CME Slides Forum - B. Rodriguez-Iturbe

INTRARENAL INFLAMMATION AND OXIDATIVE STRESS AS A COMMON FINAL PATHWAY IN SALT-SENSITIVE HYPERTENSION

Bernardo Rodriguez Iturbe, Maracaibo, Venezuela

Chair: Bernard Canaud, Montpellier, France

Ramon Romero, Badalona, Spain

rodrigueziturbe

Prof B. Rodriguez-Iturbe
Hospital Universitario de Maracaibo
Centro de Investigaciones Biomedicas
Maracaibo, Venezuela

Doctor Romero and Doctor Canaud thank you very much for the kind introduction. My task today is to cover in around 20 minutes actually the role of the immune infiltration in the kidney and salt-sensitive hypertension.

And a way to begin actually is by this very long observation that if you follow the translations that were done as early as 762 of our era, if large amounts of salt are taken, the pulse will stiffen and harden.

Of course, it’s of no news to this audience that there are a number of factors that are involved in salt-sensitive hypertension which actually implies a tendency of the, kidney to retain salt. These findings of interstitial inflammation, oxidative stress and renal angiotensin activity we think are involved in the final common pathway in the sodium retention and that conduces to arterial hypertension.

In the next 20 minutes I will attempt to guide you through the evidence supporting that intrarenal inflammation, oxidative stress and angiotensin activity are in fact a final common pathway in salt-sensitive hypertension.

When I say guide let me remind you that the guide is definite however, reality is frequently inaccurate.

With this in mind let me point out first that there are mechanisms of sustained hypertension that are driven by sodium retention and that they depend on volume-mediated effects which are essentially the production of endogenous ouabain-like steroids. We have direct pressure-independent effects due to sodium. These include myocardial hypertrophy, contractility of the vascular smooth muscle, cerebral central nervous system induces increase in sympathetic tone, increase in tissue angiotensinogen receptors and TGF beta and of course, intrarenal oxidative stress and inflammation and angiotensin II activity.

There is a long list of pathophysiological mechanisms resulting in a sustained tendency to sodium retention by the kidneys. You all know that you have genetic defects, you have systemic effects as well that include, of course, increased sympathetic tone, potassium depletion, insufficient suppression of the RAAS system etc. I’m not going to go into all the details.

You also have renal specific defects that induce A sustained tendency to sodium retention and then you have what I would call non-specific defects which include a reduced number of nephron units. Sodium driven by the direct sodium stimulation of TGF-ß production or overreproduction, reduced kallikrenin activity and the group that I put the red square around which are actually the subject of today’s talk. If you allow me to take to a simplified pathogenic scheme, if you will of sodium retention and hypertension, as I was mentioning you have systemic and specific genetic defects driving sodium retention. As a consequence you have reduced sodium excretion, sodium retention and if you have sodium retention, you have extracellular expansion with the production of ouabain-like compounds which will increase sodium concentration within the cells and activation of the sodium potassium exchanger with an increase in calcium inside and of course, you will have increased heart contractility and contraction of the vascular smooth muscle. The effect is driven by the increase in sodium and osmolarity which are some of the effects that I mentioned before. This, of course, within the kidney will increase the TGF-ß activity, expression of the receptors of the angiotensin especially of the receptor 1, increase angiotensin oxidative stress and inflammation which will impair pressure natriuresis and close the circle to reduce sodium excretion.

Now the compound effects of this sodium retention with expansion and increase in sodium osmolarity will increase the peripheral vascular system and cardiac output and hypertension. The increase of intrarenal hypertension, angiotensin and oxidative stress will fuel this process from within. Now, this I would call it tripod if you will or three legged stool feeds itself because each one of the compounds of this tripod sort of like feeds one another. You can see that immune cell infiltration of course, will increase oxidative stress both from the resident cells and from the infiltrating cells, the reactive oxygen species generated will stimulate among other things NF-kB which, of course, increases inflammation and the immune cell infiltration this is on the left side of the slide. On the other side, on the right side of the slide you see that immune cell infiltration activates or activated macrophages and resident cells will produce or increase intrarenal angiotensin activity which will in turn increase a number of molecules, of course, a transcription factor kB, adhesion molecules, MCP-1 that will increase immune cell infiltration. Finally, in the lower part that there is a connection between intrarenal angiotensin II and oxidative stress through vasoconstriction, ischemia and of course, a stimulation of NADH/NADPH system.

Essentially what we’re saying is that if you have oxidative stress, you have NF-kB activation, cytokine production, inflammation, infiltration of leukocytes, particularly macrophages and lymphocytes which will in turn increase production of reactive oxygen species, anti-oxidant depletion and oxidative stress.

This three legged stool or tripod that we are talking about are the driving mechanisms for sodium retention.

Now, once they are there you have tubular interstitial as well as glomerular effects. On the glomerular side you have loss of autoregulation, structural damage and decrease in GFR and as a consequence of that you have decreased filtered sodium and on the tubulointerstitial side you have inflammation, reduction of peritubular capillary area and the net functional effect of those things are an increase in sodium reabsorption and an impaired pressure natriuresis. Once hypertension is there then, of course, you have a negative feedback that as the pressure rises will tend to increase the filtered sodium and tend to improve or increase pressure natriuresis. However, on the other side hypertension thus increases by itself reactive oxygen species and oxidative stress so it actually fuels this three tripod legged little – that I have in there. Because there is cross-talk between glomerular and interstitium for this.

Now a high salt diet actually acts and influences all the three parts of this tripod. On the one side a high salt diet induces interstitial inflammation and fibrosis as shown by Sanders and others and stimulates NF-kB as shown by Doctor Gu and Doctor Siegel. On the renin angiotensin side will increase the expression of angiotensin receptor in arterioles and increase the production of angiotensin in proximal tubules and on the oxidative stress side the high salt diet will increase superoxide and peroxide production will increase NAD(P)H oxidase and will try or will tend to reduce nitric oxide generation by decreasing arginine transport and decreasing NOS isotypes.

So if we take each one of these three components apart I’m going to show you how interstitial inflammation induces or a reduction of interstitial inflammation improves salt-sensitive hypertension and I’m going to show you some studies with mycophenolate, mofetil mycophenolate and some studies in which we did inhibition of the NF-kB to reduce interstitial inflammation.

These models that I show you here are models in which the suppression of the intrarenal inflammation results in a correction or amelioration of the hypertension. The red arrows there show the models in which we have done some work and of course, I will be referring to them particularly but there several other groups that have studied other models that actually substantiate and support this evidence.

Let me give you an example first a standard one and this will be more or less applied for the rest of the talk. When you have salt-sensitive hypertension in experimental models what you do is to induce hypertension or induce whatever mechanism you are testing in this case by the administration of L-NAME. Then you stop the L-NAME. During the L-NAME administration the blood pressure goes up and when you stop the L-NAME in this particular model, blood pressure comes down. Then you start the high salt diet and you see that the previously treated animals with L-NAME or the animals previously treated with L-NAME the blood pressure goes up in contrast with the animals not treated with L-NAME in which the high salt diet does not increase the blood pressure.

Now the point is that if you treat these particular animals with mycophenolate during the time in which they are receiving L-NAME, the blood pressure still goes up just as the untreated animals do however, once you stop the L-NAME and give them a high salt diet, the blood pressure remains within control levels. Therefore, one can see that the prevention of inflammation there prevents a subsequent development of salt-sensitive hypertension.

Now, there are a number of models but there is essentially the same type of mechanism. This is with the angiotensin infusion showing essentially the same thing I showed you a moment before, the L-NAME which I just showed. This is the protein of an old model in which you give intraperitoneally injections of protein and show that this induces, as you all know interstitial inflammation and then when you give a high salt diet, the blood pressure goes up but if you treat these animals with mycophenolate the salt-sensitive hypertension phase is aborted, prevented.

Most recently studies with a hypertension induced by a chronic low level exposure to lead in which essentially what you can show is that you treat these animals with led and in addition to led you give them mycophenolate, blood pressure is maintained and you have, of course, the controls, the appropriate control groups. Now, let me go over one particular model which I think is interesting to bring the point home. If you have spontaneously hypertensive rats like we have here and you give them mycophenolate in two periods of time separated by a period of time in which you do not give the drug, what you find is the following: the untreated rats increase their blood pressure like shown here in the green squares but if you give mycophenolate in two periods, the blood pressure of the little pets comes right down to almost levels of the control with – levels. If you stop mycophenolate, the pressure goes up and almost meets the untreated rats over here. Then if you again repeat the mycophenolate administration, again you see the same effect. You have two periods with mycophenolate separated by one period of untreated groups that show you or brings the point home. What you find is that during the two periods and not surprisingly when you give mycophenolate, which are in the two periods over here which are this one and this one, you have reduction in lymphocyte infiltration and macrophage infiltration in the lower part and at the time when the pressures are similar in the two groups where mycophenolate was stopped then, of course, the infiltration of immune competent cells is ---.

In this study you can also demonstrate that there is a correlation between the systolic blood pressure and the infiltration of lymphocytes as shown in this particular slide.

Now, this is not only a thing you can do with mycophenolate, you can do that also with an inhibition of NF-kB with pyrrolidine dithiocarbamate. As shown here, when you use spontaneously hypertensive rats and you give them this compound that inhibits NF-kB, the NF-kB the P65 the DNA binding unit is, of course, reduced and you see in the slide at 12 and 25 weeks how the PDC treated SHRs are comparable to the WKY and as a result of this treatment you have a reduction in the infiltration of lymphocytes which are CD5 positive cells, macrophages of CD1 positive cells and you have a reduction of the oxidative stress shown in this particular case as a content of the renal MDA, malondialdehyde within the kidney. As a result of that you can see that the SHRs treated with PDTC become or stay if you will normotensive at the same levels, so that the WKY and the WKY treated with PDCT rats.

Let me go now to the oxidative stress. The oxidative stress, of course, is a reduction of the oxidative stress that improves salt-sensitive hypertension and in addition reduces intrarenal inflammation.

There is an abundance of evidence that links oxidative stress with hypertension. I’m just showing you there some of the well known data there, of course many others but hypertension induced by any means will increase oxidative stress or reactive oxygen species. A variety of anti-oxidant treatments improve hypertension.

Now, recently we have also shown that if you have a genetic deficiency, a partial deficiency of mitochondrial superoxide dismutase results in salt-sensitive hypertension. These are mice with a partial SOD2, the superoxide dismutase 2, the mitochondrial kind deficiency. You know the complete knockout mouse dies shortly after birth with a cardiac myopathy and this particular partial deficiency is characterised by mitochondrial disarrangements that are associated with what looks like senescence. But anyway these animals when given a high salt diet develop salt-sensitive hypertension and not surprisingly these animals when given a high salt diet will develop intrarenal infiltration of immune competent cells which are in fact, in this particular case macrophages related to the levels of hypertension achieved in these mice.

This is another study to drive home the same point. In this particular study where spontaneously hypertensive rats treated with an antioxidant diet that will be the T group or with a regular diet the R group or switched from one diet to another. The point here was to get a range of blood pressure and infiltrations. This allowed us to show how the infiltration of macrophages, lymphocytes or angiotensin II positive cells were in fact, correlated with the blood pressures in this group of rats.

Now let me go to the third and final part of this tripod which is intrarenal angiotensin activity or angiotensin system and I’m going to show you how there are in salt-sensitive hypertension increasing numbers of angiotensin II positive cells and I’m going to show you that not only cells but intrarenal angiotensin II activity is actually increased and I’m going to show you that in three models, in the Page (cellophane) wrapping of the kidneys, in post angiotensin II salt-sensitive to hypertension and in lead induced hypertension. Finally, I’m going to show you how the intrarenal not the plasma, the intrarenal angiotensin II activity correlates with salt-sensitive hypertension in a very exquisite manner.

Angiotensin II positive cells are present in salt-sensitive hypertension, we have shown this. And this is a slide borrowed from Doctor Mezzano showing both proximal tubular cells, as well as infiltrating cells. Actually, the infiltrating cells that I’m showing you here in a double staining preparation you can see that rhodamine-labelled macrophages below some of them are actually expressing angiotensin II on the surface in the upper fluorescein-labelled slide.

Now, Valentina Vanegas in our group was interested in the Page kidney model that as you know was described a number of years ago by Page as another model of inducing something that appeared to be like a Gold-Blatt hypertension. This meant that the --- that involves the kidney when you have cellophane wrapping of the kidney will contract the kidney and will increase plasma angiotensin and produce thereby hypertension. Well, what we found was that actually the plasma angiotensin shown in the right part of the slide is perfectly steady as the blood pressure of these rats go up and up and up. When we examine the kidneys, there is a very intense infiltration of immune cells like I’m showing you there and if you give mycophenolate to these rats, you in fact can wipe out this infiltrate and in addition to this improvement in the inflammation you get a reduction in the intrarenal angiotensin activity and as a consequence, you get a correction of the hypertension in the upper left hand square.

The third model that I was going to show you in this part of the talk was the chronic low dose lead exposure that as you know, causes hypertension and this is in the right side upper part where you have the mean intra-aortic blood pressure increased in the lead exposed rats and how when you give mycophenolate, the blood pressure is corrected to control levels.

Now, if you give to these rats mycophenolate, as I’ve shown you there, you get a decrease in angiotensin II positive cells, you get a decrease in actual renal angiotensin activity shown in the upper part showing that they go hand in hand.
The last part of this talk actually is to show you how intrarenal inflammation and intrarenal angiotensin II activity in salt-sensitive hypertension are exquisitely correlated with the hypertension. Let me first tell you that we are talking about rats here in the angiotensin II infusion model. You give angiotensin and as you know, there is intrarenal angiotensin activity that remains after the angiotensin is stopped and then you have in this particular model, as I’m showing you there, you have angiotensin II infused, you have angiotensin plus mycophenolate, you have controls with a high salt diet and controls with a normal salt diet. You can see how the plasma angiotensin actually decreases as the blood pressure goes up which is what you would expect in a salt-sensitive hypertension in which you expand the extracellular volume. Therefore, the plasma is actually decreasing as the pressure goes up however, the intrarenal angiotensin actually is increasing and this is almost like a mirror image of what you have above.
A positive correlation between renal angiotensin activity, with blood pressure and a negative correlation of the plasma one. Of course, as you would expect, macrophage infiltration in the upper part is correlated with systolic blood pressure and is correlated, possibly correlated also with a renal or intrarenal angiotensin II activity.

Well, it’s very difficult to design a study with any of these drugs, mycophenolate in hypertensive patients because it’s not ethical we have very safe and good drugs to treat hypertension. But what Josè Herrera and a group was interested is in looking at it from another angle what he studied was 8 patients that had psoriasis or rheumatoid arthritis, all of them had grade I hypertension and normal renal function. They were given by their own physicians mycophenolate not for hypertension but for the psoriasis or the rheumatoid arthritis.

Now, for three months they received this drug for their respective treatments but they agreed to have no other change in medication, no change in sodium intake, no change in protein exchange and if the referring physician felt that a change was needed, then the patient would be excluded from the study. This is what we found actually that during the 3 months of mycophenolate there was a reduction of the mean blood pressure and once the mycophenolate was stopped the pressure came straight up. This was accompanied or associated with a reduction or rather with a correlation between the levels of blood pressure and TNF-α in the urine, RANTIS in the urine, MDA concentration in the urine and TNF and RANTIS, of course, amongst them.

I just want to end up by recognising that the stories that we have shown, that I have shown you here have been made in collaboration with Jaime Herrera from the Instituto Nacional de Cardiologia in Mexico and Rick Johnson from Gainsville and Nick Vaziri from the University of California and the group of very talented people in my laboratory. Thank you very much.