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Decreased Nuclear Receptor Activity Mediates

Downregulation of Drug Metabolizing

Enzymes in Chronic Kidney Disease Through

Epigenetic Modulation

October 20, 2014

Department of Physiology and Pharmacology

Brad Urquhart, PhDAssistant Professor

Department of Physiology and Pharmacology

Department of Medicine (Nephrology, Clinical Pharmacology)

Department of Paediatrics

Lawson Health Research Institute

Imperfection of Drug Therapy

Disease

Environment

Genetics

•• AbsorptionAbsorption

•• DistributionDistribution

•• Metabolism Metabolism

•• ExcretionExcretion

Factors Mediating Factors Mediating

Drug DispositionDrug Disposition

Yeung et al. Kidney International (2014)

Chronic Kidney Disease (CKD)Stage Description GFR

(ml/min/1.72m2)

1 Kidney damage with normal or ↑GFR

≥ 90

2 Kidney damage with mild ↓GFR

60–89

3 Moderate ↓GFR 30–59

4 Severe ↓GFR 15–29

5 Kidney failure <15 or anuric

Am J Kidney Dis.

2011;57(3)(suppl 2):S32-S56

Pe

rce

nt

of

po

pu

lati

on

• Characterized by a progressive

decline in renal function over time.

• Major causes of CKD include diabetes and renal vascular

disease (including high blood pressure)

• CKD is estimated to affect between 1.9 million and 2.3

million Canadians

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Drugs and CKD

• Patients with CKD take 7-12

medications concurrently to

control a variety of co-

morbidities.

• These patients have an increased

incidence of adverse drug

events, often due to increased

blood drug concentrations.

• Many drugs are excreted by the

kidney so GFR adjusted dosing in

patients with CKD is common.

Metabolism

Renal

Bile

Drug Elimination

Wienkers and Heath (2005) Nat Rev Drug Discov. 4 (10) 825-33

Does CKD impact hepatic drug metabolism?

CYP3A

CYP2D6

CYP2E1

CYP1A

CYP2C

Shimada et al. (1994) J. Pharmacol. Exp. Ther. 270 (1) 414-23

Cytochrome P450 Mediated Drug Metabolism

Nuclear Receptor and Epigenetic Changes in CYP3A

and CYP2C Mediated Metabolism in CKD.

HypothesisHypothesis

Altered drug metabolism in CKD is secondary to decreasednuclear receptor binding to the CYP3A and CYP2Cpromoter which is associated with histone modulation.

ObjectiveObjective

1. To determine nuclear receptor binding in CKD.

2. To investigate the epigenetic modulation of hepatic drugmetabolizing enzymes in CKD.

3. To evaluate uremic toxins that may downregulatehepatic CYP3A.

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CYP3ATATAAXRE HNF-4α

HNF-4α

PXR RXR

Modified from Huss et al. 1999

-118-136 -110 -86

RNA

Pol II

RNA

Pol II

Nuclear Receptors

H3

AC AC

H4

AC AC

AC

AC

Activating

Silencing

H3K9

MeMeMe

H3K4

MeMe

“Gene On”

“Gene Off”

H4R3

MeMe

H3K27

MeMeMe

Control CKD0

20

40

60

80

Se

rum

Cre

atin

ine

(µ

M)

*

Velenosi et al. (2014) FASEB J

CYP2C11 CYP3A2

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Velenosi et al. (2014) FASEB JVelenosi et al. (2014) FASEB J

Velenosi et al. (2014) FASEB J

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Mechanism?

mRN

A

Enzym

e

CONTROL UREMIA

mRN

A

HNF-4αPXR RXR

CYPCYPAc Ac Ac

Enzym

e

RNA Pol IIHNF-4αPXR RXR

CYPCYPAc Ac Ac

RNA Pol II

Possible Mechanism For Hepatic Drug Metabolizing Enzyme DownPossible Mechanism For Hepatic Drug Metabolizing Enzyme Down--

regulation in CKD.regulation in CKD.Metabolomics

• Hundreds of uremic toxins

accumulate in kidney disease.

• Do any of them accumulate in

the liver?

• Sensitive QTOF mass

spectrometer: can quantify

and identify small molecules.

• Application to drug toxicity :

pharmacometabolomics

XEVOXEVO--G2G2--S Mass SpectrometerS Mass Spectrometer

Metabolomic InvestigationControl

Chronic Kidney Disease

Homogenize in acetonitrile

Homogenize in acetonitrile

UPLC-MS

-40

-30

-20

-10

0

10

20

30

40

-80 -70 -60 -50 -40 -30 -20 -10 0 10 20 30 40 50 60 70 80

t[2

]O

t[1]P

Scores Plot: Control vs. CKD

CKD

Control

EZinf o 2 - CKD AST-120 Rat Liver Neg Mode Control v s CKD (M2: OPLS-DA) - 2014-09-29 14:03:10 (UTC-5)

Hennop, Velenosi et al. unpublished

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6

-1.0

-0.8

-0.6

-0.4

-0.2

-0.0

0.2

0.4

0.6

0.8

1.0

-0.1 0.0 0.1

p(c

orr

)[1

]P (

Co

rre

lati

on

)

p[1]P (Loadings)

S-Plot (Control = -1, CKD = 1)

EZinf o 2 - CKD AST-120 Rat Liv er Neg Mode Control vs CKD (M2: OPLS-DA) - 2014-09-29 14:10:14 (UTC-5)

Metabolomics of Liver Samples in CKD Conclusions and Future Directions

CONCLUSIONSCONCLUSIONS

• PXR and HNF4α binding to CYP2C11 and CYP3A2promoters is decreased in CKD.

• Changes in histone acetylation are associated withdecreased CYP2C11 and CYP3A2 expression in CKD.

FUTURE DIRECTIONSFUTURE DIRECTIONS

• Metabolomic investigation to determine which uremic toxins are elevated in the liver.

AcknowledgementsAcknowledgements

Urquhart lab

• Dave Feere, MSc

• Anzel Hennop

• Andrew Kucey

• Alvin Tieu

• Dr. Ben Thomson

• Tom Velenosi

• Andrew Wong

Collaborators

• Dr. Tom Nolin (University of

Pittsburgh)

• Dr. Andrew House

• Linda Asher, RN

Dr. Ben Thomson

David Feere, MScTom Velenosi,

PhD Candidate

Cocktail:• CMPF

• Creatinine• P-Cresyl sulfate

• Indoxyl Sulfate• Guanidinosuccinic acid

• Hippuric acid• Indole-3-acetic acid

• Methylguanidine• Urea

DM

SO

Indoxy

l Sul

fate

Cock

tail

0

50

100

150

Rela

tive m

RN

A E

xp

ressio

n

* *

Velenosi et al. unpublished

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7

-2 0 2 40

50

100

150

Log [Indoxyl Sulfate] µM

Re

lati

ve

mR

NA

Ex

pre

ss

ion

(% C

on

tro

l)

Normal Uremia

-2 0 2 40

50

100

150

**

Log [Indoxyl Sulfate] µM

Re

lati

ve

mR

NA

Ex

pre

ss

ion

(% C

on

tro

l)

NO FBS Media FBS Media

Velenosi et al. unpublished

Mechanism?

I

mRN

A

Enzym

e

CONTROL UREMIA

mRN

A

HNF-4αPXR RXR

CYPCYPAc Ac Ac

Enzym

e

RNA Pol IIHNF-4αPXR RXR

CYPCYPAc Ac Ac

RNA Pol II

Possible Mechanism For Hepatic Drug Metabolizing Enzyme DownPossible Mechanism For Hepatic Drug Metabolizing Enzyme Down--

regulation in CKD.regulation in CKD.

Indoxyl Sulfate?