Slide 1

Well, I would like to thank the organisers of this symposium for inviting me to talk about the prorenin receptor and it’s relevance to cardiovascular and renal disease.

Slide 2

Ladies and Gentlemen in our classical view of the renin-angiotensin system as an intravascular system where angiotensinogen is cleaved into angiotensin-1 by circulating renin and angiotensin-1 is then converted into angiotensin 2 by angiotensin converting enzyme. Angiotensin-2 is considered as the major biological peptide that acts on the AT1 receptors and overactivation of angiotensin-1 receptor induces organ damage and that’s what we call the angiotensin-2-dependent pathway. But now we have a new component of the renin-angiotensin system which is the prorenin receptor. This receptor not only allows to focus the renin-angiotensin system in tissues at the cell surface but is also able to trigger a cascade of events that eventually may also lead to organ damage via what we would call an angiotensin-2 independent pathway. The confirmation of this angiotensin-2 independent pathway would mean that in addition to the classical angiotensin receptor blockers; ACE inhibitors and renin inhibitors maybe would need then a prorenin receptor blocker to optimise the renin blockade of the renin-angiotensin system at the tissue level.

Slide 3

The pro-renin receptor is called PRR in the international nomenclature. It’s a 350 aminoacid protein with no homology with any other protein. It is a single transmembrane domain receptor-like receptor for growth factors. It binds equally well renin and its pro-enzyme prorenin and their binding has two major consequences. First the activation of the pro-renin receptor induces ERK1, 2, phosphorylation with subsequent upregulation of profibrotic molecules such as TGF-beta, Pie1, collagens, fibronectin and also induces mitogenic effect. Second the binding of pro-renin induces its non-proteolytic activation just by a change of confirmation.

Slide 4

A while ago we showed that binding of renin and the pro-renin to the receptor induces ERK phosphorylation and this was relatively confirmed by several other groups and in particular by the group of Nancy Noble in Salt Lake City. As you can see here in rat mesangial cells an addition of renin to cells induces ERK 1, 2, phosphorylation which is absolutely not modified in the presence of losartan or enalapril indicating that this ERK phosphorylation is independent of angiotensin 2 generation.

Slide 5

Furthermore, addition of renin also increases TGF-beta 1 expression and increases Pie 1 expression as shown here by this Northern Blot. An addition of UO126 which is an uroquin inhibitor completely abolished TGF-beta1 and Pie 1 increase indicating that ERK phosphorylation is responsible for the upregulation of these powerful profibrotic molecules. The group of Nancy Nobel did even more they gave the definite proof that renin and pro-renin effects were mediated by the pro-renin receptor. What they did to knock down the pro-renin receptor by transfecting rat mesangial cells with silencing RNA and by doing so they completely knocked down the renin receptor and when they knocked down the pro-renin receptor, they completely abolished the ERK phosphorylation induced by renin as well as the completely inhibited TGF-beta upregulation of expression and this experiment definitely confirmed that renin but also pro-renin induced effect are mediated by the pro-renin receptor.

Slide 6

Now concerning the activation, the non-proteolytic activation of pro-renin, why is pro-renin so important to us?
Pro-renin is the pro-enzyme inactive form of pro-renin and there is no auto-catalytic activation of pro-renin. Pro-renin represents up to 90% of total renin in the circulation and whereas renin is only of renal origin produced in the kidney, pro-renin is synthesised in many other organs including the brain, the eye, the adrenal gland, of course the kidney, but also the glands of the reproductive system.

Slide 7

Pro-renin is inactive because the pro-segment covers the active site here in this cleft. In physiology there are only two ways to activate pro-renin. One is by proteolysis of the pro-segment here by a proconvertase that again only happens in the -- cells in the kidney and proteolysis of the pro-segment gives rise to the mature permanently open active site renin.

The other way to activate pro-renin in physiology is by binding to the pro-renin receptor and the binding induces a conformational change that also opens the active site and the active site becomes accessible to angiotensinogen but also to renin inhibitors.

Slide 8

Already a few years ago an intrinsic activity of pro-renin was already suspected. This was demonstrated very elegantly by the group of Tim – in Montreal. What they did is to recreate the human renin-angiotensin system in the pituitary gland of mice. They crossed mice, transgenic for human angiotensinogen with mice transgenic for active renin or mice transgenic for native pro-renin or mice transgenic for non-cleavable pro-renin. Pro-renin that cannot be converted into mature renin. This angiotensinogen and renin were placed under the control of the growth hormone promoter ensuring a strict secretion only in the pituitary gland of these animals. They measured the content of angiotensin 1 in the pituitary gland of these animals. They reasoned that an increase in angiotensin 1 compared to the controlled single transgenic mice would indicate the secretion and the activation of a local renin or pro-renin. So what they found that in the pituitary gland of animal transgenic for human angiotensinogen and active renin there was an increase of angiotensin 1 indicating that there was a low concentration of active renin. So these results were not surprising but what was absolutely not expected is that in the pituitary gland of animals transgenic for human angiotensinogen and native pro-renin there was also an increase in angiotensin 1 indicating that pro-renin that was secreted locally and still was in the form of pro-renin, the pro-segment and the place, this pro-renin was able to show an intrinsic activity. What was even more surprising that in double transgenic animals for human angiotensinogen and non-cleavable prorenin there was also an increase of angiotensin-1. So basically what they showed in this study was that prorenin in tissues was enzymatically active and today we would add that it was enzymatically active because locally it was bound to the prorenin receptor. The prorenin receptor is expressed in many organs at very high levels and in particular in the kidney.

Slide 9

As you can see here, by immunohistochemistry the renin receptor, you have a staining in the glomeruli, in distal cortical tubules and also in collecting cortical tubules. At higher magnification here in the glomerulus you can see the receptor in the mesangium but also in podocytes and in distal tubules here at higher magnification but this time especially at the basolateral site of the cells including the cells of the macula densa.

Slide 10

At even higher magnification in the mesangium you see that the receptor is not expressed in the endothelium but you can find it very nicely stained in the mesangium and also in podocytes. Here in smooth muscle cells of the artery again in the distal tubule cells at the basolateral site of the cells but very low or background level staining in proximal tubules and this is a comparison with ACE in the proximal tubules. As you can see here, ACE is at the apical site of the proximal tubule and the receptor is at the basolateral site of the distal and very low in proximal tubule cells.

Slide 11

But of course, the main issue is to know whether the prorenin receptor is relevant to cardiovascular and renal disease. Today we do have some arguments indicating that it maybe the case.

Slide 12

Again, the group of Nancy Noble in Salt Lake City have studied the expression of the prorenin receptor in rats made nephritic by injection of anti-Thy1 antibodies and further treated with enalapril to increase the renin secretion and what they see by immunofluorescence is that these rats have increased renin staining in the glomeruli. They also have increased renin receptor staining and that both proteins co-localise in the glomeruli of nephritic rats.

Slide 13

More recently Krebs et al have also shown that in rats of the Goldblatt model when they studied the clipped kidney they showed that they have increased renin expression in the clipped kidney that was associated with an increased expression of the prorenin receptor especially in the distal tubules but not in the vessels.

Slide 14

But these are only descriptive and you don’t know if it’s a cause or a consequence but we also have other experimental models that indicate that the prorenin receptor may have a direct role in renal and cardiovascular disease. Such as rats transgenic for the receptor in smooth muscle cells develop high blood pressure and animals with ubiquitous over expression of the prorenin receptor have increased COX-2 expression in the renal cortex and they also have glomerulosclerosis and proteinuria in the absence of high blood pressure and diabetes.

Slide 15

For example, the rats over expressing the prorenin receptor, these rats were generated in the lab of Michael – in Berlin and the prorenin receptor was placed under the control of the smooth muscle myosin heavy chain ensuring a strong expression in smooth muscle tissue and in particular in smooth muscle cells. As you can see here, by in situ hybridisation the aortas of transgenic rats have an intense labelling with a radiolabelled probe. The aortas of these rats also have increased prorenin uptake and the rats develop high blood pressure at the age of 9 months in spite of normal renin and normal angiotensin-2 concentrations.

Slide 16

But they also have a bunch of experimental data indicating an indirect role for the prorenin receptor in cardiovascular and renal disease and this time because of its ability to non-proteolytically activate prorenin. All these models were performed in the group of Ichihara and they were based on the use of the prorenin receptor blocker called HRP. They were based on the ability of this blocker to totally prevent proteinuria and glomerulosclerosis in diabetic animals and maybe their most spectacular demonstration was obtained in diabetic AT1A receptor knockout mice.

Slide 17

But why did they use the diabetic models? Because in diabetes you have an increased prorenin level compared to very low renin activity. Because already 20 years ago – et al observed in a cohort of diabetic patients that all patients with microvascular complications either microalbuminuria or retinopathy indicated here by the triangles, all these patients with microvascular complications have prorenin levels above the normal limit indicated here by the red line. Whereas patients without microvascular complications the open square have prorenin levels under the normal limits.
A few years later they confirmed this observation and they even added a prognostic value to the prorenin levels and in a cohort of diabetic children they were able to show that the children with the highest prorenin levels above the upper limit were at higher risk to develop microvascular complications than the children with prorenin in the normal range.

Slide 18

What Ichihara did was to use a peptide, a decapeptide called Handle Region Peptide, HRP which reproduces, which mimics part of the pro-segment of prorenin and this peptide would act as an antagonist of the receptor. It would block prorenin binding to the receptor therefore, it would block angiotensin generation on the cell surface and also block ERK activation mediated by the receptor. But I would like to stress here that this AT1 therapy would only act in situations with a high pro-renin because it would never inhibit active prorenin binding to the receptor because renin is devoid of the pro-segment which has been cleaved off.

Slide 19

What they did is to compare the effect of this PRR blocker to an ACE inhibitor in diabetic mice. In diabetic wild-type mice and to make a very, very long story short I would like you to focus only on 2 parameters; proteinuria and glomerulosclerosis index. So in diabetic wild-type mice they compared ACE inhibitor and this prorenin receptor blocker and what they saw is that ACE inhibitor was able to reduce proteinuria and to reduce glomerulosclerosis as expected. But surprisingly the prorenin receptor blocker was even more efficient and in AT1A receptor knockout mice so in mice were the angiotensin-2-dependent pathway is completely blunt is not existing well ACE inhibitor has no effect on proteinuria and glomerulosclerosis as expected but surprisingly again the prorenin receptor blocker was extremely efficient and these results are extremely surprising because they would cast prorenin and the pro-renin receptor as the central player in diabetic nephropathy. These results really need confirmation.

Slide 20

But recently other authors have shown that high prorenin per se is associated with high blood pressure but not with kidney fibrosis. The group of Peters et al have used rats expressing the – gene under the control of the cytochrome promoter which can be activated by xenobiotics indole-3-carbynol. They showed that when they induce the synthesis of mouse prorenin over 200-fold shown here because this is a log scale, these animals were hypertensive not a malignant hypertension but these animals were hypertensive. Even though they kept these animals hypertensive for 3 months they couldn’t see any glomerulosclerosis.

Slide 21

Their conclusion was that per se prorenin is not a profibrotic molecule you may need an additional situation like inflammation or whatever but the other possibility would also be that as a real hormone prorenin is also able to reduce, to down regulate the synthesis of its receptor and this was shown by – et al who showed that when the cells with a prorenin receptor were stimulated by renin or prorenin, they activated transcription factor called PLZF pro-leukemic zinc finger and this PLZF was able to bind a cease acting element on the prorenin receptor, gene promoter and to downregulate its expression.

Slide 22

In summary the prorenin receptor axis maybe considered as a -- garden of 2 doors one door that increases angiotensin generation at the cell surface and it’s the angiotensin-2-dependent pathway and it may also activate MAP kinase and have potential profibrotic effects and this would be the angiotensin-2 independent pathway again.

Slide 23

We now have animal models that suggest that the prorenin receptor has a role in hypertension but we don’t have convincing arguments that it may play a role in diabetic nephropathy and these issues will be soon solved by prorenin receptor tissue specific knockout mice. If the role of the prorenin receptor is confirmed, then we should really question the benefit of a blocker to optimise the tissue renin angiotensin system blockade.

Slide 24

I would like just to acknowledge all the collaborators in Paris but also the external collaboration of the groups in Germany -- and in the Netherlands, in Rotterdam and Groningen. I thank you for your attention.

Slide 25

Chairman: Thank you very much for this perfect overview. Questions? Yes please.

Question: Doctor Wiecek, Poland. It was a very nice overview but can you clarify, tell us that prorenin binds to each receptor with the handle region and then opens the active site of prorenin, how is it possible that renin which has no handle region is also capable of binding to the same receptor?

Dr. Nguyen: Yes that’s what I mentioned because basically we found that renin and prorenin were able to bind to the receptor of similar affinity and they compete for each other binding. This indicates that at least the receptor has two binding sites one for the pro-segment and one for another domain which has the renin and prorenin molecule. But obviously another domain which is not in the pro-segment.

Question: Congratulations indeed but I’m a little bit confused. From the clinical point of view you have very good results with ACE inhibitors and angiotensin-2 receptor antagonists. What do you think about aliskerin in this kind of prospective?

Dr. Nguyen: I’m sorry I didn’t get really your point. You’re asking about the effects of aliskerin or … ?

Question: Aliskerin in these situations.

Question: Of course we were interested in knowing if aliskerin was able to interfere with renin and prorenin binding to the receptor and to modify ERK activation and again to make a long story short aliskerin has absolutely no effect on renin and prorenin binding to the receptor. It has no effect on ERK activation therefore but in vivo aliskerin is able to reduce the receptor expression.

Chairman: Professor Ritz last question.

Prof. Ritz: Thank you for a fantastic update in this area. I have one part of concern with respect to the Ichihara finding that you commented upon. There is a paper out from the group of Muller and Frederick Luft who were unable to confirm the entire sequence. How do you explain this difference in laboratory methodology or the area is confusing to be polite to the outsider?

Dr. Nguyen: It’s very confusing to me too. What Ichihara claims is that the handle region peptide only works in situations where you have high prorenin and low renin therefore they choose diabetic setups but the problem is that together with Dominic Muller we found that this HRP was also able to bind cells that are devoid of the prorenin receptor. So to the best of my knowledge I would say that this HRP is not a specific antagonist to the prorenin receptor and that it acts via another pathway.

Prof. Ritz: That maybe a good explanation. Thank you.

Chairman: I’m sorry to interrupt the discussion which is fantastic one but unfortunately we are short of time. Thank you very much Madame Nguyen.