The pathophysiology and retention of gadolinium

Brent Wagner, MD

South Texas Veterans Health Care System, Department of Medicine/Nephrology University of Texas Health Science Center at San Antonio, San Antonio, Texas

Associate Professor of Medicine

Objectives

Elucidation of the mechanisms of gadolinium-based contrast agent-induced toxicity is an active area of investigation

A model has been established in rodents

The focus of this presentation is the work in my laboratory concerning the mechanisms of gadolinium-based contrast agent toxicity and how this is manifested systemically

One gadolinium-based contrast agent has been used in these experiments, Omniscan (gadodiamide/caldiamide), but the findings may be applicable for the other gadolinium-based contrast agents

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Gadolinium-based contrast agents have been linked to ‘nephrogenic’ systemic fibrosis cases

There are many different chemical formulations of gadolinium-based contrast agents used in magnetic resonance imaging

There is evidence that gadolinium is deposited in the central nervous system

The toxic effects and mechanisms of gadolinium-based contrast agents is a major gap in our knowledge

Overview

Gadolinium-based contrast agents are biologically active

Wagner B et al, Am J Physiol Renal Physiol, 2016 Wagner B et al, Adv Chronic Kidney Dis, 2017

The central nervous system toxicity warrants more study

Understanding the pathophysiology of gadolinium-induced systemic fibrosis will be critical for future discoveries

How gadolinium from different contrast agents distributes throughout the body is an active area of investigation

Little is known about the metabolism of gadolinium-based contrast agents, their biologic effects, and the implications of retained gadolinium

3

The periodic table of elements

K V Ti Ca Sc Cr Co Mn Fe Ni Ge Ga Cu Zn As Kr Se Br

Rb Nb Zr Sr Y Mo Rh Tc Ru Pd Sn In Ag Cd Sb Xe Te I

Cs Ta Hf Ba W Ir Re Os Pt Pb Tl Au Hg Bi Rn Po At

Fr Db Rf Ra Sg Mt Bh Hs Ds Fl Uut Rg Cn Uup Uuo Lv Uus

Ce La Pr Sm Nd Pm Eu Ho Dy Gd Tb Er Lu Tm Yb

Th Ac Pa Pu U Np Am Es Cf Cm Bk Fm Lr Md No

H

Li Be

Na Mg

He

C B N Ne O F

Si Al P Ar S Cl

4

‘Nephrogenic’ systemic fibrosis, a man-made disease caused by magnetic resonance imaging contrast agents

Dural thickening

Diaphragm Heart

Extremities

Trunk

Buttocks

Face

Joint contractures

Psoas Rete testes

Kidney

Extremities

Lung

Liver

Girardi M et al, J Am Acad Dermatol, 2011

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Gadolinium-based contrast agents can be acutely nephrotoxic in humans

Degeneration of tubular epithelial cells

Glomerulosclerosis

Toluidine blue stain - Kidney

Flattening of tubular

epithelial cells Calcium phosphate

H&E stain - Kidney

Cellular proliferation

Immunostain - Kidney Electron microscopy - Kidney

Akgun H et al, Archives of Pathology & Laboratory Medicine, 2006

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Quantitative whole-body autoradiography

1 hour 4 hours 24 hours 3 days 7 days

Widespread biodistribution of gadolinium-based contrast; remnants in the kidney cortex up to 7 days after a single, clinically-relevant injection in rat

Kindberg GM et al, Eur Radiol, 2010 7

Differential effects of gadolinium-based contrast agents in rats

H&E staining - Skin

Control Gadodiamide Gadoteridol

Control Gadodiamide Gadoteridol

Immunohistochemical staining – Skin fibronectin (marker of fibrosis)

Control Gadodiamide Gadoteridol

Fibronectin

GAPDH

Immunoblot - Skin

N

C

N

C

O-

O-

O

O

N -O

O

C

Gd3+ Omniscan (gadodiamide >>

caldiamide) O

C N

O

C N ProHance

(gadoteridol)

N

Gd3+

N

N N

Do C, Barnes JL, Tan C, Wagner B, Am J Physiol Renal Physiol, 2014 8

Gadodiamide administration in mice with normal renal function

Control

(2.5 mmol/kg body weight, i.p.)

Sacrifice

Mice (C57BL6)

Gadodiamide Gadodiamide (20-25 doses)

Kidney Skin

0

500

1000

Gad

olin

ium

(µ

g/g

tissu

e)

Gadolinium content, kidney

***

0

20

40

60

Gad

olin

ium

(µ

g/g

tissu

e)

Gadolinium content, skin

***

0

3000

5000

Gad

olin

ium

(n

g/g

tissu

e)

Gadolinium content, cerebellum

***

0

2000

4000

Gad

olin

ium

(n

g/g

tissu

e)

Gadolinium content, cerebrum

***

1000

4-5 weeks

9

Gadodiamide Control Gadodiamide Control

Transmission electron microscopy - Glomeruli Transmission electron microscopy - Tubules

100 nm

2 µm

Electron microscopy shows electron-dense deposits in the kidneys of gadodiamide-treated mice with normal renal function

Wagner laboratory, unpublished

10

100 nm

Li R et al, ACS Nano, 2014

Gd2O3 Gadodiamide

Transmission electron microscopy - Tubule

Transmission electron microscopy - Mesangium

Transmission electron microscopy – Phagolysosomal-

simulated fluid

Transmission electron microscopy - Water

The renal deposits resemble Gd2O3 disordered mesh-like nanowire/nanoparticle aggregates in vitro

Wagner laboratory, unpublished

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Gadodiamide induces renal fibrosis in mice

PAS staining

Control Gadodiamide

Fibronectin

Collagen IV

220 kDa Fibronectin

GAPDH

Control Gadodiamide

Gadodiamide

PAS staining - Kidney

Gadodiamide

PAS staining - Kidney

Immunoblot - Skin Wagner laboratory, unpublished

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Control

Gadodiamide

DHE fluorescence with confocal laser scanning microscopy

Gadodiamide induces oxidative stress in the mouse kidney

Hyd

roge

n pe

roxi

de (H

2O2)

ge

nera

tion

(fluo

resc

ence

of

Ampl

ex re

d/µg

pro

tein

)

0

100

200

300 * *

Amplex red assay

Cortex

Wagner laboratory, unpublished

13

0

20

40

Der

mal

nuc

lei

(per

HPF

)

Cellularity

***

Gadodiamide induces skin fibrosis in mice with normal renal function

H&E staining - Skin

Control Gadodiamide

Immunofluorescent staining - Skin

Control Gadodiamide

DAPI (nuclei)

Fibronectin (marker of fibrosis)

180 kDa

250 kDa Fibronectin

GAPDH

Collagen IV

0

0.2

0.4

Skin

fold

(m

m)

Skin fold thickness

*

Wagner laboratory, unpublished 14

Gadodiamide treatment leads to inflammation and bone marrow-derived cells to the dermis in mice with normal renal function

DAPI (nuclei)

CD45RO (fibrocyte marker)

Immunofluorescent staining - Skin

Control Gadodiamide

DAPI (nuclei)

CD163 (inflammation)

Immunofluorescent staining - Skin

Control Gadodiamide

Wagner laboratory, unpublished 15

Gadodiamide increases oxidative stress in the skin of mice with normal renal function

In situ DHE staining and confocal microscopy - Skin

Control Gadodiamide

DAPI (nuclei)

3-Nitrotyrosine

Immunofluorescent staining - Skin

Control Gadodiamide

Wagner laboratory, unpublished 16

Control Gadodiamide

Green fluorescent protein (GFP) donor

mice

Engraftment

2.5 mmol/kg i.p. for 4 weeks

1 ✕ 107 cells

Experimental design: Tagged bone marrow transplant in mice with normal renal function

Collagen IV

GFP

Control Gadodiamide

DAPI (nuclei)

Merge

Immunofluorescent staining – Kidney

Wagner laboratory, unpublished 17

GFP

CD34

Merge

DAPI (nuclei)

Control Gadodiamide

Immunofluorescent staining - Skin

Control Gadodiamide

GFP

CD45RO

Merge

DAPI (nuclei)

Gadodiamide induces the recruitment of bone marrow-derived fibroblasts to the skin in mice

Immunofluorescent staining - Skin Wagner laboratory, unpublished 18

Biopsies of patients with NSF demonstrate significant expression of the hematopoietic progenitor marker CD34

Dermal hyper-cellularity H&E staining - Skin Immunostaining for CD34 - Skin

Clinical photographs of a patient showing skin lesions

Kroshinsky D et al, N Eng J Med, 2009 19

Conclusions

Our experiments show that renal insufficiency is not requisite for fibrosis

Mechanistically, our experiments demonstrate that it is the recruitment of bone marrow-derived cells that mediate the deleterious actions

The pathologic effects of gadolinium-based contrast agents are not well-characterized

Dechelation of gadolinium is a hypothetical pathologic mechanism.

Studies concerning the biologic effects of rare earth metals in general and their retention in human organs are in the nascent stage

Gadolinium retention can be detected in humans and in our models; This allows the mechanistic study of gadolinium-induced organ injury

The science on this topic is at ground zero

We provide examples of important avenues to understanding the mechanisms of disease (lending itself to the discovery of biomarkers)

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Working hypothesis

Patient with normal kidney function

MRI Gadolinium-based contrast exposure

Gadolinium retention

Organ injury

Impaired function

Gadolinium-based contrast exposure MRI

Gadolinium-induced disease

Biomarkers Precision/Personalized Medicine

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Working hypothesis

Patient with normal kidney function

MRI Gadolinium-based contrast exposure

Gadolinium retention

Organ injury

Impaired function

Gadolinium-based contrast exposure MRI

Pre-existing conditions

(obesity, diabetes, pregnancy,

inflammation, etc.)

Gadolinium-induced disease

Biomarkers Precision/Personalized Medicine

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Acknowledgments

Hanna E. Abboud, M.D.

Viktor Drel, Ph.D.

Jeffrey L. Barnes, Ph.D.

UTHSCSA

Catherine Do, M.D.

Denis Féliers, Ph.D.

Chunyan Tan

Wagner’s Laboratory

Yves Gorin, Ph.D.

Doug-Yoon Lee, Ph.D.

NIH RO1DK102085 (PI)

Supported by : Veterans Administration Merit Award I01BX001958 (PI) Veterans Administration Career Development Award (PI)

Veterans Administration VISN 17 New Investigator Award (PI)

Northwestern

Keith MacRenaris, Ph.D. Nephrology

Jochen Reiser, M.D., Ph.D.

Rush University Medical Center Internal Medicine Department

Seema Ahuja, M.D.

UNC

Michael Jay, Ph.D. John Prybylski, Ph.D.

UTSA Miguel Yacaman, Ph.D. Josefina Arellano-Jimenez, Ph.D.

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