arsany surf

Investigating the Role of the Farnesoid X Receptor (FXR)

on Nonalcoholic Steatohepatitis (NASH)

Summer Undergraduate Research FellowshipARSANY MAKKAR

Mentors: Grace L. Guo, MBBS, Ph.D. &

Pedro Louro, MBA., QIHCMichael Goedken, D.V.M., Ph.D., DACVP.

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, pain3. Steatosis (fatty liver) = infiltration of liver cells with fat

– Necrosis = the death of cells due to disease, injury, or poor blood supply


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)


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

subs

trate

-chr

omog

en

antigen

Primary Ab (rabbit)

Secondary Ab (goat anti-rabbit)

enzyme


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


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 DietWildtype FXR Normal HFD

Liver FXR KO Normal HFD

Intestine FXR KO Normal HFD

Both FXR KO Normal HFD


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)


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


FXR/BSEP 100x FXR (N) +Control

400x BSEP Positive Control

400x HFD WT


200x HFD Both FXR KO200x WT Control

Hematoxylin & Eosin Stain – Steatosis

Summer Undergraduate Research Fellowship100x FXR Positive Control

100x HFD FXR Liver KO


Masson’s Trichrome: 200x HFD Liver KO


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?

• Highlight the key findings of your research• Typically just 1 slide


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.


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


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.

arsany surf

Documents