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
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