The pathophysiology and retention of gadolinium
Brent Wagner, MD
Associate Professor of Medicine South Texas Veterans Health Care System, Department of Medicine/Nephrology University of Texas Health Science Center at San Antonio, San Antonio, Texas Objectives
Elucidation of the mechanisms of gadolinium-based contrast agent-induced toxicity is an active area of investigation
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
A model has been established in rodents
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
2 Overview There are many different chemical formulations of gadolinium-based contrast agents used in magnetic resonance imaging Gadolinium-based contrast agents have been linked to ‘nephrogenic’ systemic fibrosis cases
There is evidence that gadolinium is deposited in the central nervous system The central nervous system toxicity warrants more study Gadolinium-based contrast agents are biologically active Little is known about the metabolism of gadolinium-based contrast agents, their biologic effects, and the implications of retained gadolinium The toxic effects and mechanisms of gadolinium-based contrast agents is a major gap in our knowledge 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 Wagner B et al, Adv Chronic Kidney Dis, 2017 Wagner B et al, Am J Physiol Renal Physiol, 2016 3 The periodic table of elements
H He
Li Be B C N O F Ne
Na Mg Al Si P S Cl Ar
K Ca Sc Ti V Cr Mn Fe Co Ni Cu Zn Ga Ge As Se Br Kr
Rb Sr Y Zr Nb Mo Tc Ru Rh Pd Ag Cd In Sn Sb Te I Xe
Cs Ba Hf Ta W Re Os Ir Pt Au Hg Tl Pb Bi Po At Rn
Fr Ra Rf Db Sg Bh Hs Mt Ds Rg Cn Uut Fl Uup Lv Uus Uuo
La Ce Pr Nd Pm Sm Eu Gd Tb Dy Ho Er Tm Yb Lu
Ac Th Pa U Np Pu Am Cm Bk Cf Es Fm Md No Lr
4 ‘Nephrogenic’ systemic fibrosis, a man-made disease caused by magnetic resonance imaging contrast agents
Dural thickening
Face Lung Trunk Heart Diaphragm Liver Kidney Psoas Rete testes Buttocks
Extremities Joint contractures
Extremities Girardi M et al, J Am Acad Dermatol, 2011 5 Gadolinium-based contrast agents can be acutely nephrotoxic in humans
Degeneration of Flattening of tubular epithelial cells tubular epithelial cells Calcium Cellular phosphate Glomerulosclerosis proliferation
Toluidine blue stain - Electron microscopy - H&E stain - Kidney Immunostain - Kidney Kidney Kidney
Akgun H et al, Archives of Pathology & Laboratory Medicine, 2006
6 Widespread biodistribution of gadolinium-based contrast; remnants in the kidney cortex up to 7 days after a single, clinically-relevant injection in rat
1 hour 4 hours 24 hours 3 days 7 days
Quantitative whole-body autoradiography
Kindberg GM et al, Eur Radiol, 2010 7 Differential effects of gadolinium-based contrast agents in rats O
C O- N N N N N ProHance C C 3+ 3+ N Gd (gadoteridol) N Gd Omniscan O- -O O O (gadodiamide >> N N C C caldiamide)
O O Control Gadodiamide Gadoteridol
Control Gadodiamide Gadoteridol
Immunohistochemical staining – Skin fibronectin (marker of fibrosis) Control Gadodiamide Gadoteridol
Fibronectin
GAPDH
Immunoblot - Skin H&E staining - Skin 8 Do C, Barnes JL, Tan C, Wagner B, Am J Physiol Renal Physiol, 2014 Gadodiamide administration in mice with normal renal function
Mice (C57BL6)
Control Kidney 4-5 weeks Skin
Gadodiamide Gadodiamide (20-25 doses)
(2.5 mmol/kg body weight, i.p.)
Sacrifice
Gadolinium Gadolinium Gadolinium Gadolinium content, kidney content, skin content, cerebrum content, cerebellum
*** *** *** *** 60 4000 5000
1000 40 3000 2000 500 20 Gadolinium Gadolinium Gadolinium Gadolinium (µg/g tissue) (µg/g tissue) (µg/g (ng/g tissue) (ng/g tissue) 1000 0 0 0 0
9 Electron microscopy shows electron-dense deposits in the kidneys of gadodiamide-treated mice with normal renal function
Control Gadodiamide Control Gadodiamide
2 µm
100 nm
Transmission electron microscopy - Glomeruli Transmission electron microscopy - Tubules
Wagner laboratory, unpublished 10 The renal deposits resemble Gd2O3 disordered mesh-like nanowire/nanoparticle aggregates in vitro
Gadodiamide Gd2O3
Transmission electron Transmission electron microscopy - Mesangium microscopy - Water
Transmission electron Transmission electron microscopy - Tubule microscopy – Phagolysosomal- simulated fluid
100 nm
Wagner laboratory, unpublished Li R et al, ACS Nano, 2014
11 Gadodiamide induces renal fibrosis in mice
Control Gadodiamide Gadodiamide Control Gadodiamide
PAS staining
Gadodiamide
Fibronectin
PAS staining - Kidney PAS staining - Kidney
Collagen IV
Fibronectin 220 kDa
GAPDH
Immunoblot - Skin 12 Wagner laboratory, unpublished
Gadodiamide induces oxidative stress in the mouse kidney
Amplex red assay
Cortex
Control ) 2
O ** 2 300
200 red/µg protein) 100
Gadodiamide 0 Amplex Hydrogen Hydrogen peroxide (H generation (fluorescence of of (fluorescence generation
DHE fluorescence with confocal laser scanning microscopy
Wagner laboratory, unpublished 13 Gadodiamide induces skin fibrosis in mice with normal renal function Skin fold Control Gadodiamide Cellularity thickness
*** *
40 0.4
20 (mm) 0.2 Skin fold (per HPF) ermal nuclei ermal D 0 0 H&E staining - Skin
Control Gadodiamide
Fibronectin (marker of fibrosis)
Fibronectin 250 kDa Immunofluorescent staining - Skin Collagen IV 180 kDa
GAPDH DAPI (nuclei)
Wagner laboratory, unpublished 14 Gadodiamide treatment leads to inflammation and bone marrow-derived cells to the dermis in mice with normal renal function
Control Gadodiamide Control Gadodiamide
CD163 CD45RO (inflammation) (fibrocyte marker)
DAPI DAPI (nuclei) (nuclei)
Immunofluorescent Immunofluorescent staining - Skin staining - Skin
Wagner laboratory, unpublished 15 Gadodiamide increases oxidative stress in the skin of mice with normal renal function
Control Gadodiamide Control Gadodiamide
3-Nitrotyrosine
DAPI (nuclei)
Immunofluorescent In situ DHE staining and staining - Skin confocal microscopy - Skin
Wagner laboratory, unpublished 16 Experimental design: Tagged bone marrow transplant in mice with normal renal function
Green fluorescent Control Gadodiamide protein (GFP) donor mice
✕ 7 1 10 cells GFP
Collagen IV
Engraftment
Merge
Control Gadodiamide
DAPI (nuclei)
2.5 mmol/kg i.p. Immunofluorescent for 4 weeks staining – Kidney Wagner laboratory, unpublished 17 Gadodiamide induces the recruitment of bone marrow-derived fibroblasts to the skin in mice
Control Gadodiamide Control Gadodiamide
GFP GFP
CD34 CD45RO
Merge Merge
DAPI (nuclei) DAPI (nuclei)
Immunofluorescent Immunofluorescent staining - Skin staining - Skin Wagner laboratory, unpublished 18 Biopsies of patients with NSF demonstrate significant expression of the hematopoietic progenitor marker CD34
Clinical photographs of a patient showing skin lesions
H&E staining - Skin Immunostaining for CD34 - Skin Dermal hyper-cellularity Kroshinsky D et al, N Eng J Med, 2009 19 Conclusions
Gadolinium retention can be detected in humans and in our models; This allows the mechanistic study of gadolinium-induced organ injury
The pathologic effects of gadolinium-based contrast agents are not well- characterized 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 We provide examples of important avenues to understanding the mechanisms of disease (lending itself to the discovery of biomarkers)
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 The science on this topic is at ground zero
20 Working hypothesis
Patient with normal kidney function MRI Gadolinium- Gadolinium-based induced disease contrast exposure
Gadolinium Impaired retention function
Organ injury
Gadolinium-based MRI contrast exposure Biomarkers Precision/Personalized Medicine
21 Working hypothesis
Patient with normal kidney function MRI Gadolinium- Gadolinium-based induced disease contrast exposure
Pre-existing Gadolinium Impaired conditions retention function (obesity, diabetes, pregnancy, inflammation, etc.) Organ injury
Gadolinium-based MRI contrast exposure Biomarkers Precision/Personalized Medicine
22 Acknowledgments Northwestern UTHSCSA UNC Nephrology Keith MacRenaris, Ph.D. Michael Jay, Ph.D. Wagner’s Laboratory John Prybylski, Ph.D. Chunyan Tan UTSA Catherine Do, M.D. Miguel Yacaman, Ph.D. Viktor Drel, Ph.D. Josefina Arellano-Jimenez, Ph.D.
Yves Gorin, Ph.D. Denis Féliers, Ph.D. Jeffrey L. Barnes, Ph.D. Seema Ahuja, M.D. NIH RO1DK102085 (PI) Doug-Yoon Lee, Ph.D. Hanna E. Abboud, M.D. Supported by : Veterans Administration Merit Award Rush University Medical Center I01BX001958 (PI) Internal Medicine Department Veterans Administration Career Development Award (PI) Jochen Reiser, M.D., Ph.D. Veterans Administration VISN 17 New Investigator Award (PI) 23