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 .