Mechanisms Mediating the Renal Profibrotic Actions of Vasoactive Peptides in Transgenic Mice
JEAN-CLAUDE DUSSAULE*,
PIERRE-LOUIS THARAUX,
JEAN-JACQUES BOFFA,
FADI FAKHOURI,
RAYMOND ARDAILLOU and
CHRISTOS CHATZIANTONIOU
*Department of Physiology, UFR Saint-Antoine AP-HP, Paris,
France. INSERM U489, Tenon Hospital, Paris, France.
Correspondence to Dr. Jean-Claude Dussaule, Department of Physiology,
Hôpital Saint-Antoine, 184 rue du Faubourg
Saint-Antoine, 75012 Paris, France. Phone: 33-1-49-28-23-14; Fax:
33-1-49-28-20-52; E-mail:
jeanclaude{at}sat.ap-hop-paris.fr
Abstract. Transgenic mice are useful tools to investigate the
mechanismsof the renal profibrotic actions of endothelin and angiotensinII.
The overexpression of angiotensinogen and renin genes inducesrenal sclerosis
independently of changes in systemic hemodynamics.The same results are
observed when the endothelin-1 gene isoverexpressed. Transgenic mice
harboring the luciferase gene,under the control of the collagen I 2
chain promoter, and madehypertensive by induction of a nitric oxide (NO)
deficiencyhave been studied. In this strain of mice, luciferase activityis
an early index of renal and vascular fibrosis. Luciferaseactivity was
increased in preglomerular arterioles and glomeruliwhen mice were treated
with N-nitro-L-arginine methyl ester,an inhibitor
of NO synthases. Bosentan (an endothelin receptorantagonist) was as efficient
as losartan (an AT1 receptor antagonist)in preventing renal fibrosis,
although it did not decrease BP.In short-term experiments, angiotensin II
produced an increasein luciferase activity that was entirely prevented by
losartanbut also by bosentan. It can be concluded that, during chronic
inhibitionof NO, the collagen I gene is activated, which contributes tothe
development of nephroangiosclerosis and glomerulosclerosis.Angiotensin II
plays a major role in this fibrogenic process,and its effect is at least
partly independent of systemic hemodynamicsand mediated by the profibrotic
action of endothelin-1.
Renal vascular and glomerular injuries are frequent complicationsof human
and experimental hypertension
(1). Stimulation of
extracellularmatrix protein synthesis may occur as an adaptation to increased
walltension and/or to the action of vasoconstrictive peptides suchas
angiotensin II and endothelin-1.
Many in vitro studies support the hypothesis that angiotensinII
regulates extracellular matrix formation independently ofits vasoconstrictive
action. For example, angiotensin II stimulatesprotein synthesis in cultured
cardiac fibroblasts (2). In
addition,endothelin-1 displays potent mitogenic properties and induces
proteinsynthesis in cultured vascular smooth muscle cells and mesangialcells
(3,4).
Moreover, in various models of hypertension, invivo administration
of endothelin receptor antagonists limitsend-organ damage but does not reduce
high BP (5).
From these data, the question of whether renal fibrosis developmentand/or
regression can occur independently of changes in systemichemodynamics has
been raised. If so, new therapeutic strategiescould be proposed to prevent
the progression of nephroangiosclerosisand glomerulosclerosis in human
hypertension.
To gain new insights into the mechanisms mediating the renalprofibrotic
action of vasoactive peptides in vivo, several transgenicmouse
models have been developed. In this review, we discussthe results of
overexpression of genes for endothelin or renin-angiotensincomponents. We
also examine data obtained using a transgenicmouse line that expresses
luciferase under the control of thepromoter of the 2 chain of the
mouse collagen type I gene (procolI 2). This model allows a reliable
and early index of renalfibrosis to be used in experimental models of
hypertension.
Overexpression of the Renin-Angiotensin Components
The renin-angiotensin system has been the focus of the largestnumber of
transgenic studies reported in the renal literature
(6).Among numerous models,
dual-transgenic mice expressing humanangiotensinogen and renin genes under
the control of the appropriatehuman promoters were developed to produce a new
hypertensivemodel (7). These
mice developed hypertension and renal fibrosis.Administration of lisinopril,
an angiotensin-converting enzymeinhibitor, significantly decreased the renal
glomerulosclerosisindex without decreasing systolic BP. These results suggest
thatactivation of the renal renin-angiotensin system induces renalsclerosis
independently of systemic hypertension. Similar conclusionscan be drawn from
another study, which applied the unilateralureteral obstruction model to a
gene-targeting mouse line inwhich angiotensin II levels were varied from none
to supraphysiologiclevels through changes in the number of copies of the
angiotensinogengene (8). In
these mice, renal collagen deposition dependedon the number of
angiotensinogen copies. Despite the fact thatsupraphysiologic levels of
angiotensin II did not acceleraterenal fibrosis development, the reduction of
copies to zeroor one limited renal collagen synthesis, compared with
wild-typeanimals (with two copies), without decreasing systolic BP. These
resultsclearly support the hypothesis that endogenous angiotensin IIproduced
locally plays a role in the formation of renal fibrosisafter ureteral
obstruction, independently of alterations insystemic resistance.
Transgenic models have also been developed to study the influenceof
endothelin on BP and end-organ damage. When endothelin-1gene overexpression
occurred under the control of appropriatepromoters, mice developed
glomerulosclerosis and interstitialfibrosis, but the appearance of the renal
lesions was not accompaniedby changes in arterial pressure
(9). Similarly, transgenic rat
modelsthat overexpressed the human endothelin-2 gene within the kidney
exhibitedglomerulosclerosis without increased BP
(10). These transgeniclines
are better models of pathophysiologic processes than arerats with
overexpression of the human endothelin-1 gene underthe control of a virus
promoter in the liver (11).
These datasupport a profibrotic action of endothelin peptides independentof
their vasoconstrictive properties. This is in accordancewith the results of
most pharmacologic studies testing the effectsof endothelin receptor
antagonists in hypertensive models
(5),with the noteworthy
exceptions of DOCA and Dahl's models
(12).
Reporter Gene under the Control of the Promoter of Procol I 2,
an Index of Renal Fibrosis
Collagen type I expression is negligible in renal vascular structuresunder
normal conditions (13). In
contrast, this protein is highlyexpressed in nephroangiosclerosis and
glomerulosclerosis
(14,15).
Forthis reason, the presence of collagen type I in preglomerulararterioles
or glomeruli is an excellent index of vascular renalfibrosis. In our
laboratory, we used a transgenic mouse linethat expresses luciferase and
-galactosidase under the controlof two sequences (-19.5 to -13.5 kb and
-350 to + 54 pb) ofthe promoter of procol I 2. Previous data
demonstrated thatthis construction contained a far-upstream enhancer
regulatinghigh levels of expression of the mouse procol I 2 gene
(16).In preliminary
experiments, we confirmed that the expressionpatterns of luciferase and
-galactosidase were correlated withthe tissue distribution of collagen
I under chronic conditionsand with that of procol I 2 mRNA after acute
stimulation ofthe collagen I gene.
This transgenic line offers specific advantages for studyingrenal vascular
and glomerular fibrosis without interfering withthe expression of the native
collagen type I gene. In particular,measurements of luciferase activity allow
mechanisms initiatingin vivo fibrosis to be investigated under
pathophysiologic conditions.This model has given us new insights into the
involvement ofangiotensin II and endothelin-1 in the renal damage inducedby
nitric oxide (NO) deficiencies
(17,18).
To inhibit NO synthesis, mice were treated with
N-nitro-L-argininemethyl ester (L-NAME), a NO
synthase inhibitor (20 mg/kg perd), for 14 wk. This dose induced a gradual
elevation of BP (Figure 1).In
separate groups of mice, the mixed endothelin receptorantagonist bosentan or
the angiotensin II AT1 receptor antagonistlosartan was coadministered with
L-NAME. Afferent arteriolesand glomeruli were isolated from the transgenic
mouse kidneys.Luciferase activity was measured in afferent arterioles,
glomeruli,renal cortical slices, aortae, and hearts. Renal histologic
analyseswere performed using Masson trichrome solution for specificstaining
of extracellular matrix proteins. We observed thatNO inhibition promoted
procol I 2 expression in isolated afferentarterioles and glomeruli
before the BP increase or the appearanceof histologic alterations
(Figure 1). In addition, procol
I2 gene activation was detected much earlier in the renal vasculature
thanin the aorta and the heart. Another important observation wasthat
bosentan completely abrogated the L-NAME-induced activationof the collagen I
gene in the renal vasculature during (8 wk)and after (14 wk) the
establishment of hypertension, withoutaffecting the increase in BP
(17). Losartan was as
efficientas bosentan in preventing renal damage induced by NO inhibition,
althoughits effect on BP was absent at 8 wk and was only partial at14 wk
(18). Several conclusions can
be drawn from these data.First, the development of renal fibrosis or its
prevention isat least partly independent of changes in systemic hemodynamics.
Thisobservation does not exclude the possibility that the variationsin the
renal vascular tone are involved in the mechanisms controllingextracellular
matrix synthesis. Second, constitutively producedNO negatively controls
collagen I formation in the renal vasculature.Third, the beneficial effect of
this autacoid is primarily explainedby its inhibitory action on the
profibrotic activity of angiotensinII and/or endothelin.
Figure 1. (Left) Luciferase activity in afferent arterioles (Afferent Arter.) and
glomeruli and systolic BP in transgenic mice treated with
N-nitro-L-arginine methyl ester (L-NAME) for
different periods of time. Values are means ± SEM. (Right)
Representative examples of extracellular matrix staining, using Masson
trichrome solution, in mice treated with L-NAME for 6 wk (normal appearance)
or 10 wk (glomerular fibrosis).
At this point in the discussion, three main questions may beasked, as
follows. How is stimulation of the renin-angiotensinsystem explained when NO
synthesis is inhibited? What are therespective roles of angiotensin II and
endothelin in extracellularmatrix production, inasmuch as losartan and
bosentan are equallyefficient in preventing renal fibrosis? Does transforming
growthfactor- (TGF-) play a role in the interaction between
endothelin-1and angiotensin II?
Interactions between NO and the Renin-Angiotensin System
The preventive effect of losartan on renal injury at the firststep of
L-NAME-induced nephropathy appears to be a paradox,because we
(19) and others
(20) demonstrated that NO
suppressioninitially inhibited renin synthesis and renal angiotensin II
production(21). One
possibility is that the activity of the residual angiotensinII was
potentiated by the absence of NO. Another explanationcould be related to the
dual regulatory action of NO on therenin-angiotensin system. In the presence
of L-NAME, the lowrate of renin production could be compensated for by an
increasedcellular effect of angiotensin II, through upregulation of AT1
receptorsor through suppression of the NO-dependent inhibition of the
intracellularangiotensin II pathway. These hypotheses are in accordance with
theresults of previous studies of vascular tissues
(22,23).
Interaction between Angiotensin II and Endothelin-1
To answer the question regarding interactions between angiotensinII and
endothelin, new experiments were performed. We incubatedrenal cortical slices
and isolated aortae in the presence ofangiotensin II. Angiotensin II produced
an increase in luciferaseactivity, which was entirely prevented by the AT1
receptor antagonistlosartan but also by bosentan. Moreover, urinary excretion
ofendothelin-1, which is considered an index of renal endothelin-1
production,was increased in L-NAME-treated mice, compared with control
animals.This excess endothelin-1 production was less pronounced whenmice
were cotreated with losartan. Similar observations weremade using other
models of renal or vascular injury in whichthe reninangiotensin system was
activated. For example, it wasreported that, in a transgenic model
characterized by overexpressionof renin and angiotensinogen, endothelin
receptor blockade hadno effect on BP but protected transgenic mice from
vasculopathyand improved their survival rates
(24). In rats,
glomerulosclerosisobserved in a model of angiotensin II-induced hypertension
wasreduced by endothelin receptor antagonists, whereas the effectsof this
treatment on BP were inconsistent
(25,26).
These results support the involvement of endothelin-1 in thestimulatory
effects of angiotensin II on renal collagen I synthesisand, more generally,
in alterations of the vascular smooth musclecell phenotype
(Figure 2). They do not exclude
the involvementof TGF-, a potent profibrogenic agent, in renal
sclerosis inducedby angiotensin II
(27). Using the same strain of
mice, we investigatedwhether TGF- played a role in the interaction
between angiotensinII and endothelin-1. Acute in vivo administration
of angiotensinII, endothelin-1, and TGF- was performed in the presence
orabsence of their respective inhibitors, i.e., candesartan (anAT1
receptor antagonist), bosentan, and decorin (a scavengerof the active form of
TGF-). Our results suggested that endothelin-1and TGF- could
stimulate the synthesis of collagen I independentlyof each other and that
they operated in a synergistic way tomediate the angiotensin II-induced
activation of procol I 2
(28).Moreover, in aortic
segments, angiotensin II activated the collagenI gene through a mechanism
involving TGF- and the mitogen-activatedprotein/extracellular
signal-regulated kinase MAP-ERK pathway,as demonstrated by the inhibitory
effects of decorin and PD98059(a blocker of the MAP-ERK cascade) on
angiotensin II-inducedluciferase activity
(29). Finally, preliminary
data indicatedthat TGF- was overexpressed in rat renal cortex after 5
wk oftreatment with L-NAME. This result is in accordance with the
observationsof Tomita et al.
(30), who demonstrated, in the
same model,that TGF- expression was increased in cardiac fibroblasts
andthat this increase was prevented by AT1 receptor antagonism.It is likely
that the renal sclerosis induced by L-NAME is explainednot only by the
activity of angiotensin II and endothelin-1but also by the induction of
TGF-. Further experiments are neededto confirm this hypothesis.
Figure 2. Likely interactions between nitric oxide (NO), endothelin-1 (ET), and
angiotensin II (Ang II) in alterations of the renal vascular smooth muscle
cell (VSMC) phenotype, the synthesis of renin, and the loops of regulation
among these three vasoactive agents.
The renal profibrotic actions of angiotensin II and endothelin-1have been
demonstrated in various experimental transgenic mousemodels. These effects
are independent of systemic hemodynamics.NO limits the fibrogenic process
induced by these vasoactivepeptides, whereas TGF- likely acts in
synergy with endothelinto favor the action of angiotensin II on the
extracellular matrix.The observation that endothelin-1 mediates at least a
part ofthe profibrotic effect of angiotensin II could have implicationsfor
the treatment of nephroangiosclerosis and glomerulosclerosisin human
hypertension.
Acknowledgments
We thank Drs. Bou-Gharios, Rossert, and de Crombrugghe (Departmentof
Molecular Genetics, University of Texas, Houston, TX), MSD-France,and
Hoffman-Laroche for providing the transgenic mice, losartan,and bosentan,
respectively. Experiments were financially supportedby INSERM and
Unité de Formation et de Recherche
Saint-Antoine.Drs. Boffa and Fakhouri were research fellows of the Fondation
pourla Recherche Mé dicale, and Dr. Tharaux
was supportedby the Groupe de Réflexion sur
la Recherche Cardiovasculaireand the Assistance
Publique-Hô pitaux de Paris.
Weisstuch JM, Dworkin LD: Does essential hypertension cause
end-stage renal disease? Kidney Int Suppl36
: S33-S37,1992[Medline]
Brilla CG, Zhou G, Matsubara L, Weber KT: Collagen metabolism in
cultured adult rat cardiac fibroblasts: Response to angiotensin II and
aldosterone. J Mol Cell Cardiol26
: 809-820,1994[Medline]
Chua BH, Krebs CJ, Chua CC, Diglio CA: Endothelin stimulates
protein synthesis in smooth muscle cells. Am J Physiol262
: E412-E416,1992[Abstract/Free Full Text]
Simonson MS, Wann S, Mene P, Dubyak GR, Kester M, Nakazato Y, Sedor
JR, Dunn MJ: Endothelin stimulates phospholipase C,
Na+/H+ exchange, c-Fos expression, and mitogenesis in
rat mesangial cells. J Clin Invest83
: 708-712,1989
Schiffrin EL: Role of endothelin-1 in hypertension.
Hypertension 34:876
-881, 1999[Abstract/Free Full Text]
Kopp JB, Klotman PE: Transgenic animal models of renal development
and pathogenesis. Am J Physiol269
: F601-F620,1995[Abstract/Free Full Text]
Kai T, Kino H, Sugimura K, Shimada S, Kurooka A, Akamatsu K,
Takenaka T, Fukamizu A, Murakami K, Ishikawa K, Katori R: Significant role of
the increase in renin-angiotensin system in cardiac hypertrophy and renal
glomerular sclerosis in double transgenic Tsukuba hypertensive mice carrying
both human renin and angiotensinogen genes. Clin Exp
Hypertens 20:439
-449, 1998
Fern RJ, Yesko CM, Thornhill BA, Kim HS, Smithies O, Chevalier RL:
Reduced angiotensinogen expression attenuates renal interstitial fibrosis in
obstructive nephropathy in mice. J Clin Invest103
: 39-46,1999[Medline]
Hocher B, Liefeldt L, Thone-Reineke C, Orzechowski HD, Distler A,
Bauer C, Paul M: Characterization of the renal phenotype of transgenic rats
expressing the human endothelin-2 gene. Hypertension28
: 196-201,1996[Abstract/Free Full Text]
Niranjan V, Telemaque S, de Wit D, Gerard RD, Yanagisawa M:
Systemic hypertension induced by hepatic overexpression of human
preproendothelin-1 in rats. J Clin Invest98
: 2364-2372,1996[Medline]
Chatziantoniou C, Dussaule JC: Endothelin and renal vascular
fibrosis: Of mice and men. Curr Opin Nephrol Hypertens9
: 31-36,2000[Medline]
Rossert JA, Garrett LA: Regulation of type I collagen synthesis.
Kidney Int Suppl 49:S34
-S38, 1995[Medline]
Yoshioka K, Tohda M, Takemura T, Akano N, Matsubara K, Ooshima A,
Maki S: Distribution of type I collagen in human kidney diseases in comparison
with type III collagen. J Pathol162
: 141-148,1992
Morel-Maroger Striker L, Killen PD, Chi E, Striker GE: The
composition of glomerulosclerosis: Studies in focal sclerosis, crescentic
glomerulonephritis, and membranoproliferative glomerulonephritis.
Lab Invest 5:181
-192, 1984
Bou-Gharios G, Garrett LA, Rossert J, Niederreither K, Eberspaecher
H, Smith C, Black C, Crombrugghe B: A potent farupstream enhancer in the mouse
pro alpha 2(I) collagen gene regulates expression of reporter genes in
transgenic mice. J Cell Biol134
: 1333-1344,1996[Abstract/Free Full Text]
Chatziantoniou C, Boffa JJ, Ardaillou R, Dussaule JC: Nitric oxide
inhibition induces early activation of type I collagen gene in renal
resistance vessels and glomeruli in transgenic mice: Role of endothelin.
J Clin Invest 101:2780
-2789, 1998[Medline]
Boffa JJ, Tharaux PL, Placier S, Ardaillou R, Dussaule JC,
Chatziantoniou C: Angiotensin II activates collagen type I gene in the renal
vasculature of transgenic mice during inhibition of nitric oxide synthesis:
Evidence for an endothelin-mediated mechanism.
Circulation 100:1901
-1908, 1999[Abstract/Free Full Text]
Tharaux PL, Dussaule JC, Pauti MD, Vassitch Y, Ardaillou R,
Chatziantoniou C: Activation of renin synthesis is dependent on intact nitric
oxide production. Kidney Int51
: 1780-1787,1997[Medline]
Schricker K, Kurtz A: Blockade of nitric oxide formation inhibits
the stimulation of the renin system by a low salt intake. Pfluegers
Arch 432:187
-191, 1996[Medline]
Verhagen AM, Braam B, Boer P, Grone HJ, Koomans HA, Joles JA:
Losartan-sensitive renal damage caused by chronic NOS inhibition does not
involve increased renal angiotensin II concentrations. Kidney
Int 56: 222-231,1999[Medline]
Dowell FJ, Henrion D, Duriez M, Michel JB: Vascular reactivity in
mesenteric resistance arteries following chronic nitric oxide synthase
inhibition in Wistar rats. Br J Pharmacol117
: 341-346,1996[Medline]
Katoh M, Egashira K, Usui M, Ichiki T, Tomita H, Shimokawa H,
Rakugi H, Takeshita A: Cardiac angiotensin II receptors are upregulated by
long-term inhibition of nitric oxide synthesis in rats. Circ
Res 83: 743-751,1998[Abstract/Free Full Text]
Luft FC, Mervaala E, Muller DN, Gross V, Schmidt F, Park JK,
Schmitz C, Lippoldt A, Breu V, Dechend R, Dragun D, Schneider W, Ganten D,
Haller H: Hypertension-induced end-organ damage: A new transgenic approach to
an old problem. Hypertension33
: 212-218,1999[Abstract/Free Full Text]
Moreau P, d'Uscio LV, Shaw S, Takase H, Barton M, Luscher TF:
Angiotensin II increases tissue endothelin and induces vascular hypertrophy:
Reversal by ET(A)-receptor antagonist. Circulation96
: 1593-1597,1997[Abstract/Free Full Text]
Herizi A, Jover B, Bouriquet N, Mimran A: Prevention of the
cardiovascular and renal effects of angiotensin II by endothelin blockade.
Hypertension 31:10
-14, 1998[Abstract/Free Full Text]
Border WA, Noble NA: Interactions of transforming growth
factor- and angiotensin II in renal fibrosis.
Hypertension 31:181
-188, 1998[Abstract/Free Full Text]
Fakhouri F, Placier S, Tharaux PL, Ardaillou R, Dussaule JC,
Chatziantoniou C: Angiotensin II interacts with endothelin and TGF-beta to
produce activation of collagen type I gene in the renal and aortic vasculature
of transgenic mice. J Am Soc Nephrol10
: 344A,1999
Tharaux PL, Chatziantoniou C, Fakhouri F, Dussaule JC: Angiotensin
II activates collagen I gene through a mechanism involving the MAPIER kinase
pathway. Hypertension 36:330
-336, 2000[Abstract/Free Full Text]
Tomita H, Egashira K, Ohara Y, Takemoto M, Koyanagi M, Katoh M,
Yamamoto H, Tamaki K, Shimokawa H, Takeshita A: Early induction of
transforming growth factor-beta via angiotensin II type 1 receptors
contributes to cardiac fibrosis induced by long-term blockade of nitric oxide
synthesis in rats. Hypertension32
: 273-279,1998[Abstract/Free Full Text]
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Top
Abstract
Introduction
2
chain promoter, and made hypertensive by induction of a nitric oxide (NO)
deficiency have been studied. In this strain of mice, luciferase activity is
an early index of renal and vascular fibrosis. Luciferase activity was
increased in preglomerular arterioles and glomeruli when mice were treated
with N
-nitro-L-arginine methyl ester, an inhibitor
of NO synthases. Bosentan (an endothelin receptor antagonist) was as efficient
as losartan (an AT1 receptor antagonist) in preventing renal fibrosis,
although it did not decrease BP. In short-term experiments, angiotensin II
produced an increase in luciferase activity that was entirely prevented by
losartan but also by bosentan. It can be concluded that, during chronic
inhibition of NO, the collagen I gene is activated, which contributes to the
development of nephroangiosclerosis and glomerulosclerosis. Angiotensin II
plays a major role in this fibrogenic process, and its effect is at least
partly independent of systemic hemodynamics and mediated by the profibrotic
action of endothelin-1. 
-galactosidase under the control of two sequences (-19.5 to -13.5 kb and
-350 to + 54 pb) of the promoter of procol I 
