ADVANCING RESEARCH. APPLYING KNOWLEDGE. IMPROVING THE QUALITY OF HEALTHCARE.
60th Annual Fall Conference and Scientific Sessions of
the Council on the Kidney in Cardiovascular Disease
Conference Abstracts
October 4–7,2006•San Antonio Marriott Rivercenter•San Antonio,Texas
the Council for High Blood Pressu
re
Research in Association with
Oral Presentations
1Nuclear Hormone Receptor LXR Induces Murine Mesenchymal Stem Cellsinto Renin Expressing Cells: Possible Origin of Juxtaglomerular Cells
Kenichi Matsushita, Fulvio Morello, Richard E Pratt, Victor J Dzau; Duke Univ Med Cntr,Durham, NCMesenchymal stem cell can differentiate into skeletal muscle, smooth muscle, cartilage, fatand bone. It may play an important role in developmental biology contributing to the genesisof various tissues. Indeed, it has been reported that juxtaglomerular cells may originate fromthe metanephric mesenchyme. In this study we examined for direct evidence that mesenchy-mal stem cells (MSCs) can be induced to become renin expressing cells. Previously we havereported that the nuclear hormone receptor liver X receptor (LXR) is a major transcriptionalregulator of renin gene expression. Accordingly, we studied the effect of LXR activation of MSCrenin gene expression. Methods: Murine MSCs were subjected to serum starvation for 16hours. We then added the LXR synthetic ligand T0901317 (10
-6
M) and investigated mRNA expression of renin, ATP-binding cassette transporter A1 (ABCA1) which is well knownupregulated by LXR activation, LXR-alpha and LXR-beta after 6 hours of pharmacologicaltreatment by using real-time RT-PCR. We further examined the renin mRNA expression byadding LXR natural ligand 22-hydroxycholesterol (10
-7
M) or cyclic AMP (10
-3
M). Results:Murine MSCs treated acutely (6 hours) with T0901317 exhibited an increase in expression ofrenin (1.9
0.4 fold change for MSCs without treatment, P
0.05). There was an increasein ABCA1 expression (3.7
0.5 fold change for MSCs without treatment, P
0.05) and nosignificant change in LXR-alpha or LXR-beta expression, suggesting that T0901317 effectivelyacted only at the post-transcriptional level. Furthermore, 6 hours of 22-hydroxycholesterol orcyclic AMP treatment also upregulated renin mRNA expression (2.0
0.4 fold and 6.0
1.8fold changes for MSCs without treatment, respectively. P
0.05). We have also overexpressedLXR-alpha in MSC using gene transfection. Upon prolonged stimulation with 22-hydroxycholesterol for 6 weeks or cyclic AMP for 2 weeks, our preliminary results show thatthe phenotype of the MSC changed to become cells containing renin granules. Conclusion: Ourresults suggest that the activation of LXR induces renin mRNA expression and renin granuleformation in mesenchymal stem cells and suggest that these cells may be the origin of juxtaglomerular cells during embryonic development.
2c-Src Inhibition Attenuates Development of Hypertension and AssociatedCardiovascular and Renal Damage in Ang II-Infused Mice throughRedox-Sensitive Mechanisms
Glaucia E Callera, Ying He, Augusto C Montezano, Univ of Ottawa, Ottawa, Canada; Alvaro Yogi, Rita C Tostes, Univ of Sao Paulo, Sao Paulo, Brazil; Ernesto L Schiffrin, McGill Univ,Montreal, Canada; Rhian M Touyz; Univ of Ottawa, Ottawa, Canadac-Src plays a critical role in Angiotensin II (Ang II)-mediated signaling. Whether this tyrosinekinase influences development of hypertension and associated target organ damage inducedby Ang II remains unclear. Here we investigated effects of Ang II on blood pressure (BP) andrenal and cardiac fibrosis in mice treated with the c-Src inhibitor, CGP 077675 and in micedeficient in c-Src (c-Src
-/-
and c-Src
/-
). The systolic blood pressure (SBP) increase inducedby Ang II infusion (400 ng/kg/min, 2-weeks) in c-Src
/
mice was significantly reduced by CGP077675 (25 mg/Kg/day). Ang II-induced hypertension was blunted in c-Src
-/-
and c-Src
/-
mice(167
8 vs 137
8 and 105
7 mmHg). Ang II-induced increase in plasma TBARS (control,14.4
0.6 vs Ang II, 19.6
0.7 nmol/mL) and NAD(P)H oxidase-mediated vascular generationof
.
O
2-
(3,5-fold) were reduced by CGP 077675 (p
0.05). c-Src inhibition also inhibited theincreased phosphorylation of ERK1/2 (Ang II, 86
8 vs Ang II
CGP, 61
3, arbitrary units) andJNK (Ang II, 126
19 vs Ang II
CGP, 43
1, arbitrary units) in aorta and resistance(mesenteric) arteries. Ang II infusion failed to increase MAPK phosphorylation and
.
O
2-
generation in c-Src
/-
, c-Src
-/-
mice (p
0.05). The greater expression of proliferating cellnuclear antigen (PCNA), a marker of cell growth, in mesenteric arteries from Ang II-infusedc-Src
/
mice was inhibited with CGP 077675 treatment (Ang II, 130
9 vs Ang II
CGP,104
4, arbitrary units). PCNA protein expression was similar in c-Src
/-
mice infused with AngII and vehicle. Cardiac and renal collagen content induced by Ang II was significantly lower inin c-Src
-/-
and c-Src
/-
mice and in CGP 077675-treated mice. In conclusion, inhibition of c-Srcactivity by CGP077675 attenuates Ang II signaling and ameliorates oxidative stress, BPelevation and cardio-renal fibrosis by Ang II. Studies in c-Src- deficient mice support theseresults. Our findings identify c-Src tyrosine kinase as a novel target to blunt Ang II -dependenthypertension and associated cardiovascular and renal damage.
3Enhanced Susceptibility to Ang II-Induced Hypertension and Impaired AngII Metabolism in ACE2-Deficient Mice
Susan B Gurley, Thu H Le, Robert Griffiths, Nisha Phillip, Timothy A Haystead, Thomas MCoffman; Duke Univ, Durham, NCIn order to clarify the physiological roles of ACE2, lines of mice with targeted disruption of the
Ace2
gene have been generated. However, phenotypic features of the different ACE2-deficient(KO) mouse lines have been variable, especially with regard to blood pressures (BP) andcardiovascular (CV) functions. One potential source of this variability might be heterogeneity ofgenetic background. Thus, we generated inbred ACE2 KO mice by sequential back-crossing ofthe
Ace2
null mutation onto C57BL/6 (B6) or 129/SvEv (129) background for more than 6generations. Both lines of inbred ACE2 KO mice are viable, have normal reproduction, and lack any gross anatomical or structural abnormalities. Because of these mice have normal cardiacdimensions and function, we could examine the role of ACE2 in BP regulation without thepotential confounding effects of impaired heart physiology. Baseline BPs were measured in the ACE2 KO lines with radiotelemetry. Mean BPs were not significantly different betweenB6-
Ace2
/y
and
-/y
mice, and 129-
Ace2
/y
and
-/y
mice (128
2 vs. 132
3 mmHg, n
6 allgroups). As in vitro studies suggest that ang II may be a substrate for ACE2, we examined theeffects of ACE2 deficiency on susceptibility to ang II-dependent hypertension, using the inbredlines.
Ace2
/y
(MWT) and
-/y
(MKO) mice were infused subcutaneously with angiotensin II (1000ng/kg/min) with an osmotic pump, while BP was monitored by radiotelemetry. With ang IIinfusion, BP increased significantly in both MWT (133
2 to 169
6 mm Hg; p
0.0005) andMKO mice (131
2 to 195
6 mm Hg, p
0.0002), but the magnitude of this increase wasalmost 2-fold greater in the MKO animals (
64 mm Hg) compared to MWT (
36 mm Hg). After2 weeks of ang II infusion, BPs were significantly higher in the MKO mice than WT (p
0.01).To examine the mechanism for the more marked BP response to ang II in the ACE2-deficientmice, we measured kidney ang II levels after ang II infusion using MALDI-TOF massspectrometry. Renal ang II content was nearly six-fold higher in MKO mice compared to MWT(0.28
0.05 vs. 0.05
0.002 relative intensity; p
0.0008). In summary, we find no effect of ACE2-deficiency on basal BP regulation or CV function. However, ACE2 protects against angII-dependent hypertension by regulating ang II levels in the kidney.
4Exaggerated Blood Pressure Variability Aggravates Hypertensive CardiacRemodeling through the Angiotensin II-Mediated Inflammation
Hiroshi Kudo, Hisashi Kai, Narimasa Takayama, Takahiro Mori, Yusuke Sugi, Daisuke Fukui,Kiyoko Takemiya, Ayami Ikeda, Masayoshi Futamata, Hideo Yasukawa, Nobuhiro Tahara,Mitsuhisa Koga, Yumiko Kawai, The Cardiovascular Rsch Institute, Kurume, Japan; Yoshitaka Hirooka, The Dept of Cardiovascular Medicine, Fukuoka, Japan; TsutomuImaizumi; The Cardiovascular Rsch Institute, Kurume, JapanIt was demonstrated that hypertensive patients with large blood pressure (BP) variability hadgreater risk of cardiovascular events and exaggerated end-organ damages. However, thepathogenesis is currently unknown. The aims of this study were to create a new rat model ofchronic hypertension with exaggerated BP variability and to investigate the effects ofexaggerated BP variability on hypertensive cardiac remodeling and the underlying mechanism.For this purpose, we performed bilateral sino-aortic denervation (SAD) in spontaneoushypertensive rats (SHRs). Seven weeks after SAD or sham operation (week 7), 24-hour BP wasmonitored telemetrically in SAD
SHRs and sham
SHRs. Although mean BP was similar in thetwo groups, SAD
SHRs showed greater BP variability parameters, such as standard deviationand covariance of mean BP, compared with sham
SHRs. At week 7, both groups showedconcentric LV hypertrophy, and SAD enhanced it by 1.3-fold. SAD
SHRs enhanced myocytehypertrophy and myocardial fibrosis by 1.4-fold and 4.7-fold, respectively, relative tosham
SHRs. Perivascular macrophage infiltration was evident in SAD
SHRs, but not insham
SHRs. SAD remarkably upregulated expressions of myocardial angiotensinogen andmonocyte chemoattractant protein-1 (MCP-1). To determine the role of angiotensin II in the BPvariability-induced cardiac remodeling, non-depressor dose of angiotensin II receptor blocker,candesartan, was orally given to SHRs everyday from 1 week after SAD operation(SAD
Cand
SHRs). At week 7, although the small dose of candesartan did not affect BPvariability, the SAD-enhanced LV and myocyte hypertrophy was significantly reduced and theSAD-induced myocardial fibrosis was almost abolished. Moreover, in SAD
Cand
SHRs, theSAD-induced MCP-1 upregulation and macrophage accumulation were almost reversed to thelevels of sham
SHRs. In conclusion, exaggerated BP variability aggravates hypertensivecardiac hypertrophy and fibrosis through chronic activation of inflammatory process. And,angiotensin II would play a key role in the activation of the inflammatory process, independentlyof the presser effect.
5Kidney-Specific Enhancement of Angiotensin II Initiates Renal Injury inGene-Targeted Mice
Hiroyuki Kobori, Yuri Ozawa, Yuki Suzaki, Tulane Univ Health Sciences Cntr, New Orleans,LA; Curt D Sigmund, Univ of Iowa, Iowa City, IA; L. G Navar; Tulane Univ Health SciencesCntr, New Orleans, LA We recently reported that concomitant increases in proximal tubular angiotensinogen (AGT)mRNA and protein participate in increased intrarenal angiotensin (Ang) II leading to progressiverenal injury in AngII-infused rats. However, it has not been established if selective increases inintrarenal AngII can be responsible for renal injury. Using a transgenic mouse model in whichhuman AGT (hAGT) is expressed only in the kidney, experiments were performed to determineif selective renal overproduction of AngII elicited by stimulating hAGT present only in the kidneyin the presence of human renin (hR) will cause renal injury. We used 3 groups of mice: 1) singletransgenic (A, N
14) expressing hAGT only in the kidney regulated by kidney-specificandrogen regulated protein promoter, 2) double transgenic (D, N
13) expressing hRsystemically in addition to hAGT only in the kidney, and 3) wild type mice (W, N
12).Exogenous hAGT protein is inactive in A mice because endogenous mouse renin cannot cleavehAGT to AngI due to a high species-specificity. All mice were monitored from 12 to 18 wks ofage with free access to a regular diet and water. Systolic BP was progressively increased from116
/-5 mmHg (12 wks) to 140
/-7 (18 wks) in D mice during this period. This increase wasnot observed in A or W mice. Intrarenal hAGT mRNA and protein were similar in A and D mice;however, hAGT mRNA or protein was not detectable in kidneys of W mice. While plasma AngIIconcentrations were similar among the 3 groups, kidney AngII levels were increased in D(216
/-43 fmol/g) compared with A (117
/-16) and W (118
/-17) mice. Interstitialcollagen-positive area, stained by PicroSirius Red, was significantly increased in D (0.52
/-
e26
Hypertension Vol 48, No 4 October 2006
0.06%) compared with A (0.36
/-0.05) and W (0.34
/-0.03) mice. Interstitial macrophageinfiltration, evaluated by CD68-positive cell number, was significantly increased in D (46
/-5cells/mm
2
) compared with A (20
/-3) and W (19
/-2) mice. Afferent arteriolar wall thickness,stained by alpha actin, was significantly increased in D (3.31
/-0.41
m) compared with A (2.21
/-0.12) and W (2.16
/-0.11) mice. These data indicate that the selective renaloverproduction of AngII initiates renal injury in the gene-targeted mice even before thedevelopment of marked hypertension.
6A Novel Regulatory Effect of AT1 Receptor-Interacting Molecule onVascular Smooth Muscle Cells
Koichi Azuma, Kouichi Tamura, Masashi Sakai, Yuko Tsurumi, Toyoichiro Shigenaga, YutakaTanaka, Motoko Ozawa, Miyuki Matsuda, Tomoaki Ishigami, Yokohama City Univ, Yokohama, Japan; Marco Lopez-Ilasaca, Harvard Univ, Boston, MA; Masatsugu Horiuchi,Ehime Univ, Shigenobu, Japan; Satoshi Umemura; Yokohama City Univ, Yokohama, Japan Activation of tissue AT1 receptor (AT1R) signaling plays an important role in cardiovascularhypertrophy and remodeling and the C-terminal domain of AT1R is involved in the receptorinternalization and its downstream pathway. We previously cloned a novel molecule interactingwith the C-terminal domain of AT1R, ATRAP (for AT1R-associated protein), using the yeasttwo-hybrid strategy. In this study, we tested the hypothesis that vascular smooth muscle cells(VSMC) express ATRAP and that ATRAP modulates Ang II-induced responses in VSMC. Weidentified that the ATRAP mRNA and protein were endogenously expressed in VSMC, and founda substantial co-localization of ATRAP and AT1R in intracellular compartments in AngII-stimulated VSMC. Overexpression of ATRAP by adenoviral gene transfer significantly inhibited Ang II-mediated increases in TGF-
mRNA expression (
p
0.05, n
6) and TGF-
productioninto the medium (
p
0.05, n
6). Furthermore, this phenomenon was accompanied byinhibition of Ang II-induced activation of BrdU incorporation (
p
0.05, n
6). These resultsindicate that ATRAP significantly promotes internalization of the AT1R and attenuates AngII-mediated proliferative response and synthesis of extracellular matrix in VSMC, and maysuggest a novel strategy to inhibit vascular remodeling.
7Human G Protein-Coupled Receptor Kinase Type 4
(hGRK4
) Wild-TypePrevents Salt Sensitivity While its Variant, hGRK4
486V, Promotes SaltSensitivity in Transgenic Mice: Role of Genetic Background
Zheng Wang, Laureano D Asico, Crisanto S Escano, Georgetown Univ Sch of Medicine,Washington, DC; Robin A Felder, Univ of Virginia Health Sciences Cntr, Charlottesville, VA;Pedro A Jose; Georgetown Univ Sch of Medicine, Washington, DCThe hGRK4
486V variant, either by itself or in conjunction with other hGRK4
variants (65Land 142V), is associated with salt-sensitive hypertension (Clin Chem.2006;52:352), asevidenced by its development in hGRK4
486V transgenic mice (but not in hGRK4
wild-typetransgenes) generated in our laboratory. The influence of genetic background on the expressionof salt sensitivity was studied by introducing the transgenes into mice with varying gene ratiosof salt-resistant
SJL
and salt-sensitive C57BL/6J (
B6/J
) backgrounds. The mice (6–8 mo old)were maintained on normal (0.9%) or high (6%) NaCl diet for 3 weeks. With normal NaCl intake,aortic BPs (measured via the femoral artery under pentobarbital anesthesia) were similar intransgenic and non-transgenic mice. However, with high NaCl intake, BPs differed among thegroups, and influenced by genetic background (Table). BPs of non-transgenic littermates (NT)with at least 12% SJL background were not affected by high NaCl. In contrast, high NaClincreased BPs of 486V mice, despite having 25% SJL background (P
0.001). The ability ofhigh NaCl to elevate BPs of 486V mice decreased as the SJL background increased to 56%.High NaCl increased BPs of NTs with more than 94% B6/J background. However, hGRK4
wild-type prevented the salt-sensitive hypertension of B6/J mice (P
0.001). We conclude thathGRK4
prevents, while the 486V variant promotes, salt-sensitive hypertension in transgenicmice. The extent of salt-resistant or salt-sensitive genetic background is crucial in uncoveringthe roles of hGRK4
wild-type and 486V variant in salt sensitivity. Inconsistencies in theassociation of candidate genes with hypertension may be explained by differences in geneticbackground.
Mice% Background Normal NaCl (0.9%) High NaCl (6%)P
#
SJL(salt-esistant)B6/J(salt-ensitive)SBP(mm Hg) NSBP(mm Hg) NSJL 100 0 97
0.6 4 97
2.2 5 0.87
B/6J
0
100
103
1.2 4
123
1.8
* 5
<
0.001
NT 25 75 100
0.9 4 100
3.7 6 0.97NT 12 88 105
2.9 4 102
7.7 5 0.76NT
6
>
94
102
1.8 8
122
1.2
11
<
0.001GRK4
6
>
94
101
2.3 7 103
2.3** 7 0.5
486V 25
75 100
0.5 2
125
4.6
*** 9
0.03486V 56
44 96
3.1 6
110
3.6
**** 5
0.01
#P 0.9% vs 6% NaCl, ANOVA, Newman-Keuls (6% NaCl): *P
0.01, SJL vs B/6J; **P
0.001, NT(
94%B6/J) vs GRK4
; ***P
0.001, NT(25% SJL) vs 486V; ****P
0.05, 486V (25% SJL) vs 486V (56% SJL).SBP
systolic blood pressure, Data are M
SE.
8Association between Variants of the Human
GSTM
Gene Family andHypertension
Christian Delles, Univ of Glasgow, Glasgow, United Kingdom; Ana C Braga-Marcano, PatriciaB Munroe, Barts & The London Med & Dental Sch, London, United Kingdom; SandoshPadmanabhan, John D McClure, Nick J Brain, Univ of Glasgow, Glasgow, United Kingdom;Morris J Brown, Univ of Cambridge, Cambridge, United Kingdom; Nilesh J Samani, Univ ofLeicester, Leicester, United Kingdom; David Clayton, Univ of Cambridge, Cambridge, UnitedKingdom; Martin Farrall, Univ of Oxford, Oxford, United Kingdom; John Webster, AberdeenRoyal Infirmary, Aberdeen, United Kingdom; John M Connell, Univ of Glasgow, Glasgow,United Kingdom; Mark J Caulfield, Barts & The London Med & Dental Sch, London, UnitedKingdom; Anna F Dominiczak; Univ of Glasgow, Glasgow, United Kingdom
Background:
Glutathione S-transferases (GSTMs) are involved in cellular defences againstoxidative stress. The human
GSTM
gene cluster consists of 5 genes,
GSTM4
,
2
,
1
,
5
and
3
. Wehave shown that the rat orthologue,
Gstm1
, is a positional and functional candidate gene forrodent hypertension. The aim of the current study was to test the hypothesis that this discoverycan be translated to man.
Methods:
First analysis was performed in 138 white subjects inwhom exons, flanking introns, 3’ and 5’ regions of
GSTM4
,
2
,
5
and
3
were sequenced todetect single nucleotide polymorphisms (SNPs) within the
GSTM
gene family and to examinelinkage disequilibrium (LD) between these SNPs. We have then chosen 10 SNPs across the
GSTM
gene cluster and genotyped 1151 families (3453 individuals) from the MRC BRIGHTStudy for these SNPs and for the common
GSTM1
deletion genotype. Analysis was performedby Transmission Disequilibrium Test (TDT) using FBAT Software.
Results:
We detected 18, 5,6, and 5 SNPs in
GSTM4
,
2
,
5
and
3
, respectively, with minor allele frequencies of 5% orgreater. There were two distinct LD blocks, one between
GSTM4
and
2
and one between
GSTM5
and
3
. In the BRIGHT TDT Study we detected a highly significant association betweena SNP in the 3’ region of
GSTM5
(rs11807) and hypertension (z
2.698,
P
0.007) andsignificant associations for two more SNPs within the
GSTM5/3
LD block (rs11101992:z
2.443,
P
0.015; rs3814309: z
2.215,
P
0.027). The
GSTM1
deletion was also over-transmitted to affected offspring (z
2.339,
P
0.019).
Conclusions:
The
GSTM1
deletiongenotype and three SNPs in the
GSTM5/3
LD block are associated with hypertension as aqualitative trait. This is an example for successful translation of findings from a rodent modelto human cardiovascular disease.
GSTM
s, and in particular
GSTM1
,
5
and
3
, are robustcandidate genes for human essential hypertension. Genetic variants in
GSTM
s may lead toreduced defences against oxidative stress and thereby to endothelial dysfunction and relentlessprogression of cardiovascular disease.
9Effect of Age and Angiotensinogen 235 Genotype on Renal Plasma FlowResponsiveness to Angiotensin II
Tejas V Patel, Gordon H Williams, Naomi D Fisher; Brigham and Women’s Hosp, Boston, MA
Objective
: In essential hypertensives(HT), blunted renal plasma flow (RPF) response to Ang IIis associated with the Angiotensinogen AGT235TT genotype, suggesting a pathologic increasein intrarenal Ang II. RPF and its responsiveness to Ang II fall with age. We sought to determinethe interaction of age and genotype on RPF responsiveness in both HT and normotensives (NT).
Method
: A total of 315 subjects in high sodium balance had RPF response to Ang II (3ng
●
kg
-1
●
min
-1
) measured by PAH clearance. Subjects were divided into
45 yrs (N
83 HT, 65 NT) and
45 yrs (N
145 HT, 22 NT) for age analysis, as 45 was the median age.
Result
: Age and AGT235 genotype independently predicted RPF response to Ang II. For both HT and NT, the AngII induced fall in RPF was significantly lower in older than in younger subjects (p
0.03 and0.004, respectively). Among HT, this fall with age was seen in subjects carrying both the AGT235MM and MT genotypes (p
0.005, Fig. A). However, AGT235TT homozygotes did notdemonstrate a fall in RPF responses with age (p
0.72, young vs. old, Fig. B), as youngersubjects already showed blunted responses. Among NT, all AGT235 genotypes had a significantfall in RPF response to Ang II. This association between age, RPF response to Ang II andgenotype was not seen with 3 other genes in RAAS: ACE I/D, ATR1 and aldosterone synthase.
Conclusion
: In HT but not NT, the AGT235TT variant is associated with a blunted RPF responseto Ang II that does not fall with age. This suggests that young HT with the AGT235TT genotypewarrant a more aggressive management to preserve renal function. This is the first report ofage and genotype interaction, which may have important implications in managing essentialHT. #
10Disruption of Natriuretic Peptide Receptor A Gene Increases AdrenalAngiotensin II and Aldosterone Levels
Di Zhao, Naveen K Somanna, Elangovan Vellaichamy, Kailash N Pandey; Dept of Physiologyand Hypertension and Renal Cntr of Excellence, Tulane Univ Health Sciences Cntr & Sch ofMedicine, New Orleans, LA The disruption of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene (Npr1)leads to elevated arterial blood pressure, cardiac hypertrophy, congestive heart failure, andsudden death in mice lacking NPRA. ANP-NPRA signaling is known to counteract therenin-angiotensin-aldosterone system (RAAS). We studied whether Npr1 gene copy number
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