1. Investigating the Role of the
Farnesoid X Receptor (FXR)
on Nonalcoholic Steatohepatitis (NASH)
Summer Undergraduate Research Fellowship
ARSANY MAKKAR
Mentors: Grace L. Guo, MBBS, Ph.D.
&
Pedro Louro, MBA., QIHC
Michael Goedken, D.V.M., Ph.D., DACVP.
2. Summer Undergraduate Research Fellowship
Background – NASH (nonalcoholic steatohepatitis)
• Fatty, inflamed liver not due to alcohol consumption
• Common cause of chronic liver disease.
– Up to 1/3 of Americans have some form of liver disease,
– Ideal treatment yet to be found,
• no specific drug therapy
Characteristics
1. Fibrosis (scar tissue) = the thickening/scarring of connective tissue,
usually as a result of injury
2. Inflammation = redness, swelling, heat, pain
3. Steatosis (fatty liver) = infiltration of liver cells with fat
– Necrosis = the death of cells due to disease, injury, or poor blood supply
3. Summer Undergraduate Research Fellowship
Background – FXR (Farnesoid X Receptor)
• A receptor that controls the
production and excretion of bile
acids
– Bile acid levels must be tightly
regulated
• too much = liver tumor & cirrhosis
• too little = inadequate lipid and
cholesterol levels
• made from cholesterol
– FXR & bile acids
• ↑ BA FXR ↓ BA synthesis
• ↑ BA FXR ↑ excretion via Bile
Salt Export Pump (BSEP)
4. Summer Undergraduate Research Fellowship
Background – IHC (Immunohistochemistry )
• IHC is the process of
detecting proteins in cells via
targeted antibody-antigen
binding and staining reaction
– Immunology
• Using antibodies to
detect proteins using
their unique molecular
characteristics
– Chemistry
• Antibody bound to
enzyme* that activates
chromogen (visible)
– Histology
• Microscopic anatomy of
cells/tissue to know
where chromogen
flagging the proteins are
antigen
Primary Ab (rabbit)
Secondary Ab
(goat anti-rabbit)
5. Summer Undergraduate Research Fellowship
Objective & Hypothesis & Experimental Approach
Objective
– Determine how FXR plays a role on NASH
Hypothesis
– An absence of hepatic, intestinal, or whole body FXR will result in liver
injury and be able to be detected with IHC markers in a NASH mouse
model.
– Different KO groups will cause different severities of liver injury
• FXR whole body KO > FXR Liver KO > FXR Intestine KO
Experimental Approach
– Feeding mice a high fat diet (HFD) will parallel a NASH model.
– Mice with FXR “knocked out” of the liver, the intestine, or both liver and
intestine, will model organ-specific lack of FXR regulation
6. Summer Undergraduate Research Fellowship
Experimental Design
• C57 mice (susceptible to diet-induced obesity)
• Four genotypes fed either normal or HFD for 6 months
followed by necropsy and collection of livers for injury
severity scoring, special staining, and IHC (FXR, BSEP)
• 10% PBS-buffered formalin-fixed tissue for 24hrs.
Group Diet
Wildtype FXR Normal HFD
Liver FXR KO Normal HFD
Intestine FXR KO Normal HFD
Both FXR KO Normal HFD
7. Summer Undergraduate Research Fellowship
Experimental Design – Staining
• Normal Stain
– Hematoxylin & Eosin: blue nuclei / red-pink cytoplasm
• shows anatomy/pathology
– lipid accumulation, necrosis, inflammation, biliary hyperplasia
• Special Stains
– Masson’s Trichrome: abnormal collagen deposition (fibrosis)
– Picro Sirius Red: collagen I & III (fibrosis)
– PAS (Periodic Acid-Schiff): glycogen & other polysaccharides
• IHC Stains: *WERE NOT USEFUL IN SCORING*
– *FXR: inhibition BA synthesis
– *BSEP: movement of bile salts
– Fibrin: inflammation damage
– Caspase 3: ultimate protein in apoptosis (programmed cell-death)
8. Summer Undergraduate Research Fellowship
Results
• Normal HFD mice had lipid accumulation (steatosis) but no signs of NASH.
• KO groups on regular diet did not have noteworthy have steatosis or injury.
• KO groups on HFD had some signs of NASH (necrosis, inflammation, biliary
hyperplasia, and fibrosis)
– Liver KO > Liver + Intestine KO > Intestine KO
• IHC results were inconclusive:
– BSEP was not specifically in canaliculi but also on cell surfaces and
macrophages
• Assay was deemed effective on positive control tissue.
– FXR nuclear staining in controls & KO mice were comparable
– Fibrin showed no noteworthy difference in staining intensity between
different groups. While there was sinusoidal staining in expected
affected groups, similar patterns were seen in some controls.
– Apoptosis staining in controls & KO mice were comparable
Mouse Group Result
Normal diet No steatosis nor injury
WT HFD Steatosis but no injury
KO HFD Some signs of NASH (necrosis, inflammation, biliary
hyperplasia, fibrosis)
Liver KO > Both KO > Intestine KO
*BSEP Not specific in canaliculi but also on cell surface/macrophages
Assay was deemed effective on positive control tissue
*FXR Controls & KO mice were comparable
*Fibrin No noteworthy difference in staining intensity between groups.
While there was sinusoidal staining in expected affected
groups, similar patterns were seen in some controls.
*Apoptosis Controls & KO mice were comparable
13. Summer Undergraduate Research Fellowship
Discussion
– Why the IHC didn’t work?
• IHC is not cook book. It is complicated and doesn’t always work at first
• Worked on some of our positive samples but not on any of the test samples
• ALL test samples had similar background, comparison of collection/fixation
variables/techniques of positive controls to test samples may optimize IHC
– The HFD groups with a KO resembled the NASH model
• KO groups have injury signs
– Lipid accumulation does not necessarily result in liver injury
– Storing fat for later use is physiologic but can lead to pathologic processes in conjunction
with other disease variables
– Kidney BSEP staining was specific to proximal convoluted tubules
• Shouldn’t be in kidney at all
• Worked well for liver tissue on exact same slide
• Is the AB also picking up antigen specific to proximal convoluted tubules?
14. Summer Undergraduate Research Fellowship
Future Directions
• Discuss bile acid levels with Dr. Grace Guo to see the
relationship between BA & liver damage.
• Discover the mechanism of how FXR regulates inflammation.
• Harvest major GI organs* and score results rather than just
the liver after knocking out FXR.
– major organs: kidneys, livers, pancreas, spleen, intestines, stomach.
• Replicate project with a 1-month study to test for “true” acute
inflammation.
15. Summer Undergraduate Research Fellowship
Acknowledgements
Special thanks to
– Pedro Louro, Marianne Polunas & Dr. Michael Goedken for their
constant support academically and in staining.
– Dr. Grace Guo & Bo Kong for FXR antibodies.
– Dr. Lauren Aleksunes & Jamie Moscovitz for BSEP antibodies.
– National Institutes of Health (R01GM104037)
– Rutgers University, SURF
16. Summer Undergraduate Research Fellowship
References
• "Overview." 000664. The Jackson Laboratory, n.d. Web. 19 July 2016.
• Eroschenko, Victor P., and Mariano S. H. Di. Fiore. "Liver." Di Fiore's Atlas of Histology with
Functional Correlations. 12th ed. Philadelphia: Lippincott Williams & Wilkins, 2000. 367-76.
Print.
• Chavez, Junn D., Jr. Basic Immunohistochemistry. Fremont: Lab Vision Corporation, n.d. Print.
• "Associate Degree Nursing Physiology Review." Associate Degree Nursing Physiology Review.
Nursing, Allied Health and Other Health-related Educational Grant Program, n.d. Web. 19
July 2016.Taylor, Tim.
• "Liver." InnerBody. HowToMedia, 1999. Web. 17 July 2016.
• Fickert, Peter et al. “Farnesoid X Receptor Critically Determines the Fibrotic Response in Mice but
Is Expressed to a Low Extent in Human Hepatic Stellate Cells and Periductal
Myofibroblasts.” The American Journal of Pathology 175.6 (2009): 2392–2405. PMC. Web.
20 July 2016.
• Kim, Insook et al. “Differential Regulation of Bile Acid Homeostasis by the Farnesoid X Receptor
in Liver and Intestine.” Journal of Lipid Research 148. (2007): 2664-2672. 20 July 2016.
• Gadaleta, Raffaella M. et al. “Bile acids and their Nuclear Receptor FXR: Relevance for
Hepatobiliary and Gastrointestinal Disease, Biochimica et Biophysica Acta (BBA) -
Molecular and Cell Biology of Lipids, 1801.7 (2010): 683-692.
• Modica, Salvatore, Raffaella M. Gadaleta, and Antonio Moschetta. “Deciphering the Nuclear Bile
Acid Receptor FXR Paradigm.” Nuclear Receptor Signaling 8 (2010): e005. PMC. Web. 26
July 2016.
Editor's Notes
The bile acid receptor (BAR), also known as farnesoid X receptor (FXR) or NR1H4 is a nuclear receptor that is encoded by the NR1H4 gene in humans.
So, what is NASH -- 1/3 of Americans have liver disease & 1/3 of those
Fibrosis = collagen
Harmful stimuli such as pathogens or damaged cells.
Cirrhosis = a chronic liver disease marked by degeneration of cells, inflammation, and fibrous thickening of tissue.
NAFLD = Nonalcoholic fatty liver disease (NAFLD) is a medical condition that is characterized by the buildup of fat (called fatty infiltration) in the liver. There are two types of fatty infiltration of the liver:
●Nonalcoholic fatty liver (NAFL) is a generally benign condition in which the fatty infiltration is simple and there is no inflammation
●Nonalcoholic steatohepatitis (NASH) in which there is fatty infiltration along with liver inflammation (steatohepatitis)
Another experimental approach to treating NASH is the use of newer antidiabetic medications—even in persons without diabetes. Most patients with NASH have insulin resistance, meaning that the insulin normally present in the bloodstream is less effective for them in controlling blood glucose and fatty acids in the blood than it is for people who do not have NASH. The newer antidiabetic medications make the body more sensitive to insulin and may help reduce liver injury in patients with NASH. Studies of these medications—including metformin, rosiglitazone, and pioglitazone—are being sponsored by the National Institutes of Health and should answer the question of whether these medications are beneficial in NASH.
a receptor that tightly regulates BA
FXR plays a big role because BA can cause liver disease.
Bile Acid deficiency is associated with bacterial overgrowth
Chronic inflammation is associated with enhanced risk of tumor formation & cirrhosis (degeneration of cells, inflammation, and fibrous thickening of tissue)
Cholesterol is virtually insoluble in aqueous environment, so it is incorporated in bile.
Cholestasis is impaired bile secretion and flow. Intrahepatic accumulation of bile acids may occur and cause fibrosis, inflammation, and cirrhosis.
and how we approached this is through IHC,
composed of 3 parts, immunology (antibodies) chemistry (enzyme+substrate reaction) histology (miscroscopic anatomy) - this shows where the protein is expressed
Analogy = tetris, connecting to blocks
Enzyme lables: horseradish peroxidase & alkaline phosphatase (HRP + AP)
Chromogens: 3-amino-9-ethylcarbazole (AEC) + DAB
DAB = stable dark-brown
AEC = unstable bright-red
Unlabeled 1º to Ag
Enzyme-labeled 2º to 1º
Substrate-chromogen to enzyme
Rabbit-anti-caspase3/fibrinogen
Antigen = whatever we are looking for.
Rabbit-anti-IHC
Mouse on mouse = backgrounds.
Rodents, human+primates, large animals.
NOW THAT I'VE GIVEN YOU A LITTLE BACKGROUND, let's talk about the project.
liver, intestine, or both, will cause liver injury in HFD. In order to resemble NASH/ FXR regulation...
this can potentially serve as a therapeutic marker for NAFLD or obesity.
C57 mice (susceptible to high-fat obesity)
High-fat-diet mice were fed 60% calories from fat.
AND EACH OF THESE mice were fed normal diet for first 2 months HFD for 6 months.
10% PBS-buffered formalin-fixed tissue for 24hrs.
3 types of stains: H&E, mike was able to score this and gave us ---------
Biliary hyperplasia = fibrosis around the bile ducts, scarring of connective tissue
2 main antibodies that we looked at.
WT HFD no NASH no liver injury.
What I said earlier: FXR whole body KO > FXR Liver KO > FXR Intestine KO
Some sort of role that the intestine plays in regulated FXR that kept the both from being the most damaged.
BSEP not specific enough to conduct a score
The results between the control and KO mice were comparable so we couldn’t really differentiate between that.
Here are the canaliculi. It is transferring the bile from the hepatocytes into bile duct (portal triad) which then takes it into the intestine.
Effective on positive controls ---- but on test samples they are on cell surfaces and macrophages
mRNA in kidney, & mRNA subcanalicular cytoplasm in liver
Digestive bile-secretory organ
Lobules have a central vein & a peripheral portal triad.
Sinusoids carry blood INTO the central veins
Hepatocytes secrete bile INTO bile canaliculi TO bile ducts
Lipid Score of 0 Lipid Score of 4 microvisicular lipid and macro
All FXR groups had comparable staining to this picture
Postiive control and HFD, nuclear staining is still all the same
deep blue is collagen.
Score: 2
fibrosis in sinusoids. some collagen is expected in the portal triad region as it is the connective tissue scaffolding
Bile Salt Transporters are in the ileum & kidney. So maybe the Ab was recognizing an epitope on the antigen that is also in the kidney. Maybe the BSEP is picking up other epitopes that are involved with other bile salt transporters.
Other bile salt transporters include the organic anion transporting polypeptides OATPs (SLC21A) and the multidrug resistance-associated proteins 2 and 3 MRP2,3 (ABCC2,3). Bile salt transporters are also present in cholangiocytes, the renal proximal tubule, and the placenta
American Physiological Society, Bile Salt Transporters: Molecular Characterization, Function, and Regulation
(descriptive analysis)
Measure inflammation
Absense of FXR would impact inflammation
