CME Slides Forum - W.E. Mitch

A joint Congress by ERA-EDTA and ISN

THERAPEUTIC INTERVENTIONS FOR PROTEIN-ENERGY WASTING IN CHRONIC KIDNEY DISEASES

William E. Mitch, Houston, USA

Chair: Christian Combe, Bordeaux, France

Joel D. Kopple, Santa Monica, USA

mitch

Prof. William E. Mitch
Department of Nephrology
Hospital Edouard Herriot
University Lyon 1
Lyon, France

Thank you very much for the introduction and the opportunity to tell you about some of our work over the past years. I’ll start off by showing something that’s familiar to all of you.

Here are some data from Joe Coppell and his colleagues published many years ago in which they assessed the abnormalities of patients with anaemia specifically those being treated by hemodialysis shown in yellow and compared them to age and gender match control subjects looking at body weight, body fat and an estimate of muscle mass, the so-called mid arm muscle circumference. What the slide indicates is that there is a serious problem for patients with uraemia because they have lost body weight and this is comprised of both an abnormality on body fat, as well as loss of muscle mass.

So the question is why does this occur? We addressed this many years ago and I’ll just show you one experiment to give you insights into how we evaluated the problem. Basically what we did was to create rats and more recently mice with CKD by removing most of the kidney and then feeding the animals a very high protein diet, so as to induce the same blood chemistries that one would find in patients. Then after this had been induced we paired the rats with sham-operated and fed them for at least 2 weeks with a high protein diet, so that at this stage of the experiment we had rats with a BUN of 92 mg/dL and a serum bicarbonate that averaged 16 mmol. Specifically they had uraemia, a high BUN and metabolic acidosis. So when we took the muscles from these animals and measured the rate of protein breakdown in these muscles that were isolated, we found that there was a sharp increase in the rate of muscle protein breakdown shown in red compared to the value in sham-operated pair fed control animals shown in yellow.

The interesting thing about this experiment however, was that these abnormalities were eliminated simply by correcting metabolic acidosis. Specifically when we mixed bicarbonate in with the feed of these animals, the rate of protein breakdown in muscles in rats that were uremic and fed bicarbonate was exactly the same as their pair fed sham-operated controls.
So the conclusion from this slide is obvious namely that’s there no reason to keep the animals or by inference patients with CKD acidotic because it will stimulate protein breakdown and the loss of muscle mass.

Now the problem with this is, ok what’s the stimulus for this? What we did was to examine the same model and measure the intracellular pH and what we found was that it’s not abnormal. So that the muscle intracellular pH was not different in those animals that were uremic and acidotic compared to sham-operated pair fed controls.
When we examined this a little bit further and looked at other conditions that stimulate protein breakdown in muscles; sepsis, trauma, inflammation, cancer, as well as high doses of glucocorticoids, what we found was that each and every condition not only stimulates protein breakdown but is characterised by insulin resistance.
So the question was, can defects in insulin and IgF1 signalling cause muscle protein wasting in uraemia, as well as other catabolic conditions?

So the way we approach this is to examine animal models of diabetes either streptozotocin-induced as a model of acute type 1 diabetes or db/db mice as a model of type 2 and you can see in this slide the red fibre soleus muscle, the white fibre EDL muscle and the mixed fibre plantaris muscle in every case protein breakdown was higher in muscle of diabetic animals suggesting that defects in insulin signalling could indeed cause abnormalities in muscle metabolism that are characterised by stimulation of protein breakdown.

Now, I don’t have time to go through all of the different proofs of this but what we concluded is shown in this slide namely that there is activation of the ubiquitin proteasome system. The way this system works is that somehow or another in cells a protein substrate that’s going to be degraded through an ATP-dependent reaction involving 3 enzymes is conjugated to a small protein called ubiquitin. Ubiquitin is named ubiquitin because it’s present in all cells. After conjugation to a protein there’s a chain of ubiquitins created that allows it to be recognised by this very large multi-subunit comprised molecule called the proteasome. What this molecule is able to do is it straightens out this coil chain of protein, knocks off the ubiquitin which can be recycled, injects it into the central core so that it’s degraded to peptides and aminoacids.

So we believe that this abnormality in protein breakdown is due to activation of that molecule and more specifically if there’s impaired IgF1 or insulin signalling what happens is that there’s a decreased activation of the intracellular signalling pathway with a reduced activity of PI3K, a decrease in Akt-P and this allows this fore kid transcription factor to enter the nucleus and to make the different enzymes that activate the ubiquitin proteasome system. Now, what we also found is shown here that whenever there was a decrease in phosphorylated Akt that there was activation of another enzyme, caspase 3 that contributes to accelerated protein breakdown. Whenever there is plenty of insulin in IgF1 signalling everything is reversed. Now PI3K, phosphoinositide 3-kinase increases, more Akt-P synthesis occurs, this is phosphorylated and cannot get into the nucleus and therefore, muscle hypertrophy occurs.

So what has this got to do with the models I’ve shown you so far? Well, interestingly if you recreate the ubiquitin proteasome system in a test tube and you add actin or myosin, they’re quickly broken down but if you add actomyosin, the coiled substrate of muscle or myofibrils, they are not broken down. This implies that another enzyme is breaking up the complex structured muscle so the ubiquitin proteasome system can degrade it.

We believe that the mechanism for this other enzyme is there is activation of caspase 3. Specifically, since uraemia and many of these other conditions are characterised by inflammation and high levels of tumour necrosis factor that activates caspase 3, cells stress such as acidosis will activate caspase 3, degrade protein.

The way we went about doing this is to purify actomyosin and incubate it with recombinant caspase 3 and what you find is that at different levels of purified actomyosin there is degradation of the actomyosin and it always leaves this characteristic actin fragment that is 14 kD in size.

So, if we took rats with chronic renal failure as I showed you or different models of diabetes, you can see that there was more of this 14 kD actin fragment in the muscle of the animals with these catabolic conditions suggesting that this was the initial step in protein breakdown.

So does this have anything to do with patients? Well, one experiment we did was to look at patients with severe osteoarthritis who were undergoing hip replacement and the leg that had the atrophy was compared in a muscle biopsy to an age and gender matched controlled subject and you can see there was more of this 14 kD actin fragment.

So if we measure the rate of protein breakdown in muscle and this actin fragment simultaneously during surgery in a number of these patients, you can see that as protein breakdown increased, there was more and more of this actin fragment at least in this small group of patients suggesting that whenever protein breakdown was high that perhaps the caspase 3 activation was playing a role.

Has this got anything to do with patients with uraemia? Well, fortunately we were able to collaborate with my colleague Joe Coppell here who was doing an experiment in patients with hemodialysis and what he did was to obtain a muscle biopsy before beginning a period of different types of exercise including exercise with a bicycle-like apparatus that he labelled as endurance exercise. He then trained these individuals for 18 weeks and then took another muscle biopsy. So compared to the amount of the 14 kD actin fragment in a biopsy from a normal adult there were high levels initially and this was followed by lower levels.

If we looked at a number of these individuals, you can see that the dialysis patients before beginning any exercise had 48.9% higher values compared to normal if they were doing the bicycling or strength exercise, a combination or no abnormalities. None of these values were statistically different.
But after the training period a second biopsy showed that the bicycle-like training reduced the actin fragment almost down to the same level as in normal adults. There was no significant decrease with strength training which was a bit of a surprise and when we combined the two, there was a decrease but it was much less in magnitude than bicycle training by itself. Of course, with no training there was no significant difference.

So we concluded that caspase 3 activity leaves a characteristic 14 kD actin fragment in muscle and that muscle wasting from kidney disease, inactivity or I haven’t shown you other types of injury increase the amount of this fragment. Perhaps with a lot larger groups of patients and under different conditions this could become a biomarker of muscle protein loss.

So the last part of my experiment are some new data we’ve just come upon which ask the question, can the proteolytic activity of this very large proteasome be increased in different conditions?

So the way we did this is to take muscle cells, grind them up, make a preparation of proteasomes and then add recombinant caspase 3 and what the slide shows is that in cells that were activated to stimulate caspase 3, the caspase 3 increased the proteasome activity and this could be eliminated by blocking caspase 3 activity with a relatively specific inhibitor of this aminoacid chain.

We believe that the reason for this is because there are alterations in this set of subunits in this very complex picture of the proteasome. Specifically if there is cleavage of Rpt 5 subunit, then the Rpt 6 subunit flips over and blocks entry into the proteasome in this fashion.

So we asked the question, ok in muscle cells and in conditions such as uraemia and diabetes is there evidence that there is cleavage of our Rpt 5 subunit or some of these other subunits? In the first set of experiments we did we examined what happened in mature C2C12 myotubes and you can see under control conditions there was this level of Rpt 2 subunit, Rpt 5 and Rpt 6.

When we stimulated the cells to activate caspase 3, there was degradation of Rpt 2 and Rpt 6. This was blocked by the inhibitor of caspase 3 DEVD. So, we then cloned Rpt 6 and Rpt 5 and removed the site where caspase 3 was cleaving this particular subunit.

So here is the wild type and after caspase 3 you can see that this band of Rpt 6 has been degraded and in the cells in which we removed the caspase 3 cleavage site there was no such reaction.

If we now looked at the ability of caspase 3 to activate the proteasome, you can see that cells infected just with the vector had an increase in caspase 3. This was eliminated in those in which there was mutant Rpt 2, mutant Rpt 6 or the combination of them. In this case caspase 3 was unable to activate the proteasome.

So we conclude that what happens under normal circumstances in undifferentiated immature cells that Rpt 5 is cleaved that allows Rpt 6 to flip over and block entry but in mature cells if we activate caspase 3, this subunit is broken and now there is free access to the catalytic sites of the proteasome.

So has this got anything to do with other patients? Well, it turns out that in a group of studies done by Arny Ferrando that if you just put normal adults to fasting or prolonged bed rest or those subjected to flight as astronauts or different types of critical illnesses or severe injuries, you get net protein losses due almost entirely to activation of the proteasome and increased protein breakdown.

But perhaps more pertinent to all of us here in the audience it was shown that if you took elderly people that is individuals over 65 years old that after 10 days of absolute bed rest these individuals lost body weight and lean body mass. If you took younger adults, those who were astronauts over 28 days of complete bed rest, they had similar findings. So the point is that just putting elderly patients to bed activates these proteolytic systems and causes loss of lean body mass and muscle mass.

So, these are my colleagues that participated in all of these experiments and I always like to end up with a slide like this.

When you present new data to an audience, Arthur Schopenhauer pointed out that first the finding is reviewed as ridiculous, secondly it’s vehemently denied and thirdly, it usually comes out as being self-evident. Thank you for your attention.

Chairman: Just one comment you should know that it was just recently announced that Doctor Mitch has won the Peters Award of the American Society of Nephrology and it involves not only this work but this type of work over a long career and I’d like to congratulate you.

Chairman: So this paper is now open for discussion. Regarding the proteasome and its role in muscle protein metabolism, do we have any data of the metabolism of patients who are treated by proteasome inhibitors for myeloma or whatever?

Question: The question is somebody uses proteasome inhibitors in patients. There is one study from Japan that had a hint that this was beneficial but it was a very small group of patients. I believe people have been concerned that if you inhibited the proteasome, many other problems would arise. Myself, I believe that you could accomplish the same benefits by correcting insulin IGF1 signalling. So thiazolidine types of drugs might …

Prof. Mitch: I understand that my question was, do we have any data on the evolution of the nutritional status of patients treated by bortezomib for other conditions?

Question: These are very interesting data. I have a couple of questions. First of all this system as far as I know does not act in the liver. Is that correct? Is there a proteasome system in the liver?

Prof. Mitch: Yes but it doesn’t breakdown the bulk of proteins, it mainly acts on transcription factors and short lived proteins.

Question: I see. The second issue is that your comment that the activation of the proteasome system has been observed in fasting animals or people?

Quetion: Suggests that the activation of the proteasome system is playing an intrinsic role in the metabolic response to starvation. We tend to think of this in someway as being bad but a lot of what the proteasome does is probably critical to survival.

Prof. Mitch: I think the argument has been that during fasting what is necessary is to provide glucose for certain cells that are obligatory for glucose: white cells, the renal medullar and at least initially the brain. So what’s happening is that the proteasome is breaking down muscle protein to give aminoacids which are going to the liver and being used to form glucose.

Question: I don’t know what god’s purpose was in doing all of this so I’m not quite sure what the purpose is but it would seem to me that there are a lot of other functions of the release of protein from muscle during starvation which are critical to life for example the need for new antibody production, the need for wound healing which are often present in an organism that in fact is unable to acquire food. They are often injured or sick and therefore, they often need new aminoacid sources for healing.

Quetion: Can you block the proteasome activity in bed rest if you infuse glucose?

Prof. Mitch: Unfortunately, that experiment hasn’t been done, it’s a good one but I think to the extent that you activate insulin and hence promote insulin signalling I would predict that you would reduce proteasome activity but it would be not because you’re providing glucose but because you’re stimulating insulin.