 All right, thank you for the introduction. Can everybody hear me? Make sure this thing's being tall as tough sometimes. All right, thanks everybody for showing up this morning. I'm here to talk about my research I've been doing during my doctoral thesis and now as a postdoc and hopefully it makes a lot of sense. I'll try to make it as clear as I can. I'm a basic science researcher. So sometimes I talk to a lot of academics so it can be a little challenging to communicate the things I do to everybody else but hopefully it can fall along. I'll try to make it as clear as I can. All right, so starting off, so autosomal dominant polycystic kidney disease, a very long name. So I'll be calling it either PKD for short. So if you see that acronym show up, that's what I'm talking about. So it's a monogenic disease. So that means it's caused by a single gene mutation. There's a handful of different PKD types of diseases that are caused by many other mutations but the one that I'm primarily interested in is the one that's associated with the PKD1 gene or the PKD2 gene. These account for the majority of the cases that we see in PKD or ADPKD. This thing I feel like is right in my way. All right, so the first one is the PKD1 protein or the PKD1 gene that codes for this protein, polycystin one. It has a lot of functions which makes it really challenging to study. So nobody really totally understands everything that it does yet but some of the more interesting things that we're, we know that it does is it affects cellular proliferation and now more interestingly in metabolism. So it seems to be central to a lot of different effects in the cell. This disease, it affects a lot of people. So one in 400, there's some of the estimates. So about 600,000 people in America alone. This is about 10 million worldwide. It's slowly progressing and that's an important point to all my research is that it doesn't show up and manifest itself until later in life. People were diagnosed usually early on in adulthood because of a familial, they'll know that they have PKD, somebody in their family, they'll be tested but then they have renal function fine for many years and then at some point they eventually succumb to renal failure and then they need a kidney transplant or lifelong dialysis and this is really the issue of this problem is that it doesn't show up for a long time so people don't really worry about it and all of a sudden they're stuck on dialysis and at that point you only have one to two years in which you can survive at that point before needing a kidney transplant or dying, one of those two. The other thing is that cis formation is still poorly understood so that's where I get to come in so I get to do a lot of my work from that. But so why do we study PKD? The main reason that we study this is because it is very expensive on our healthcare system. Most of the dialysis is coming from kidney failure, chronic kidney disease and one of the big percentages of this is from PKD so it can cost up to $850,000 a year in some instances just to keep people on dialysis. This cost goes up as kidney function declines and this is because people have a more difficult time working. Their families need to take care of them. Taking people to dialysis is a very expensive thing to do. They have to go multiple times a week. They sit there for hours at a time so it's very costly to the individual and their families. Right now, just recently, the Tulvapden is the first approved PKD therapy. This is about $5,700 a month if you wanna take this drug but it has a lot of side effects and because it's a vasopresent agonist, you have to pee all the time. So if you wanna go to the bathroom multiple times a day and for a minimal effect on decreasing the progression of the disease, that's the way to go but it doesn't look like that's a great option for people. But as I mentioned, because it's slowly progressing, if we can just figure out a way to slow down the progression then we effectively cure the disease because if you can just spread the disease to go to your 80s, 90s, it's pretty much a cure at that point. So trying to extend it towards the end of life would be the goal. So here's a kind of a graph that shows the progression of PKD. It starts off pretty early on with just small cysts that form throughout the kidney. It starts to progress more and more as people get older and is also these other manifestations of the disease. People develop hypertension. They have some other aneurysms can be a secondary effect of the disease because of the blood pressure regulation but also kidney function starts to decline and so people start to form kidney stones, infections in the kidney, all sorts of other manifestations of the disease. And you can see at the very end there, the kidney is almost completely covered with cysts and if you imagine, if you take your fist and you think about a fist as being the normal size of a kidney and then you take your hands and you go about this far, that's about the size of the kidney becomes. So somewhere about a size of a football or larger by the time that it's actually manifested to the end stage of PKD. So this slowly progressive nature of the disease kind of gives us a little idea about how it might be actually functioning. So you start off with a normal copy of this PKD-1 allele and then at some point you end up with a mutation in the gene line or the germ line and that is a, now you have this heterozygous mutation and then nothing happens but at some point we believe that there is a secondary mutation in this somatic mutation that would be in the cyst itself and then finally nothing happens until you injure the kidney and then at that point a cyst seems to form and this is backed up by a lot of experimental research which shows that treating kidneys in this context with a mercuric chloride which is not a very common thing people run into in their everyday life or ischemia reperfusion where you actually cut off the blood supply to a kidney and then let it go back in or removing one of the kidneys and letting the other one grow in compensation that will all trigger cyst formation if you have both of these genes mutated. None of these seem to be things that happen to people normally so it's kind of confusing like what is actually the trigger for cyst formation in humans and this is where we got our ideas that go forward from here. So I wanna introduce this idea of micro crystals and kidney stones. So micro crystals, when I was referring to those it's talking about these subclinical precipitates in the kidney and so these are gonna be made up of lots of different substances primarily oxalate, uric acid or phosphate those are most common those are really attracted to calcium so they form these little tiny crystals and when they get big enough they form a kidney stone so that's what we kind of know when it actually manifests as something that we recognize and we know that diet is affects how much of these stones are formed. So from an evolutionary question is how do kidneys deal with these micro crystals normally because we form them all the time we can look at urine and we can find out that they're there so there must be some way that we deal with them and so we're thinking that as a hypothesis maybe these cysts are just formed because of injury or inappropriate response to crystal formation. So looking at how this kind of fits in with PKD we know that crystals are formed in the general population and if you look at urine you'll find them up to a quarter of every urine that you look at you'll find crystals in there they are more common as individuals age they're common more in PKD patients and they're also more common in males and we know that males have a more severe PKD phenotype. So these all kind of mirror the progression of the disease. We also know that from some animal research that if you treat animals with citrate they seem to get better so the disease doesn't progress as quickly and before this was kind of unexplainable but we also know that citrate is a key later of calcium so it pulls excess calcium out of the urine so this might be a way to prevent crystal formation. So I've treated some PKD rats with citrate myself and found that we saw these under normal conditions these black spots these are, this is a stain for oxalate crystals you can find these crystals in these lists throughout the kidney but then when we treat with citrate they're almost completely gone and that's just after one week of citrate treatment so this is kind of supporting that maybe this is one of the way this is working. So our model is if you look up at the top you can kind of see a normal tubule where the fluid flows through without any impairment at some point a crystal form and then block fluid flow we think at this point that this is when it activates some of these cellular pathways that are involved in tubule dilation they're activated in order to kind of clear up the crystal and then when the crystal goes away these pathways turn off and the tubule goes back to normal but under a PKD context where they have loss of these polycystins they would then go on to form a cyst. So we think that maybe tubule dilation is the normal process to get rid of crystals and that this is defective in polycystic kidney disease. Some support to this is a disease called primary hyperoxyleria where humans they have a mutation that causes them to form a lot of calcium oxalate crystals. These little shiny spots those are calcium oxalate crystals are really easy to see under a polarizing microscope so we can just look at them and you see that there's plenty of them in these primary hyperoxyleric patients and when we look at some of the signaling pathways so these red marks here these are a marker for the protein mTOR so we look at these everywhere it's red that means that mTOR is active we see that in PKD these cyst lining cells are very strongly activated for mTOR and also in the primary hyperoxyleric patients so we're showing that this is a similar mechanism by which these cells are hyperactive for mTOR and we also know that when we inhibit mTOR this is research done in our lab as well that if you inhibit mTOR in a PKD mouse model that the cysts do not progress as fast and they actually kind of look almost normal in adults. So in wild type rats so this is one of the first experiments we did we just inject animals with a dose of sodium oxalate so they start to form all of these crystals very quickly. After six hours you can see a lot of these crystals are already starting to form 24 hours the tubules begin to dilate so these crystals are becoming less apparent and after three days you see a lot of tubule dilation and very few crystals left in the kidney and after seven days the tubules look almost completely normal again so they've cleared out the crystals and are recovered after a full week of this process. We see that when you treat with these animals with these oxalate crystals they have again activated mTOR and if you treat with the inhibitor rapamyacin which blocks the activity of mTOR these tubules they still dilate but not to the same extent and the mTOR signal is cut off. When we looked at a bunch of different tubule segments so it's not important what the two markers are for the different tubule segments all you need to look at is that they don't dilate and we treat with the rapamyacin so this is showing that mTOR seems to be intimately linked to this dilation process so this is kind of in support of our model that it is these overactive mTOR signaling that leads to tubule dilation and cis formation in PKD. So these are normal wild type animals so we wanted to test this in a PKD model instead of the acute injection where we treat them with just one time with sodium oxalate we treat them with ethylene glycol ethylene glycol leads to the formation of calcium oxalate crystals but this is over a longer period of time so we start with week three so as soon as they're weaned they now start drinking a water filled with ethylene glycol I wouldn't encourage that and then after five weeks we look at the kidneys. So at eight weeks old you already can see that these kidneys are much larger they have a lot more cysts and they're polarizing microscopy you can see there's a ton of these little micro crystals all over the kidney. Interestingly females they don't form crystals and they also do not get more cystic so this is a good negative control for our experiment so this is showing that this is not an effect of oxalate alone but actually because of the crystal formation. To back this up even further and show this is not a calcium oxalate specific effect we treated another model of PKD with high phosphorus diet so now they form these calcium phosphate crystals and they too become more cystic but different from the previous model females are the ones who are more susceptible to calcium phosphate crystal deposition and they become more cystic. So this is again showing that this is not just a some other artifact of our treatment but actually crystal specific. These crystals also activate mTOR and so they were just showing here that these crystals are deposited in these active sites of where cysts are forming. All right so that's the first part of my talk so that's showing what I wanna remind everybody is that mTOR is important in PKD we already knew that and so that was just the kind of the central player in our hypothesis so we just showed that it's active also when we treat these animals and how they form crystals. We're arguing that crystals activate mTOR and that's what's leading to tubular dilation and I showed that if you inhibit mTOR activity you can inhibit that dilation effect and that we showed that cyst formation is exacerbated by crystals in different models of PKD. All right so these data they kind of give us an idea that maybe just treating the citrate could be an effective treatment for PKD so if we can prevent these crystals maybe that will be a effective treatment and it also supports our initial hypothesis so a lot of good stuff there let's keep on going. So next one ADPKD and metabolism so I mentioned that ADPKD is a that mutation in Pleistocene I kind of controls a lot of different functions in one of those metabolism and it looks like the more we do research on ADPKD we find that it's actually a disease of metabolic inflexibility and these PKD cells they prefer glucose over any other fuel substrate so they actually have a lot of similarity to what we call the Warburg effect in cancer cells and we know that if we treat cells these PKD cells with a lot of different ways to disrupt glucose metabolism they'll become better so if you treat with different things either block glycolysis or increased fatty acid oxidation you can actually rescue a lot of these animals and there's a lot of research already done on that so one of the things we're thinking about is maybe glucose deprivation is a way to use as a therapeutic window so I think people know where that's going so the Warburg effect if I just remind everybody is that in normal conditions cells are able to either use oxidative phosphorylation so that means they use oxygen and they can burn up glucose that way by through the mitochondria or through fatty acid oxidation or they can do anaerobic glycolysis where they just create lactate from pyruvate but this is kind of the only pathway that's available to cancer cells or cells that are proliferating they seem to be very dependent on glucose and they almost primarily use all their energy from glucose and prefer to do nothing with the mitochondria so you end up seeing a lot of damaged mitochondria in these dependent cells so our question was can we just use the body's own physiology to kind of regulate cyst growth and so is glucose withdrawal sufficient way to do this so we're looking at targeting the different metabolic pathways in PKD the one thing we looked at first is beta hydroxybutyrate and so everybody's probably familiar with that compound by now through the ketogenic diet it's also a very important signaling molecule besides it's energetic functions and in PKD all these different pathways were kind of regulated by beta hydroxybutyrate and so it's got so many different it's handed so many different pathways it's kind of silly like nobody's thought of this already so in our research in our lab we'd done an experiment on caloric restriction in PKD mice and we found that when you reduce their caloric intake by 20% they got much better but one of the issues with this treatment is that or in many of the studies that are done on caloric restriction is kind of the experimental design so animals are given all their food once a day so they give them like you restrict food you imagine the next time you get food you'd be pretty hungry so they eat all their food very quickly so they're actually getting a long period of time in which they do not consume any calories so this is actually probably a time restricted feeding model or intermittent fasting model not a caloric restriction so being able to figure out is this due to caloric restriction alone or to some other fasting effect is kind of the goal of this next project so what we just saw this is kind of highlighted here you know if you give a mouse's cheese he's gonna eat it all and if you give him a mouse's cheese once a day he's gonna run away and eat it all right away and then he's gonna be stuck waiting for that cheese to come back and he knows it's coming but he's gonna have to wait so that's kind of the paradox here is like what is actually going on is this time restricted feeding or is this caloric restriction so time restricted feeding if you're not familiar is just giving all of the food within a designated feeding window this is ad libitum so our experiment we gave them eight hour feeding window 16 hours the rest of the time they were fasted they had all the water they wanted and during this feeding fasting period they're gonna have an increase in beta hydroxybutyrate at least that's the goal so we did measure that and it was the case so the way this works is the animals are allowed food access ad libitum either all the time as the controls or during the dark period because mice and rats are nocturnal so that made the experiment a little more challenging for the researchers we had to go in at night and do all this work so it was a little more challenging but it paid off as it actually works so we gave them all their food and that eight hour window in the dark period and then we looked at their kidneys and we found that the time of feeding animals you can see that they still have cysts but the kidneys did not grow at the same rate and the cysts were actually much smaller when we looked to see what their caloric intake was we found that the amount of calories they were eating was similar to their ad libitum controls and so this was not an effect due to caloric restriction we also looked at change in body weight for males and females they also mimicked each other for the both groups and so we could conclude that this effect was not due to caloric restriction so I think that's very promising we also found that mTOR was a decreased in these animals so this is one of the effects we assumed would be the case we saw that in these cyst lining cells that you can see the ad libitum animals have the very strong signals that's seen in green and the time of shift of feeding animals do not what we also found was that there was a decrease in these myofidroblasts so these are really important for scarring the kidney one of the problems that happens in PKD is that they form a lot of fibrosis so the fibrosis is really where you lose kidney function because that replaces normal kidney with this scarred kidney tissue that does not have any function and once it's there it's kind of there forever and these green cells their job is to lay down collagen so they sit there and they just excrete collagen and they exacerbate the disease and on the time of shifted animals they're almost completely absent so that we think that this might be one of the things that underlies what actually is going on in these animals why they don't get more cystic and we measured that so we just measure the number of cells that's that graph there just shows that there's a huge decrease in the number of these smooth actin positive cells that's SMA and we saw just at the phenotypic level there's a reduction in the collagen deposition so that red stain there is the fibrosis and it's almost absent in the time restricted animals so next we tested a ketogenic diet and we wanted to find out if we just gave animals a high fat ad libitum diet so no restricting the time they eat here this is purely just gonna be just a ketogenic diet all they want we found that they also had a reduction in the amount of cystic reform in this model though there was a they did not grow they did not grow normally we think that this is probably due to the low protein content of the diet but we do know in humans this might not be the same effect cause there are children that have that have been successfully treated with the ketogenic diet for many years there is some growth defect but it's not at the same level you see in these mice and rats so we think that this might be secondary so we're not really too concerned about this other than that they're phenotypically normal and they also ate the same amount of calories relative to their body weight so this was something this was not just a caloric restriction thing this is probably due to some other effect the same thing we saw here we saw that these animals also had a reduction in the mTOR signaling relative to the ad libitum normal chow animals and we also saw again a reduction in these smooth actin cells so these are probably again the same effect that these cells are probably very glucose dependent and we're just starving them out that's probably one of the things we think is going on and again fibrosis is again knocked very far down this would be great if you could see this kind of progression in the human so in summary for the second part both the ketogenic diet and time restricted feeding were successful and able to treat this disease or reduce its progression each one of these diets was not due to caloric restriction so we were able to show that just by how much the animals actually ate and it seems that both these dietary interventions are effective at reducing fibrosis which is kind of the end problem of all of these PKDs that you just lose kidney function and so this might be able to preserve kidney health longer when these treatments are successful in humans and so kind of future implications and directions here is that maybe it's possible to treat PKD with dietary intervention because people will find out very early on in life that they have this disease people know that there is an option for using citrate for instance or foods that might be low and either oxalate, phosphorus or other things that might be forming crystals or stones they might be able to slow the disease progression and then that will keep them healthy for many more years. Right now we're currently collaborating with some people in Germany that we're gonna start doing a clinical trial just for safety but that's gonna take a while to find out so we're gonna see if any of these interventions are feasible in a human context so hopefully people can follow along. It's always a concern whenever you have a dietary intervention that can people follow a dietary strategy so we're trying to figure out what is the dose needed here like cause ketogenic can be hard for people not eating is very hard for people so anything that makes it more palatable might make it more successful. So with that I would like to thank all our collaborators there's a lot of them and this is a picture of Santa Barbara this is kind of you can see my building in the back there so sometimes it's hard to get work done if you're just staring out the window but in thinking about going outside and there's research to be done so with that thanks everybody and I'll take your questions. Thank you so much Jacob we have plenty of time for questions so if you guys would like to step up to the mic you're welcome. I know that's so that's you know where the scope of all you know there's so many different ways to combine this together and yes that would be that would be a way we're gonna do I would love to do that experiment it's always just about like timing money and you know all that kind of stuff and we're thinking that at this point it might only be a marginal increase to considering how efficacious both of the treatments were and that's another thing is like generally doing experiments you don't want to show like a 5% or 6% increase you want to get those big effects and both of these diets had such a big effect on the cystic progression like there was like a 20 to 30% reduction in the cystic progression in these both of these models and that's pretty profound so I think that if we were to combine them we might not get much more effect so I think that would be a great experiment maybe somebody will do it. So absent in a human absent of taking that very difficult to come by stained slide out of a human so what would you suggest for diagnostics blood tests MRI can you help with how to in a general sense diagnose the function of the kidney? Are you saying like how do they how do they do kidney function now for a kidney patient? How would you recommend a someone who has intermittent kidney pain and stone problems to proceed with diagnosing their kidney situation? Well so there's a couple different ways they measure kidney function so it'd be like estimated glomerular filtration rates or the EGFR that's the kind of the standard way people do kidney function the people that have PKD they go in maybe once or twice a year or you know every other year to get measurements if you wanted to find out like how far it's progressing they'd have to do MRI or CAT scan to see those things both of those are expensive so people generally don't do them they just kind of measure what their kidney function is and right now people don't even think about kidney stones as an issue like they know that people get them that have PKD they're not thinking about preventing them or thinking about them as actually causing you know worsening of the disease so it's kind of like it's I know I just don't know people if you wanted to do it you'd have to go through great expense yourself to just look at all your numbers make sure that your kidney function is maintaining and that's all they have right now they really don't have any other What was the name for that kidney function test? Estimated glomerular filtration rate so it's just a calculation based off of some different numbers they get from your urine and blood and they can figure out how you're doing it they can do a direct measurement but that's generally not done Thanks for sharing your research with us I thought it was a really clear presentation which is sometimes hard to come by with like pure science I was wondering maybe I didn't catch it but how long did you test the time-restricted feeding window and then if you maybe doubled that time do you think that the disease rats would catch up with the treated disease rats would catch up with the untreated in terms of fibrosis Yeah okay so we treated them the same timeframe I didn't put that in there sorry week three to week eight so the animals are weaned at week three so that's kind of like the earliest we'd want to start doing this treatment and then week eight is just a convenient time point that's when they are generally considered adults I don't think that they would progress further if we kept them on this regimen because most of the cysts are formed early on so that they kind of like progress, progress and then they kind of stop and the kidney function starts to go down but the cysts don't necessarily grow more of them so we want to do this in adults that's kind of the next experiment we're trying to see if this is actually can regress some of these cysts and that's kind of always been the big issue is like a lot of these treatments like you can treat animals early on and they don't progress but can we actually treat somebody who has advanced PKD that would be really the best thing ever because once fibrosis sets in that's generally considered irreversible but there are mechanisms that the body has to reverse fibrosis just how do you encourage those or promote those in a damaged kidney and so if we could, if that happened we would be done. Yeah. Hi Jacob. Hello. You know I read a lot of kidney articles. A few thousand of them and my take on kidney research is that a huge majority of it is either in rats or monolayers of either dogs or human cells not in vivo and humans necessarily but it sounds like you're talking about designing studies in humans at this like you guys are now transitioning with your German group for human studies right? Yeah. Okay and then there's a couple of categories here there's people at risk for PKD who don't express a disease yet and the prevention mindset that you mentioned earlier and then you're just saying about well we know they're already in disease state to diet work but then it sounds like we're talking about at least three interventions possibly citrate salts certainly in the prevention stage without any other dietary changes like supplements versus are you doing any studies that include an arm that includes just citrate salts? No, the citrate stuff has never been explored unfortunately that there's a lot it's interesting because that research was done in the 90s there was a George Tanner was he did all this research and he showed that the disease was that like these animals did not progress the same as their controls and they tried it in a different PKD model a mouse model and nothing happened and the reason I think now we probably we would conclude that that's because they don't form kidney stones so mice just are non stone formers so we can't even use those as models so rats are the classic crystal stone form model and so that's why we had to use those and he was using rats so it's probably was the same thing I showed at the beginning where you treat with citrate the crystals go away but they couldn't explain it like they didn't know why that was the case and so that research kind of just died there they couldn't get anybody to pick it up and also citrate is free and you know it's no patent and it's cheap anybody can get it so there's no drug company that will support research for that and it's just difficult to do it for that reason because doing a human trial would be very expensive on citrate and speaking of expensive I'm thinking about in human trials what's your timeline that you would need to follow up patients either in a prevention would be longer than a treatment style intervention yeah so it'd be I think two years is around the that's enough usually the marker because PKD progresses at a consistent like a steady rate in adults so if you can just show that you have no progression that's all you're looking for if you maintain kidney function and progression doesn't go down or the progression is not increasing that's all they look for so like with this Tolvapten treatment that's all the metrics that we're looking at was like does the kidney function stay the same or is total kidney volume staying the same and just small changes that apparently gets you approved so it seems like it wouldn't be that tough to do the experiment so in a human trial why wouldn't it be easy or I mean it's expensive outside of the expense in the headache of another group why wouldn't one of the control or treatment groups be a citrate group without dietary intervention well I think it's also just like access to patients too so like the cohorts that so you wouldn't want to work with people that aren't normals they would all be these are all PKD patients right so like where people were working with they have a cohort of PKD patients that they can that are ready to go into a trial and that's generally the other hard part of performing any clinical trials you need to have people that can sign up for the trial and so these patient databases are kind of guarded by different gatekeepers and so getting access to those another challenge so you know to do multiple arms it just increases not just the cost but just the number of patients you need and then your controls too also have to change so that's also another problem can I do just a little advertisement for kidney health as a nutritionist our biggest nutrient deficiency is potassium nobody talks about it everyone talks about magnesium and how deficient we are in that those both come in citrate forms I highly recommend that everyone use those fascinating research Jacob and really promising results thank you congratulations apologies ahead of time for the kind of scattered form of this unquestioned but I'm intrigued by the critical role of mTOR and I'm more aware of mTOR inhibition through protein restriction so it may not matter to you guys given your results but how do you tease out the effects of glucose deprivation and protein limitation how do you know what's really inhibiting your mTOR and how does this tie into what your actual macros were on your version of the keto diet right so this is always this is always a big question right like how do you know where the effect is coming from on mTOR so for the time restriction feeding it almost certainly the protein restriction is the effect that is how mTOR is being inhibited I mean that's pretty well researched that you know fasting inhibits mTOR and that's through like leucine deprivation right so I think that's pretty clear and the ketogenic diet you know they're getting a they're getting enough they're getting protein they're getting all of the protein they need to survive so mTOR should be ostensibly active in these animals right so it's not going to be off but there's something else that is causing the cyst to form so in this model we're thinking that that's where the glucose deprivation so that's how we're trying to tease apart glucose deprivation versus protein restriction so time restriction to feeding that would be protein restriction glucose deprivation would be the ketogenic model because mTOR should still be on right at some level it has to be on you make proteins right but downregulated certainly in the feeding window case well so down so downregulated well you'd also expect that when you restrict protein you have mTOR inhibition but then you have like a an accentuated activity following when you refeed right so I think that's one of the other things is you get this compensatory mechanism where after mTOR is you know is downregulated for that 16 hour window when you refeed you get a much stronger mTOR signal for it's like punctuated and so the punctuated thing is probably where it's healthier that's probably just where most times we should live in that punctuated level rather than that just on all the time because it also the mTOR it's such a complicated signaling pathway because there are a lot of other inputs that mTOR is regulated by like AMPK like we know a lot of this is through AMPK signaling so in the context of the ketogenic diet you're probably regulating mTOR more directly through AMPK being active so also you know mTOR exists in two different complexes it has this mTOR 1 and mTOR 2 complex and you're probably shifting you know where mTOR is residing between these two complexes when you're changing what the macronutrient ratio is and so that's another thing that we don't know exactly like how that plays into everything because the mTOR 2 complexes were involved in regulating autophagy in the cellular repair pathway and so is that another component of this it's like probably 16 hours usually is not long enough to get into autophagy itself but might be playing a role a minor role longer duration fast would be more potent activators of autophagy and so part of my I didn't talk about any of this but I'm doing research now looking at longer duration fast because we found that very good at slowing down the disease progression as well so that's kind of the next step is like looking at like can we just completely fast these animals for long periods of time and get the same outcome and on that issue the difference in their metabolism plays a role too because if you're doing feeding windows of only 8 hours it may be equivalent to a 3 day fast in a human as I understand it yeah mice and rats have a much faster metabolism so there is a big difference in trying to figure out what would be the same thing in a human mice you know you fast them for a single day they lose like 10 to 15% of their body weight and they also get to BHP levels of like 3 to 4 millimolar in one day which is humans would never get that level of ketosis in a day and you know the rats it can take several days to get up to like 2 millimolar so it's again like they're so different it makes doing the experiments a little more challenging for the translational side but it's the efficacy of these things is really good so we're hoping that maybe it will be translatable thank you thank you so much Jacob so okay we're going to wrap up here we're now off to lunch and primal play and then sessions will begin again at 150