Now I'm going to talk to you about something that I was asked to talk about, which is bile acid signaling in genetic cholestatic syndromes. I am a pediatric hepatologist, so this is my role. But I really want to use this as a jumping off point for understanding the response of the liver to bile acid retention. I am a consultant for several companies, but I am not going to talk about anything clinical here. I really want to focus things that may seem very basic to this audience, but I think is absolutely necessary to start the process, that we really want to just talk about whether the obstruction is extrahepatic and intrahepatic, like in biliary atresia, our most fibrotic disease in liver, full stop. Is it just the cholangiocytes here with ABCB4 deficiency? Or is it just the hepatocytes here, which I think is our most beautiful, easy one, right? You can't get bile acids out of the liver, out of the hepatocytes, so it's just hepatocellular retention. And you can see the histology is, of course, vastly different, but there are also gray areas between these that we see in various liver diseases. Genetically, I'm going to really just talk about two diseases. I'm going to talk about ABCB11 disease. Richard Thompson and his group cloned this in 1998, and that is the sole bile acid transporter across the canalicular membrane. For some reason, there's no redundancy in this system. It's one or none. And so we really have an easy way to work with this. And it just transports conjugated bile acids. And the other one is the ABCB4 transporter, which is solely responsible for transporting phosphatidylcholine into bile. And these are the two that I'm recognizing here with this histology. So I'm really going to talk about two things, and I wanted to get some new ideas here. One, what are the consequences to the liver if you have problems in these three different realms? And the other, and this is, and I'm glad that there are people here in the audience who've done the work, that there's really some new thinking in cholestasis, that we all think we know this, and we really don't know this, and that there's a lot of new things with intravital microscopy, stable isotope measurements that I think we need to start paying attention to. So I'm going to talk about that at the end. So I'm going to actually talk first about this really cool stable isotope study with 11C-cholosarcosine. The molecule is here on the left. It is cholil with sarcosine, and here's the 11C. And if you inject this, it is a bile acid. It doesn't go through the enteropatic circulation, but it gets taken up by the liver. It gets excreted into bile. And if you're cholestatic, you'll see. So this is from a Danish group just published in 2017, and basically you use PET-CT. The 11C compound lasts for 20 minutes. You've got to have everything close by and everybody on board, I guess. So what did we learn from these studies? I think it's really fascinating. Normal bile flows at about 0.26 mL per minute in adults. They even can measure dwell time with a half-life for conjugated bile acids. And in a normal liver, it's about two and a half minutes. So the enteropatic circulation, when bile acids get back up to the liver, it takes two and a half minutes for it to go flow into bile. I found this number absolutely necessary to know. And if you're cholestatic, it's of course longer, but there's longer dwell time. So flux, dwell time, these are things that we don't talk about in hepatology, but I really think that they have some relevance. And the gradient is tremendous between these different fluid status. So from portal blood to liver, it's a two to three-fold in normal livers. But back up into bile, this is a steep uphill gradient up to about a thousand-fold more. So this is an active transporter that must work. So what I'm showing on the right is one of their scans from a normal patient. And what is on the left is the blood in red and blue. It's really not that important. And this is the 11C cholesarcosine in liver over time. It peaks in about 10 minutes and goes down, and then here it shows up in bile. This we sort of know, but I think I've never seen it described like this. They also did it in a few cholestatic patients. And here's one of a 20-year-old with PSC, serum bile at levels of close to 50 micromolar. Look what happens to the 11C cholesarcosine here. It doesn't go up. It stays. And it barely gets into bile. So I think that this is not just a basic physiology story. This is one for us as hepatologists to really recognize that there are differences and that there is dwell time that we need to start paying more attention to. But as I'll go into later that there's supposedly an adaptive mechanism and part of that is reflux back from the cholestatic hepatocyte into sinusoidal bile and they could look at the backflow. And in fact, in healthy patients versus cholestatic patients, you can see some backflux from the cholestatic liver back into the bloodstream. This is something I just want to alert this audience from this SIG. I think this is really a new area that we need to start paying more attention to. So what about ABCB11 deficiency, bile as a transporter? Here is a small mock-up of FXR regulated pathways. FXR activates BCEP promoter and it inhibits the synthesis and also inhibits the uptake of the NTCP and increases the exporters, the ost alpha beta. In fact, there are 7,800 sites in liver chromatin for FXR. It's really an RXR, FXR heterodimer. So it's more than just these sites. FXR is really acting on a whole other number of sites in the hepatocyte. So this is sort of what makes you feel good about the liver. It knows that it's supposed to handle bile acids, excrete them, get them back out. But we know that it doesn't work so well. And so here in ABCB11 deficiency, this is what happens to the liver. The adaptive mechanisms can't work as well as we hope. So that there is really ongoing bile acid toxicity. And there's a wide variety of BCEP function and expression even in this room. So you could think about added cholestatic insults to that, that there's a variability of patient to patient. And just for completeness, there's really other diseases I want you to think about. Another full stop, retention of bile acids, PFIC-1 which works to help modify how BCEP works. But remember, bile acid synthesis defects. Here there's a retention not of the end product, conjugated bile acids, but of the intermediates which are toxic and they keep being fed forward because the end product is FXR mediated and should downregulate synthesis. It doesn't. That's why colic acid is a therapy for this particular set of disorders. FXR deficiency sounds simple, but it really ends up being looking like BCEP because the BCEP promoter is dependent on RXR, FXR plus. And osbeta deficiency with Paul Dawson and I was part of this as well, just described a little while ago. We're not quite understanding how this works, but if you can't get rid of bile acids this way, there is some cholestasis and diarrhea involved in that patient. So one of the consequences I want to talk about is cancer. And so where can it best be explained? I think in the BCEP deficient patients, those that have truncating mutations, and Richard Thompson and others have shown this, that in truncating mutations where you have absolutely no BCEP expression, these are kids who are at risk for hepatocellular carcinoma. We don't see this in our realm very often except in cholestatic disease. This is a concern. And for me, this is the strongest bile acid to cancer link in liver that we can think about. If the only problem in this hepatocyte is getting rid of conjugated bile acids, how do you get cancer? And so this is really interesting. It's relevant for the clinicians. You need to know your genetics a tad and know that if you have a truncation mutation, you need to then follow these patients differently from those with BISENS mutations. And so really, it's quite profound, you know, 13 months youngest detection, et cetera. So as I mentioned, truncation variants are associated with cancer and knowledge alters our plans for monitoring. So let me talk about the phospholipid story. So this is where bile acids are still being secreted into the canaliculus, but phospholipids aren't. And it's not a hepatocellular disease as it was for ABCB11. It's a cholangiocyte disease. So you have a hepatocellular gene product that is leading to a cholangiocyte pathology. And so here's one of a kid that was biopsied by us two weeks of age with ABCB4 deficiency. And you can see the profound pericholangiolar fibrosis in this and the complete intracellular disarray here in the cells. Great. Oh, I see. So you can't see when I move this. Can you? Oh, I'll use the pointer next. Okay. So here's some other diseases. So in this, I can't quantify how AlgaeO works into this, because part of it's developmental and yet the cholangiocytes are probably still not normal as well. But it really does lead to variable degrees of bile acid retention. So here's a disease called cholangiocytic fibrosis, as we know, ARPKD and Corolli disease, which is one where we see a cholangiocyte disorder, and Joubert, and newly ones of DCDC2 of neonatal sclerosine cholangitis and others. These are really nicely grouped into a set of diseases known as cholangiociliopathies. So the cilium of the cholangiocyte is this fantastic micro organelle that is both a sensor and a deliverer of information. And these are ciliary expressed proteins. Very hard to fully categorize these. You know, the membrane percentage of a cilium to the whole cholangiocyte is quite tiny. So there's not a lot of these proteins there, but we need to keep exploring those. So what happens with the cholangiocyte that I think is quite distinct from the hepatocyte when it gets irritated, as in here with the ABCB4 deficiency, which is replicated in the MDR2 knockout mouse. This is the mouse that Paul just alluded to, and I'm going to copy one of the slides and highlight one aspect of it. When the bile ducts get damaged, they really are different. These are antigen-presenting cells. They secrete chemokines and cytokines and a whole host of molecules that then recruit all of the wound response that goes on in liver. So having damaged cholangiocytes really does change the parenchyma, and there's a number of people who are studying how can you inhibit the stellate cells, the fibroblasts, et cetera, that are being recruited quickly in this particular disorder. So I want to put this slide in on purpose to echo what Paul went through. This is, again, the work from Alex Mithke and where they did the ABCB4 knockout mouse. And I'm just highlighting this aspect of it. If you treat this mouse with the bile acid uptake inhibitor in the ileum, the liver bile acid concentrations in the upper left here, I can't see that much anyway. So the upper left in the knockout, in the blue dots, goes down dramatically in the presence of ileal bile acid uptake inhibitor. This is an intact enteropetic circulation, so it works, and these are the normals. So it's a pretty profound reduction of the retention of bile acids in the liver. And the serum ones come down, too. This is a consequence, and as Paul pointed out, I wanted to repeat the histology. You really can make them better in a couple weeks. So this is really, I think, in our world, great evidence that you can use the gut to start helping the liver, and it's really with a nice non-absorbable particular agent. But this one has risks. So here we're talking about ABCB4. Here, retention of bile within the cholangiocytes is damaging them, and that you can be at risk for both cholangiocarcinoma, and what I find interesting here, also hepatocellular carcinoma. So this is also a monitoring risk. And in our world, our biggest disease is biliary atresia, as I mentioned, all the fibrosis. What I think is different here is we really have bile that's flowing in, and here you see a bile plug within abnormal, very damaged cholangiocytes that have recruited a ton of peribiliary fibrosis, all in a short period of time. It's more than just distal obstruction. It's distal obstruction with abnormal cholangiocytes, and we know they're trying to find ways that we can understand this. I think the pathology that goes on in biliary atresia also in broad brush, also may be at play for panbiliary with PSC. We and others are looking at the genetics of a subgroup of biliary atresia kids who have splenic malformations, and they have left-right abnormalities. So if anyone's got genetic causes of biliary atresia, it's this group, and so we found this gene earlier in the year. Manuel Gonzalez and others have worked with CFC1. These are really where we think the future is going with biliary atresia and cholangiocyte biologies. And again, Allergy Ill Syndrome. I still have trouble wrapping my heads around this with the impaired development. So to put these all together, what we have here for bile acid mediating pathogenesis is that you have some degree of injured bile ducts on the left with retention of bile acids and this massive recruitment of cells and having secreted factors, and it really does lead to apoptosis and other damage to the cells themselves with recruitment of the immune and fibroblast. So in the last bit of the talk, I want to talk about what I think is new thinking, and I'll try to rush through this if I can. And there's really two realms here. One, looking at the apical domain, also the canalicular domain, which these so-called microinfarcts, and tight junctions with the bile blood mixing. So the first one, and I'm just giving you a smattering. I really would point you to several papers. This is a very nice overview from Peter Janssen and groups where they were looking at cholestatic livers with new microscopy. And just to show you that in a BDL mouse, you can now envision, and here's two different markers, EBB4 and K19, normal and cholestatic over here, that you can see the dilation of the cholangiols, and you can also obviously see what we see clinically at the bottom here is the ultrasound. On the right-hand side is more sophisticated intravital imaging in a bile duct ligated times three days, and I want to introduce you to another molecule of interest, which many of you know, is this cholelysylfluorescine, and I'll talk about this with other groups as well. Here in green, the cells are in red. In a BDL, we can see that the canaliculi really do get bigger quick, and so you can see actually starting some disruption of hepatocytes. And this group has really made this nice paper just this year in hepatology, and the title really tells it all, a biomicroinfarction cholestasis are initiated by rupture of the apical hepatocyte membrane. And so here, I'm going to give you just some small matter, and I'm sorry I can't point well with the mouse here, but I'll try to walk you through this with words. On the left is sham. The red is a marker for hepatocytes. It really also measures mitochondrial membrane permeability, and CLF is that cholelysylfluorescine that I mentioned before. After just three days of BDL, you actually start to get several hepatocytes on the right in that sort of linear green row that are retaining bile. Not every one, but some of them, and it is sort of a neighboring, you know, one domino falls and then the next domino falls nearby, but not the dominoes further down the row. So there is more and more damage that one can actually start to see in real time with intravital microscopy. And so I'm not going to show any movies, but I'm going to show you some slices through this, and at the top of this slide, they're showing a schematic of what's really happening below in the slide. Okay? So the left is nothing's going on there. On the right, they introduced the cholelysylfluorine, and you can see in the green swath that's going from left to right, that's in the sinusoids. Where the bile is going in the sinusoids first. That's within half a minute. And then by two minutes, and this is after BDL, you start to see one of these cells, and this is a cell, the one that's darker in the middle there, is now the one that's filling with bile. Not the adjacent ones, but the other one's filling. And you can see then, it's hard to see on this screen, but there's no need to really drop the lights, but there is evidence time-wise that it's not just coming from the sinusoid coming in, it's actually primarily coming in from the canalicular space. So this is the other thing I'd like you to pay attention to in the future, is that there's a lot of intravital microscopy, where this particular cell on the far right, the big green one, is one that is filling from the apical surface. And so they have, even within one day after BDL, and what's showing from left to right on the top here is an area that's circled in the white circle that you can see the canalicular parts are filling, and then the cell is filling, and then just bursting open. But this took time, this was 50 or 60 minutes, and schematically is at the bottom is where I think our brains go, is normal, a little bit of apical rupture, and then a lot of apical rupture, and spilling into the sinusoid. So why is this important? Well, one is, I think it tells us that cell to cell things are different, and that it can happen rapidly. The second thing is, when we're measuring serum bile acids, this is what we're measuring. Yes, they're shunting, etc., but for large amounts, this is probably what we're measuring. Okay? And the second one I want to talk about is tight junction disruptions. This is really some beautiful work that Paul Monga and his group has done, and it really is based actually on some clinical data as well, that there are two tight junction proteins, Claudin 1 and TJP 2. TJP 2 is also ZO2, that when these are disrupted in patients, you develop acholestasis. Okay? And so what Dr. Monga's group has done is that the TJP 2 mouse is, I think, embryonic lethal, or it's just early lethal, so we can't really work with it, but they made this beta-catenin knockout mouse, which really disrupts this part of the tight junction. So whatever bile is here is now maybe going to be going paracellularly, and here this is slightly different molecules, but still the cholelisoflurane, and then a different one for blood, and so on the top right, this is normal liver in a mouse where you can see blood in the sinusoids, and the liver is starting to turn a little green. They get less green in the middle one by three minutes, but there you see the canaliculi fill, and they still fill by six minutes. So this is what happens normally. So what happens when you knock this one out? It's not good. And on the right-hand side is probably best to start. This is one that's a knockout that's just in hepatocytes, and you can see this unbelievably great mixing of blood and bile when the tight junctions, and again, when we have thought about this, we first say, oh, yeah, we know this. We really don't know this, and it really does happen, and this can lead to what's on the left, ductular reactions, as well as, and that's in the full knockout, which has a quite more severe phenotype, mostly cholangiocytes, and fibrosis. So this is where I think we're going, and to summarize here is I really think that there are four things when we talk about bile acid signaling here. From the upper left is sort of our standard. Huiping talked about this with FXR, et cetera, transcriptional regulation. On the bottom left, the reactive cholangiocytes. This is absolutely relevant for a number of liver diseases. The tight junction disruptions that I talked about here on the top right and the canalicular membrane disruption, these are the things that I think are going on when we talk about bile acid signaling. Everything else is hundreds of different kinases, transporters, et cetera, but all of this is really happening in the liver is now in the hepatocyte, and I don't know how to measure this, in the cholangiocyte in these diseases, is really trying to respond to this. So to summarize is really what's next. I really think we're going to have better delineations of bioflux. I like to think about things really in these three different realms, as do many of us here in the room, but I think that we're not able to tease apart the distinct molecular consequences. There could be single cell sequencing that's going to help us here, but I think it's going to end up being more than that. There's a lot of intracellular signaling and changes in membranes that really just change the function of proteins. I think bile blood mixing is really an interesting idea here that it really is leading to recruitment of cells that shouldn't expect to see this, and frankly, based on the MDR-2 knockout mouse and the response to IBAT inhibition and seeing what's going on with the amount of bile acids that are being retained, one of the therapeutic approaches that we'll get into is really to just do our best to try to reduce hepatic bile acid accretion. I know it sounds quite simplistic, but it's not really where we're going. We talk about fibrosis measures. We talk about anti-inflammatory issues, but if the root cause of much of this disease is bile acid accretion, we need to work on this and so far it's just the transporters at our disposal. Thank you. Thank you, Dr. Carpin. Before I introduce the next speaker, I was going to say we have seats in the front for those who are standing in the back. Please come to the front. All right. Our next speaker is Dr. Marina Silveira from Yale University, and she'll talk to us about clinical trial design for bile acid modulators.

bile acid signaling

genetic cholestatic syndromes

liver response

ABCB11 deficiency

intravital microscopy

cancer risks