An update from the Kidney Exercise Team on the muscle tissue research

Some of the Leicester Kidney Exercise Team (Emma = 3rd from right)

I got a lovely surprise email today from Emma Watson, one of the scientists working with the Leicester Kidney Exercise Team, who were one of the recipients of the money we raised via the Pedalling to Paris jaunt last year.

She was mailing with an update to what’s been happening with the muscle tissue I donated via biopsy last year, and some info on how the funds they received from us were spent. She must have spent a long time writing this report, and I found it fascinating and asked if I could share it with you. Also, I repeat this at the bottom, but as scrolling is such a hardship, I’ll do the same here – if anyone vaguely in the Leicester area feels like contributing a bit of leg muscle (I did, it was easy!) then it would help them a lot.  Email Emma Watson on ec174@leicester.ac.uk for details!

Anyway, here’s the report!  It’s fairly heavy on the science, but most people should be able to absorb something from it.  Over to Emma!



An update on research performed on your muscle sample

Thank you again Dan for the sample you so kindly gave us and the huge effort in raising money. This is a small synopsis of what we have been doing with it so far.  This research still has two years to go, so hopefully I can send you a more thorough report then.

Experiments

The first thing we are interested in is whether the muscle cells from a kidney patient are able to respond to anabolic stimuli (signals that usually make muscle grow – like insulin, growth hormone, feeding and exercise) to the same extent as our healthy controls.  When these anabolic factors bind to the muscle cell they activate a cascade of events within the cell that starts the process of protein synthesis that will ultimately result in larger muscles.  This cartoon explains it quite well. In this case the ligand will be something like insulin and the response will be protein synthesis.

 

What we can do is look at the extent to which these proteins in the cytosol are turned on.  The way that we do this is to add some of these factors to your muscle cells for a set period of time, take them off the plastic they are growing on and run them over a gel that will separate out all the proteins within the cells based upon their size.  We can then put antibodies on the gel that will bind only to the proteins we are particularly interested in which will fluoresce if the protein is there and is switched on. The amount of fluorescence is directly proportional to the activity of the protein.  What we end up seeing is a black band – the dark the band the more active the protein, which looks something like the picture below.

Here P = Patient and HC = Healthy Control.

Each of these annotated experiments is looking at the effect of insulin (I) or different concentrations of IGF-1 (a growth hormone) 0.4, 10 and 100 on signaling to protein synthesis via 2 different molecules, Akt and P70S6k – the darker the band the more protein will be made eventually in the cell.  From what you can see here the patients don’t seen able to mount the same level of response as the controls – it’s quite striking!

So what I went on to do next was to see how much the uraemic environment affected the activity of these proteins.  So as well as adding the anabolic factors to the cells, we also added some of the patients’ blood back on the patients cells and the controls’ blood back onto the control cells.  Then we ran the cells on the same type of gel and this is what we have seen so far…

 

What this basically shows that when you add Insulin (I) or IGF-1 at various concentrations 0.4 or 100, the amount of activation you get is not reduced when you also add serum (s) from that same patient. This might mean that the problem resides in the muscle cells themselves and is not coming from the blood.

Another important aspect of muscle health is how well the muscles are able to repair themselves after an injury.  Muscles experience small microtears all the time which is completely normal and is just the result of daily living as well as more considerable damage as the result of exercise.  In healthy individuals there is a well-defined process of how this happens. Firstly immune cells infiltrate the muscle from the blood and remove any damaged tissue.  They then activate the skeletal muscle stem cells that sit around each muscle fibre to infiltrate the replace the tissue that has been removed. Hopefully this is explained a bit better in this diagram:

This is an important step – interestingly our muscles are what we call post-mitotic.  This means once mature, they exit the cell cycle and are therefore no longer able to grow and divide so most other cell types in our body do. So if we need to grow our muscles or replace damaged tissue, we are only able to do this if we can activate these stem cells.  There is some evidence from animal studies in Chronic Kidney Disease that these patients have a depleted pool of these stem cells, and are not able to activate them as well. What this ultimately would mean is that if patients do ever injure their muscle, they are not able to heal it as well and instead it is replaced with fibrotic tissue that is not functional.  Patients then start to get weaker and less able to perform normal daily tasks like going upstairs.

However, our results shown that these pools are not depleted in CKD:

So we are next going to see how easily they are activated compared to the healthy controls.

Cell Stretch

We can experimentally mimic exercise in culture and we built a machine to do this using the money that you raised.

What this allows us to do is to grow the muscle cells on a 6-well plate that has flexible bottoms to them.  We seat this plate onto of the sliver posts amd shut the door.  It might be hard to see in the picture, but there is a black c shaped arm that goes over the top of the sliver posts – this arm mechanically moves up and down and in doing do pushes the flexible bottom wells over the sliver posts.  This creates a stretch over the well.  We do this in cycles so that for 30 minutes there is a 2 second stretch and a 4 second release.  This is a lengthening type of contraction – an eccentric contraction in humans, which is when the muscle is generating force when lengthening instead of shortening and creates the most muscle damage of all the types of muscle contraction and so is a good model to use – it’s the most aggressive.

What this allows us to do is to investigate some of the odd results we have seen in our patients in more detail.

If someone with normal kidney function was to go and lift some weights in the gym, we would usually see this…

 

So here, muscle biopsies were taken from the volunteers at baseline and 24h after the very first bout of resistance training – the black bar. They then went off and did 8 weeks of resistance training and a third biopsy was taken 24h after their last training session – the gray bar.

Now, we have repeated this experiment in CKD patients and see this…

So patients have a complete suppression of Akt in the middle bar here – which is comparable to the middle bar in the graph above. However, the good news is that this seems to be corrected by training – seen by the last bar here.

What we don’t know is what has happened in between baseline and “untrained”.  It might just be that the time course of this response has shifted and by taking the biopsy at 24h we have missed the peak of the response – so this is where your piece of kit comes in!!  Ideally to look at this we would get a patient to exercise and then take samples immediately after, 2hours after, 5hours after etc to see what is happening. However, we can’t do this, but what we can do is stretch the cultured cells in the flexible 6 well plates and then collect each well at a different time.  So we can collect the cells from well 1 before they are stretched so this is our comparator, then stretch them for 30minutes and collect the cells from well 2 straight away.  The plate is then put back into the incubator and the cells from the remaining 4 wells are collected at 1hr, 3hrs, 7hrs and 24hrs. This can then provide us with the same information as taking serial biopsies from patients.  This is what we have found so far…

Here 1 = Pre stretch, 2 = immediately post stretch, 3 = 1h post, 4 = 3h post, 5 = 7h post and 6 = 24h post.

So it looks like CKD patients have a greater response immediately after stretch, but this falls back to baseline much quicker than in the controls. This may suggest that the anabolic environment that is created by exercise disappears much faster in CKD.


 

And that’s the end of that!  Thanks so much to Emma for sending it over, it’s fascinating to see what’s happened to my leg tissue, and also what the money we raised has been spent on!

If anyone is interested in taking part in this study, they are still looking for participants.  The biopsy was easy – about as painful as a blood test, and leg very slightly sore for a couple of days, nothing really. They are after both healthy people and kidney patients – if you ‘re interested, drop Emma Watson a line on ec174@leicester.ac.uk.

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