular Capillariesin Dahl S Rats

A B

High saltControl

C

High salt + anti TGFb therapy

2 photon verificationof TGF b effectof TGF b effect

Fitc dextranLow mw dextranAlbumin blue

Dahl S high salt 20X

Evidence Linking P4504A to salt sensitive Hypertension in Dahl S Rats

• The formation of 20‐HETE is decreased in the kidney in Dahl S rats compared to other normotensive and hypertensive strains of ratsother normotensive and hypertensive strains of rats.

• 20‐HETE production in OM is reduced in Dahl S rats which contributes to elevated l Cl b ti i TALH 20 HETE d ti i d d i l lloop Cl‐ reabsorption in TALH.   20‐HETE production is decreased in glomerulus but vascular production does not seem to lower than other strains. 

• Chronic treatment with Fibrates increases the renal formation of 20‐HETE, improves pressure natriuresis, and reduces blood pressure in Dahl S rats.

• The CYP4A region on chromosome 5 cosegregates with hypertension in a Dahl S X Lewis F2 cross.

• CYP4A genes are located on rat chromosome 5. Do they contribute to Hypertension in Dahl S rats?

Consomic rats are single chromosome substitutions

MCW: NHLBI program forGenomic applications - PGA

SSBN1

SSBN4

SSBN8SSBN8

SSBN13SS diseased

SSBN16BN normal

SSBN20

Note: the strain sequenced—is this BN

Blood pressure targets captured in the SS.BN Consomic strains

Mattson et al. AJP Renal Physiol 295: F837‐F842, 2008.

Telemetry Confirmation of renoprotective phenotype in SS.5BN rats

Days on High Salt Diet‡ P<0.05 vs. other strains

CYP4A expression in the kidney of SS and SS.5BN rats

Chronic administration of HET0016 reverses the phenotype of SS.5BN rats

**

*

14 Days on High Salt Diet

Sequencing of CYP4A3 cDNA

2140bp

13 1536CDS

430 503 958 1111/2

TAAATCT….ACTTCATTACCATG

E1 E8E5E2 E3 E4 E6 E7 E9 E10 E11

C430T A503G(Arg-Trp) (Asn –Ser) (Leu – Leu)

A1111G

Ref SeqNM 175760 SD ccagcaatc… gtcaatataat ………… .attcatgttt… gataccttacacc

SS1 ccagcaatc… gtcagtataat ………… .attcatgttt… gatgccttacaccSS2 ccagcaatc… gtcagtataat ………… .attcatgttt… gatgccttacaccSS3 t t t t t tt t ttt t ttSS3 ccagcaatc… gtcagtataat ………… .attcatgttt… gatgccttacaccSSBN51 ccagcaatt.. gtcaatataat .………… attcatgttt… gataccttacaccSSBN52 ccagcaatt.. gtcaatataat .………… .attcatgttt… gataccttacaccSSBN53 ccagcaatt.. gtcaatataat .………… .attcatgttt… gataccttacacc

Creation of GFP transgenic rats using Sleeping Beauty transposon

MCW, Guertz and JacobAdd3, Dusp5 in FHH, 4A1 in SS rats

MAP and Proteinuria‐ SB Trasposon‐ CYP4A Transgenic rats vs Dahl S rats

* vs Dahl SSJr, p<0.05

Zinc finger nuclease KO technology (MCW, Guertz and Jacob)

Zinc finger motifsFok1  nuclease domain Agouti Coat Color Phenotype

PNAS, 2006 103(44): p16370‐16375Nature Biotech, 2008.  26(6): p 702‐708

MCW, Omeara and Jacob

34

KO Add3, Dusp5 in FHH.1BN congenic, 4A2 in SS.BN5, MMP, TGFb in SS

The FHH rat

The FHH rat is a genetic model of hypertension‐induced renal disease Proteinuria‐ QTL chr 1 Focal glomerulosclerosis Systolic hypertension (late) Pulmonary Hypertensiony yp Bleeding disorder Coat Color

Kidney Int Suppl 45: S2‐S5, 1994

Substitution of Chromosome 1 from the BN rat restores the autoregulation of RBF in FHH rats

AJP Renal Physiol 290: F1213‐F1221, 2006.

Pgc is elevated in FHH rats

75FHH

*la

ryg)

65

rula

r cap

ilur

e (m

m H 55 *

Glo

mer

Pres

su 45

100 120 140 160

35

AJP Regul Integr Comp Physiol 276: R855‐R863, 1999.

RPP (mm Hg)

MMP‐2 and TGF‐β1 levels are increased in the kidneys of FHH rats at 21 weeks

mg) 550

600

mg)

8

10

els

(pg/

m

450

500

els

(pg/

6

P-2

leve

400

450

F-1

leve

4

MM

300

350(6) (6)(6) TG

F

0

2

(6) (6)(6)

FHH AR+Control FHH AR+Control

Dilutional Palb measurements in SD and FHH rats

100

110

ores

cenc

e

90

% F

luo

70

80

FHH (8,82)

Time (seconds)0 20 40 60 80 100 120 140 160

60

FHH (8,82)SD (12,206)

Time (seconds)

Infusion of FITC albumin in FHH and FHH.1BN congenic rats

10x

40x

FHH AR+

Unbiased Discovery Based Approach: Unbiased Discovery Based Approach: 

ConsomicConsomic rats are single chromosome substitutionsrats are single chromosome substitutionsConsomicConsomic rats are single chromosome substitutionsrats are single chromosome substitutions

MCW NHLBI fMCW: NHLBI program forGenomic applications – PGA2001

SSBN1

SSBN4

SSBN8SSBN13

SS diseased

SSBN16BN normal

Albuminuria in FHH treated with LNAME

* * *

*

* **

*

Mattson et al. AJP Renal Physiol 293: F1905‐F1914, 2007.

Transfer of Chrm 1 restores autoregulation of RBF in the FHH rat

AJP Renal Physiol 290: F1213‐F1221, 2006.

Genetic map illustrating the introgressed regions in FHH.1BN AR+ congenic strains

Left: indicates the location of genetic markers used to genotype the animals on chromosome 1 in FHH rats. The closed and open filled bars refer to Fawn hooded-hypertensive (FHH) and Brown Norway (BN) genomes, respectively.Right: indicates known candidate genes in the 2.6 Mb region of interest. AR= Auto regulation; + present; - present

FHH and FHH.1BN congenic rats

g)

140 FHH (6)A (4)

dex 1.0

mm

Hg

130

A (4)B (6)C (9)D (5)

FHHAD

lato

ry In

0.6

0.8

ol (1

00

120E (5)

B

Aut

oreg

u

0.4 † † †

f con

tro

100

110 CE

RB

F A

0.0

0.2

FHH A B C D E

% o

f

90

100

FHH(6)

A(4)

B(6)

C(9)

D(5)

E(5)

RPP (mmHg)90 100 110 120 130 140 150

Restoration of myogenic response in AR+ FHH.1BN congenic strain E 

60 mmHg60 mmHg

22

riole

m

)

18

20

140 H rent

art

eram

eter

(14

16

18

140 mm HgA

ffer

dia

12

14FHH (4) FHH Ca+2 free (4) AR+ (4)AR+ Ca+2 free (4)

Perfusion pressure (mmHg)40 60 80 100 120 140 160

10

Restoration of RBF autoregulation mitigates proteinuria and renal injury

FHH AR+

*

*

300

400 FHH (10)

AR+ (6)

4*

*

tion

(mg/

day)

200

300

†r inj

ury

scor

e

2

3 *

Prot

ein

excr

et

100

†

Glo

mer

ula

1

2

Age (weeks)

10 12 14 16 18 20 22P

0

0FHH(4)

AR+(4)

Decision tree for the prioritization of candidate genes for transgenic studiesfor transgenic studies.

SNP analysis in coding region of Add3 gene:  37 SNPs

19 SNPs in Dusp5 gene

FHH and FHH.1BN congenic rats

(AR+ D1Rat20G17B‐6)

Rab‐38 ‐ responsible for the re‐uptake of filtered  protein i h i l b lin the proximal tubules

Autoregulation of RBF + +‐‐

“Candidate region”

Autoregulation of RBF

Proteinuria

+ +‐‐++ ‐ ?

Rab38 Sequencing

SequencingSequencing Western BlottingWestern Blotting

FHH.BNRab38

SequencingSequencing Western BlottingWestern Blotting

FHHFHH

Rab 38 regulates reuptake and processing of filtered albumin

Reasons for lack of progress in preventing diabetic nephropathynephropathy

• Problem:No rodent model of diabetic nephopathy exhibits progressive renal disease and lesions resembling those seen in man. Cell models not particularly informative. 

• Solution:We combined the genome of the FHH rat that develops renal disease but not diabetes with that of the GK rat that develops type II diabetes but not renal diseaseGK rat that develops type II diabetes but not renal disease. 

Glucose Intolerance in T2DN Rats600

/dL) 500

*

** *#

#

#

#

12 months

a (m

g/

400*

*

*

**#

#

9 months

12 months

lycemia

300**

*#

#

3 months

6 months

Gl

200

BN

Time of IPGTT (minutes)0 30 60 90 120

100

Progressive Proteinuria in T2DN Rats

Male BN

Male T2DNmimic

300

* #

ay)

200

250

ria (m

g/da

150*

Proteinu

r

50

100

* #

* #

Age (months)

01 3 6 9 12

(10)(6) (9)(7) (21)(6) (7)(7) (36)(7)

g*‐ Different from age‐matched BN

# ‐ Different from previous age in group‐matched

A B

Diabetes model

ce

100

110 T2DN 6 months (4,69) SD (12,206)

* * ** *ce

100

110 STZ (8,135) SD (12,206)

* * * * *

B

% F

luor

esce

n

80

90

*

* ** * * * * * *

% F

luor

esce

nc

80

90* *

* * * *

0 20 40 60 80 100 120 140 160%

60

70

0 20 40 60 80 100 120

%

60

70Expected Δ

Expected Δ

Time (seconds)0 20 40 60 80 100 120 140 160

Time (seconds)0 20 40 60 80 100 120

Glomerular Basement Membrane in  12 month T2DN Rats

Effect of tight diabetic control on diabetic nephropathyff f g p p y

Vehicle InsulinVehicle Insulin

MMP-2 and TGF-β1 levels in T2DN rats

s ein) 150

200

*

s ein)

1000

1200

*

MP2

leve

lsm

g of

pro

te

100

GF-

leve

lsm

g of

pro

te

400

600

800

MM

(pg/

m

0

50

TG(fg

/m0

200

400

03 month

T2DN(4)

6 monthT2DN

(4)

03 month

T2DN(4)

6 monthT2DN

(4)

Type-2 diabetes

Effects of XL784 on proteinuria in T2DN rats) 4 0 0

V e h ic le (1 2 )L is in o p r il (1 0 )X L 7 8 4 , 5 0 m g /k g (1 0 )

mg/

day)

3 0 0

4 0 0 X L 7 8 4 , 5 0 m g /k g (1 0 )L is in o p r il + X L 7 8 4 , 5 0 m g /k g (1 1 )X L 7 8 4 , 1 5 0 m g /k g (1 0 )

etio

n (m

2 0 0

3 0 0

***

in e

xcre

1 0 0

0 0

**

*******

Prot

ei

0

**

* * * * * * **

T im e (m o n th s )C o n tro l 1 2 3 4

Effect of an ACE Inhibitor and MMP on diabetic nephropathy

Vehicle Lisinopril XL784

diabetic nephropathy

Acknowledgements

Richard Roman, Ph.D.

CollaboratorsCollaboratorsHoward Jacob, Ph.D.Jozef Lazar, Ph.D.Jan Williams, Ph.D.

Roman LaboratoryRobin Dycee Reddy PadabbiReddy PadabbiFan Fan, M.D.Marilyn BurkeJin ZhangAndy Chen