Mechanisms of hypertension and diabetic nephropathy .Mechanisms of hypertension and diabetic nephropathy

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Mechanismsofhypertensionanddiabeticnephropathy

Richard J Roman Ph DRichardJ.Roman,Ph.D.

DepartmentofPharmacology

Theeconomicburdenofkidneydisease

HypertensionandDiabetesaretheleadingcauseofendstagerenaldisease.66%ofnewcases.

Numberofpatientswithchronicrenaldiseaseisexplodingcurrentlyestimatedat30millionor13%ofthepopulation.

Currently,approximately500,000Americanshavebeendiagnosedwithkidneyfailure.ThenumberofAmericanswiththisadvancedstageofthediseaseisexpectedtogrowto785,000by2020.p g y

Theannualcostoftreatingkidneydiseaseis$42billion,25%ofMedicarespending.

Thereisacriticalneedfordevelopmentofnewtherapiestosloworreversetheprogressionofchronickidneydisease.

http://www.asnonline.org/facts_and_statistics/kdhealththreat

Impaired autoregulation and 20Impaired autoregulation and 20--HETE, MMP2,TGFHETE, MMP2,TGF-- 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

MaculaDensa

EfferentarterioleAfferentArtrioleDistalTubule

AfferentarterioleDistaltubule

GlumerulusMaculaDensa

Effectof20HETEonTGF

18

21

riole

m) 15

18

entArte

iam

eter

(um

12

ofAffere

(m)

Di

9

ametero

20HETE in tubule (10 m)

NaCl

NaCl

NaCl ET

E0

Dia 20HETEintubule(10m)

10mM

Na

80mM

Na

10mM

Na

80mM

NaCl+

20HE

T

Control

60mmHG 120mmHG

Effectof20HETEonMyogenicResponse

16

e

12

14

Arteriole

amet

er(u

m)

10

12

ferentA

Dia

8

20-HETEterofAf

0

6

60 120 60 120 60 120

20 HETE

HET-0016

Diamet

(m)

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

MMP2andTGF1levelsinDahlSratsfedi h l l hi h l dieitheralowsaltorhighsaltdiet

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

XL784reversibilitystudyinDahlSrats

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- in Dahl S Rats

Effect of TGF- 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

Figure1B

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

H20H20

6% BSA

FITC-Dx250

4% BSA

FITC-Dx250

6% BSA

FITC-Dx250

H20 H20

FITCDextran250kD

LabelingandIsolationofGlomeruliandImaging

SD glomerulus albumin and dextran labeled

Live Image of glomeruli

Figure3A

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

Figure5B

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 Expected

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

EvidenceLinkingP4504AtosaltsensitiveHypertensioninDahlSRats

Theformationof20HETEisdecreasedinthekidneyinDahlSratscomparedtoother normotensive and hypertensive strains of ratsothernormotensiveandhypertensivestrainsofrats.

20HETEproductioninOMisreducedinDahlSratswhichcontributestoelevatedl Cl b ti i TALH 20 HETE d ti i d d i l lloopCl reabsorptioninTALH.20HETEproductionisdecreasedinglomerulusbutvascularproductiondoesnotseemtolowerthanotherstrains.

ChronictreatmentwithFibratesincreasestherenalformationof20HETE,improvespressurenatriuresis,andreducesbloodpressureinDahlSrats.

TheCYP4Aregiononchromosome5cosegregateswithhypertensioninaDahlSXLewisF2cross.

CYP4Agenesarelocatedonratchromosome5.DotheycontributetoHypertensioninDahlSrats?

Consomic ratsaresinglechromosomesubstitutions

MCW: NHLBI program forGenomic applications - PGA

SSBN1

SSBN4

SSBN8SSBN8

SSBN13SS diseased

SSBN16BN normalSSBN20

Note: the strain sequencedis this BN

BloodpressuretargetscapturedintheSS.BNConsomicstrains

Mattsonetal.AJPRenalPhysiol295:F837F842,2008.

TelemetryConfirmationofrenoprotectivephenotypeinSS.5BN rats

Days on High Salt Diet P

CYP4AexpressioninthekidneyofSSandSS.5BN rats

ChronicadministrationofHET0016reversesthephenotypeofSS.5BN rats

**

*

14 Days on High Salt Diet

SequencingofCYP4A3cDNA

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

CreationofGFPtransgenicratsusingSleepingBeautytransposon

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

MAPandProteinuria SBTrasposon CYP4ATransgenicratsvsDahlSrats

* vs Dahl SSJr, p

ZincfingernucleaseKOtechnology(MCW,GuertzandJacob)

ZincfingermotifsFok1 nucleasedomai