Nephrology Dialysis Transplantation 2005 20(10):2050-2053; doi:10.1093/ndt/gfi143
Published by Oxford University Press on behalf of ERA-EDTA [2005].
Translational Nephrology
‘As time goes by’: angiotensin II-mediated transactivation of the EGF receptor comes of age
Gunter Wolf
Department of Internal Medicine III, University of Jena, Germany
Correspondence and offprint requests to: Gunter Wolf, MD, Department of Internal Medicine III, University Hospital Jena, Erlanger Allee 101, D-07740 Jena, Germany. Email: gunter.wolf{at}med.uni-jena.de
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The renin–angiotensin system (RAS) and progression of renal disease
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Clinical studies have provided ample evidence that inhibition
of the RAS with ACE inhibitors and/or AT
1-receptor blockers
is an effective strategy in slowing the progression of chronic
renal disease [
1–3]. Although it was initially thought
that inhibition of the RAS is renally protective through haemodynamic
mechanisms (for example by reduction of hyperfiltration), it
is now clear that angiotensin II (ANG II) is a multifactorial
cytokine exhibiting growth stimulatory, proinflammatory and
profibrotic effects [
4–6]. These more pleiotrope effects
of ANG II on the kidney were initially greeted with some skepticism
[
5]. At a time when the specific receptors for ANG II have only
just been cloned and the ANG II-receptor blocker, saralasin,
was only recently replaced with more specific agents, these
reservations were not surprising [
6].
ANG II binds to various receptors [6]. The AT1-subtype is involved in many of the deleterious effects of the peptide. Signalling through the AT1-receptor (a receptor with seven membrane-spanning domains) involves G-proteins (Gq) leading to activation of phospholipase C and the subsequent generation of diacylglycerol and inositol trisphosphate, which in turn stimulate protein kinase C and increase intracellular calcium [3]. In addition, activation of various protein kinases such as extracellular signal kinases 1,2 (Erk 1,2), the phosphatidylinositol 3-kinase (PI3K)-dependent kinases Akt, and the mTOR/S6 kinase pathway are necessary for ANG II-mediated growth responses including cardial and renal hypertrophy [7–9]. Although activation of some of these kinases, for example Erk 1,2, could be explained by protein kinase C, stimulation of other signal kinases is difficult to understand because the AT1-receptor lacks intrinsic tyrosine kinase activity.
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Relationship between ANG II and the EGF receptor
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Earlier findings suggested a close relationship between ANG
II and epidermal-growth factor (EGF)-mediated cellular effects.
Norman
et al. [
10] studied the potential interaction between
ANG II and EGF in proximal tubular cells already in 1987. They
found that ANG II itself was not mitogenic for the cells, but
it amplified the proliferative effect of EGF and the dose-response
curve of EGF-stimulated mitogenesis was shifted to the left
in the presence of ANG II. However, ANG II had no effect on
the binding of EGF to its putative receptor and also did not
influence receptor down-regulation. Norman
et al. [
10] concluded
that ‘ANG II potentiates EGF-induced mitogenesis at one
or more postreceptor steps’. In 1990, we described in
a cultured murine proximal tubular cell line that ANG II-pretreatment
further enhanced EGF-induced cell division by
40% [
6]. Although
there were similarities between EGF and ANG II in the induction
of immediate early genes, EGF stimulated mitogenesis whereas
ANG II induced hypertrophy in proximal tubular cells [
6,
11].
These early findings provide evidence of a potential interaction
between ANG II and EGF-mediated signalling.
In 1996 Axel Ullrich's group discovered a pathway showing how G-protein coupled receptors such as the AT1-receptor could cause phosphorylation and activation of the EGF receptor [12]. ANG II was initially not studied, but the investigators found that endothelin 1, lysophosphatic acid and thrombin all activate G-coupled receptors without a kinase domain, phosphorylate tyrosine residues on the EGF receptor and thereby activate other downstream kinases [12]. These findings were rapidly extended to ANG II. For example, in cardiac fibroblast ANG II stimulates tyrosine phosphorylation of the EGF receptor, a process named transactivation [13]. Further studies revealed that stimulation of G-protein coupled receptors leads to EGF-receptor phosphorylation through activation of metalloproteinases (MMP). After ligand binding to the G-protein coupled receptors, MMP is activated inducing cleavage of pro-heparin-binding EGF (HB-EGF), which liberates a soluble HB-EGF that binds to and activates the EGF receptor [14,15].
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A novel mechanism of ANG II-mediated transactivation of the EGF receptor
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The study by Lautrette
et al. [
16] provided an important new
twist to this fascinating story. The investigators studied transgenic
mice overexpressing a dominant negative form of the EGF receptor
[
16]. These mice, which could not activate the EGF receptor
and downstream kinases, were protected from renal lesions (e.g.
glomerulosclerosis, mononuclear cell infiltration, tubular fibrosis
and atrophy) induced by 2 months of ANG II-infusion compared
with normal non-transgenic mice that bear a functioning EGF
receptor. ANG II-infused EGF receptor mutant mice exhibited
significantly less proteinuria compared to ANG II-treated wild-type
mice, but tail-cuff measured arterial blood pressure was apparently
identical in both groups. These results indicate that the damaging
effects of ANG II-infusion are mediated by EGF receptor transactivation
rather than by systemic hypertension. Lautrette
et al. [
16]
next studied potential mechanisms by which ANG II transactivates
the EGF receptor during renal injury. Incubation of a rat liver
cell line with ANG II induced secretion of transforming growth
factor-
(TGF-
), a potential ligand of the EGF receptor. TGF-
is released from a larger integral membrane precursor called
pro-TGF-
. An important enzyme in cleaving TGF-
from its transmembrane
precursor is TACE (tumor necrosis factor
converting enzyme),
first identified in the processing of membrane-bound precursors
of tumor necrosis factor
[
17]. A pharmacological EGF receptor
antagonist as well as specific TACE inhibitor prevented ANG
II-mediated kinase activation in rat hepatocytes. Lautrette
et al. [
16] then tested whether similar mechanisms are operative
in ANG II-induced renal injury
in vivo. First, ANG II infusion
stimulated a marked increase in tubular (ascending limb of Henle's
loop and distal tubule) TGF-
protein expression without concomitant
mRNA increase, suggesting that more membrane-bound pro-TGF-
is converted to active TGF-
. In parallel, immunostaining for
TACE but not mRNA expression increased in ANG II-infused mice
at the same tubular localization as TGF-
. It appears that ANG
II-infusion leads to a TACE translocalization from the perinuclear
compartment to the cell surface. Treatment of ANG II-infused
animals with a TACE inhibitor blunted ANG II-induced TGF-
accumulation,
EGF-receptor phosphorylation and renal damage without influencing
blood pressure. Similar effects were also observed in TGF-
‘knockout’
mice in which ANG II failed to transactivate the EGF receptor.
Since ANG II infusion is a rather unphysiological model of kidney
injury, the authors finally studied a renal ablation model in
mice (75% reduction of renal mass). Immunohistological and western
blot analyses revealed that both TGF-
and TACE, but not EGF
protein levels, increased 2 months after ablation at a time
when renal injury developed. Treatment with an AT
1-receptor
antagonist prevented these changes, indicating that renal ablation
has led to intrarenal RAS activation as previously described
[for review see 3]. In summary, these elegant experiments suggest
the following signal transduction pathway (
Figure 1). ANG II
binds to G-protein coupled AT
1-receptors leading to a redistribution
of TACE from the cytoplasm to the cell surface. How this works
is currently unclear. Cell surface-associated TACE has an increase
in half-life time and cleaves adjacent membrane-associated pro-TGF-
to release active TGF-
. TGF-
, in turn, binds to the EGF receptor
in an autocrine or paracrine manner and activates the receptor-associated
kinase cascade leading to phosphorylation of mTOR/S6 kinase,
PI3K/Akt and Erk 1,2 (
Figure 1). This quite complicated signal
transduction pathway goes from the outside (ANG II) to the inside
(TACE), again to the outside (TNF-
) and finally returns to the
inside of the cell (EGF-receptor phosphorylation). Nature is
complicated.
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Anything wrong with this landmark study?
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I have some doubts in believing that this is the only mechanism
by which ANG II mediates renal damage. Previous studies have
shown that ANG II or its degradation products (e.g. angiotensin
IV) bind to receptors other than AT
1 and could mediate proinflammatory
and profibrotic effects through these receptors [
18,
19]. It
is currently unclear whether ANG II-induced TACE translocalization
and expression is only mediated by AT
1-receptors. In addition,
it remains to be established whether the TACE inhibitor used
may also inhibit other enzymatic systems. For example, assuming
that the TACE inhibitor interferes with other matrix-degrading
proteins, disruption of the basement membrane, the first process
in initiation epithelial–mesenchymal transition that is
important in interstitial fibrosis [
20], would be prevented.
In addition, EGF receptor transactivation has been linked to
the release of HB-EGF and activation of another enzyme metalloprotease
(ADAM12) in ANG II-stimulated cardiac hypertrophy [
21,
22], The
potential role of HB-EGF in the current renal injury model requires
further study because TACE may also release HB-EGF. Finally,
inducing chronic renal injury and making physiological measurements
(e.g. blood pressure measurements) is a challenging task in
mice [
23]. Measurements of blood pressure with the tail-cuff
method could be difficult [
23]. In contrast to findings by Lautrette
et al. [
16] who observed a similar degree of ANG II-induced
hypertension in wild-type mice compared to TGF-
‘knockout’
animals, application of antisense oligonucleotides against the
EGF receptor significantly reduced hypertension in ANG II-infused
rats indicating that EGF-receptor transactivation is also involved
in ANG II-mediated blood pressure regulation [
24]. Since there
is increasing evidence that growth factors such as transforming
growth factor-ß or EGF may be involved in regulation
of renal haemodynamics [
25,
26], it would be interesting to know
whether treatment of ANG II-infused mice with the TACE inhibitor
influences renal haemodynamics.
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What do the novel findings mean for the practicing nephrologist?
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One could argue that the mechanism of inhibition of the RAS
that slows progression of renal diseases does not matter as
long as ACE inhibitor and AT
1-receptor antagonists do their
job. However, this treatment strategy is far from being perfect
and patients are still reaching end-stage renal failure [
27].
Experimental models have provided convincing evidence that high
doses of ACE inhibitors or AT
1-receptor antagonists could induce
regression of previously deduced fixed structural renal changes
such as fibrosis [
28,
29]. The study by Lautrette
et al. [
16]
identified novel potential targets to treat chronic renal disease.
It would be worthwhile to study pharmacological inhibition of
TACE to decrease release of ANG II-mediated TNF-
and transactivation
of the EGF receptor in patients with chronic renal diseases.
Presumably, such treatment could provide therapeutic benefit
beyond dual blockade of the RAS with ACE inhibitors and sartanes.
Such innovative strategies are urgently needed to deal with
the future deluge of patients with chronic kidney disease.
Sam, the piano player in Michael Curtiz's 1942 classic ‘Casablanca’, sings in ‘As Time Goes By’: ‘And no matter what the progress, or what may yet be proved, the simple facts of life are such they cannot be removed’. Although Sam originally addressed the more romantic facts of life, he was wrong in terms of renal disease progression. The simple pathophysiological fact of progression could be efficiently halted by RAS blockade and experimental evidence suggests that the simple facts can even be removed. A better understanding of the many pathophysiological effects of ANG II including the novel observations made by Lautrette et al. [16] convincingly contribute to such a progress.
Conflict of interest statement. None declared.
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The renin-angiotensin system...
Relationship between ANG II...