![Chapter 1
Restoration of Podocyte Phenotype in Culture
Eishin Yaoita
Abstract
The most distinctive characteristic of podocytes in the kidney is the presence of interdigitating cell processes
with nephrin and podocin that are concentrated at sites of cell–cell contact. Unfortunately, these defining
features are easily lost in culture. We previously reported culture conditions that can restore the differ-
entiated phenotypes in primary cultures of rat podocyte. Since then, some of the materials used have been
discontinued or improved. In this chapter, we therefore provide our most recent protocol for the restora-
tion of the podocyte phenotype in culture.
Key words Podocyte, Glomerulus, Primary culture, Rat, Interdigitation, Dextran sulfate, All-trans-
retinoic acid, Laminin
1 Introduction
Podocytes in the kidney exhibit a unique 3D morphology consist-
ing of interdigitating primary processes and foot processes with slit
diaphragms. These cells play a critical role in maintaining the glo-
merular filtration barrier, with podocyte injury being a defining
cause of glomerulosclerosis [1]. Therefore, podocytes have
attracted much interest, with many studies having been performed
using various culture systems. Podocyte cell lines are most com-
monly used [2–4]. These cell lines, however, express low levels of
podocyte-specific genes and show a polygonal morphology without
interdigitating processes when at confluence [5]. No one has suc-
ceeded in inducing the interdigitation of cell processes using
immortalized cells. In addition, the podocyte origin of these cell
lines is controversial as cultured parietal epithelial cells of the Bow-
man’s capsule (PECs) express many of the same proteins used to
define podocytes [6].
Conversely, primary cultured podocytes, especially those
derived from isolated glomeruli, are often assumed to retain their
phenotypes in vivo because of the short-term culture. Although
glomeruli are conventionally isolated by serial sieving, podocytes
Tim D. Hewitson et al. (eds.), Kidney Research: Experimental Protocols, Methods in Molecular Biology, vol. 2664,
https://doi.org/10.1007/978-1-0716-3179-9_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023
3](https://image.slidesharecdn.com/43787-250307183841-773e2ee8/85/Kidney-Research-Experimental-Protocols-3rd-Edition-Edition-Tim-D-Hewitson-22-320.jpg)
![are damaged by this process [7], meaning that most of the cell
outgrowths are derived from PECs [8, 9]. To address this,
Takemoto et al. devised a new way of isolating glomeruli to mini-
mize podocyte damage and PEC contamination, a process involv-
ing the perfusion of kidneys with magnetic particles followed by
collagenase digestion [10]. Using glomeruli isolated by this meth-
odology, we were able to generate primary podocyte cultures [11]
and succeeded in establishing culture conditions that restore podo-
cytes to a physiological morphology with interdigitating cell pro-
cesses and high expression levels of podocyte-specific markers
[12]. The recapitulation of in vivo phenotype is in itself a robust
validation of podocyte phenotype in these cells. The established
culture system has also been shown to reproduce well the patho-
logical changes of podocytes seen in vivo [13]. In this chapter, our
current protocol for podocyte culture is provided.
4 Eishin Yaoita
2 Materials
Use sterile materials. Store reagents at 4 °C (unless indicated
otherwise).
2.1 Animals Male rats 8–10 weeks old (see Note 1).
2.2 Perfusion of
Kidneys
1. Anesthetic agent, dissecting instruments.
2. Phosphate-buffered saline (PBS): Sterilize by autoclave.
3. Carbonyl iron powder (CS grade, φ6.0–7.0 μm) (BASF SE,
Ludwigshafen, Germany): 20 mg/mL PBS. Sterilize by
autoclaving.
4. 10 mL syringe.
5. 21G or 23G butterfly needle.
6. 90 mm petri dish.
2.3 Isolation of
Glomeruli
1. Hanks’ balanced salt solution with calcium and magnesium
(HBSS).
2. Collagenase A: 100 mg/5 mL HBSS. Subdivide into small
portions, and store at -20 °C.
3. Deoxyribonuclease I (DNase I): 100 mg/10 mL HBSS. Sub-
divide into small portions, and store at -20 °C.
4. Magnetic particle concentrator.
5. 100 μm cell strainer, 70 μm cell strainer.
6. 90 mm petri dish, 14 mL round bottom tube, 50 mL tube.
7. Syringe plunger with a flat tip.](https://image.slidesharecdn.com/43787-250307183841-773e2ee8/85/Kidney-Research-Experimental-Protocols-3rd-Edition-Edition-Tim-D-Hewitson-23-320.jpg)
Kidney Research Experimental Protocols 3rd Edition Edition Tim D. Hewitson
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- 5. Kidney Research Experimental Protocols 3rd Edition Edition Tim D. Hewitson Digital Instant Download Author(s): TimD. Hewitson, Nigel D. Toussaint, Edward R. Smith (Editors) ISBN(s): 9781071631782, 1071631780 Edition: 3rd Edition File Details: PDF, 18.72 MB Year: 2023 Language: english
- 6. Kidney Research Tim D. Hewitson Nigel D.Toussaint Edward R. Smith Editors Experimental Protocols ThirdEdition Methods in Molecular Biology 2664
- 7. M E T H O D S I N M O L E C U L A R B I O L O G Y Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK For further volumes: http://www.springer.com/series/7651
- 8. For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by- step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.
- 9. Kidney Research Experimental Protocols Third Edition Edited by Tim D. Hewitson, Nigel D. Toussaint, and Edward R. Smith Department of Nephrology, The Royal Melbourne Hospital, Parkville, Melbourne, VIC, Australia, Department of Medicine (RMH), University of Melbourne, Parkville, Melbourne, VIC, Australia
- 10. Editors Tim D. Hewitson Department of Nephrology The Royal Melbourne Hospital Parkville, Melbourne, VIC, Australia Department of Medicine (RMH) University of Melbourne Parkville, Melbourne, VIC, Australia Nigel D. Toussaint Department of Nephrology The Royal Melbourne Hospital Parkville, Melbourne, VIC, Australia Department of Medicine (RMH) University of Melbourne Parkville, Melbourne, VIC, Australia Edward R. Smith Department of Nephrology The Royal Melbourne Hospital Parkville, Melbourne, VIC, Australia Department of Medicine (RMH) University of Melbourne Parkville, Melbourne, VIC, Australia ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-3178-2 ISBN 978-1-0716-3179-9 (eBook) https://doi.org/10.1007/978-1-0716-3179-9 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
- 11. Preface The inherent complexity of kidney structure and function often poses unique problems for research in kidney biology and disease. Through necessity therefore, an organ-specific approach has evolved over time where specialist techniques have been developed and adapted. In this third edition of Kidney Research – Experimental Protocols, we include novel techniques as well as improvements and refinements to established protocols. In doing so, our emphasis has been on protocols that address new and emerging research questions in Nephrology. The first section (Part I) addresses various in vitro, in vivo, and ex vivo models of kidney disease. An initial collection of current in vitro kidney cell culture methods is provided (Chapters 1, 2, 3, and 4). While such 2D cell culture models are valuable tools for examining mechanisms in a controlled environment, being based on a single cell type means that they cannot reproduce complex cellular interactions, homeostasis, and microenvironment. Therefore, in companion chapters, authors also provide state-of-the-art protocols for orga- noid generation (Chapters 5 and 6), in vitro microfabrication models (Chapters 7 and 8), and precision cut kidney slices (Chapter 9) to replicate essential components of in vivo kidney physiology in vitro. These protocols emphasize the utility of such approaches in toxicity screening and drug development. Also included is a cell culture model of tubule cyst formation (Chapter 10). Finally, the similarity of the zebrafish genome to that of the human, and its ease of manipulation, makes the zebrafish a useful single nephron model of glomer- ular disease (Chapter 11). In Part II, a variety of new imaging protocols are provided for direct observation of kidney tissue in studies of kidney disease. These include a technique based on magnetic resonance imaging for the measurement of sodium stores in tissue (Chapter 12), a newly recognized and poorly understood phenomena where imaging is particularly useful for assessing the excess sodium accumulation that occurs in people with chronic kidney disease (CKD). Also presented is a new microscopy technique for examining tissue collagen using second harmonic generation (Chapter 13), which is both quantitative and able to provide a high-resolution view of collagen fiber organization previously only available with electron microscopy. Likewise, a high-resolution adaptation of confocal microscopy is provided (Chapter 14), while the use of a ferritin tracer for imaging glomeruli (Chapter 15) completes Part II. The final section, Part III, consists of recent advances in analytical and functional assays. Protocols for in situ hybridization and spatial transcriptome sequencing are provided in Chapters 16 and 17, respectively. Both approaches offer important morphological context to gene expression within intact tissue. Much current interest relates to the role of mito- chondrial function in disease. To address this, Orozco-Ibarra et al. outline imaging and functional protocols for examining and measuring mitochondria activity (Chapter 18). In this section, Mullins et al. also detail a solution to the vexed problem of measuring glomerular filtration rate in mouse experiments (Chapter 19). Mineralization disorders in v
- 12. CKD are common and measurement of calcific calciprotein particles (C RhoGTPase activity (C biology. poorly understood. Therefore, two chapters provide tools for ation propensity (Chapter 20) and the formation of circulating hapter 21). The final chapter describes a protocol for measuring hapter 22), an important element of signal transduction in kidney vi Preface We are indebted to the generosity of the authors in sharing their protocols and insights. Nephrology is a specialty where discovery science has delivered a great deal. We hope that the protocols will help provide the necessary tools for that to continue. Parkville, Melbourne, VIC, Australia Tim D. Hewitson Parkville, Melbourne, VIC, Australia Nigel D. Toussaint Parkville, Melbourne, VIC, Australia Edward R. Smith
- 13. Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi PART I IN VITRO, IN VIVO, AND EX VIVO MODELS OF KIDNEY DISEASE 1 Restoration of Podocyte Phenotype in Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Eishin Yaoita 2 Propagation, Culture, and Characterization of Renal Fibroblasts. . . . . . . . . . . . . . 13 Tim D. Hewitson and Edward R. Smith 3 Isolation of Rat Glomeruli and Propagation of Mesangial Cells to Study the Kidney in Health and Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Tim D. Hewitson and Edward R. Smith 4 In Vitro Models to Evaluate Molecular Permeability of the Kidney Filtration Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Dan Wang and Nicholas Ferrell 5 Generating Human Glomeruli from Pluripotent Stem Cells for Disease Modelling and Drug Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Lorna J. Hale and Melissa H. Little 6 Large-Scale Production of Kidney Organoids from Human Pluripotent Stem Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Veronika Sander, Aneta Przepiorski, Neil A. Hukriede, and Alan J. Davidson 7 Construction of a Multitubular Perfusable Kidney-on-Chip for the Study of Renal Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Brice Lapin, Sarah Myram, Manh-Louis Nguyen, Giacomo Gropplero, Sylvie Coscoy, and Stéphanie Descroix 8 A “Kidney-on-the-Chip” Model Composed of Primary Human Tubular, Endothelial, and White Blood Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Julian A. Marschner, Lucas Martin, Gregor Wilken, Maria Elena Melica, and Hans-Joachim Anders 9 Standardized Protocol for the Preparation of Precision-Cut Kidney Slices: A Translational Model of Renal Fibrosis . . . . . . . . . . . . . . . . . . . . . . 123 Michael Schou Jensen, Camilla Merrild, Rikke Nørregaard, Peter Olinga, and Henricus A. M. Mutsaers 10 Culture of Three-Dimensional Madin–Darby Canine Kidney (MDCK) Cysts for In Vitro Drug Testing in Polycystic Kidney Disease. . . . . . . . . . . . . . . . . 135 Sayanthooran Saravanabavan and Gopala K. Rangan 11 Zebrafish Model to Study Podocyte Function Within the Glomerular Filtration Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Nina Sopel and Janina Müller-Deile vii
- 14. viii Contents PART II IMAGING IN KIDNEY RESEARCH 12 Magnetic Resonance Imaging (MRI) Analysis of Tissue Sodium Concentration in Chronic Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 Kylie Martin, Vijay Venkatraman, Angela Agostinelli, Braden Thai, Daniel St€ ab, Tim D. Hewitson, Sven-Jean Tan, Nigel D. Toussaint, and Patsy Robertson 13 Determination of Interstitial Collagen Deposition and Related Topography Using the Second Harmonic Generation-Based HistoIndex Platform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Sadman Bhuiyan, Robert E. Widdop, and Chrishan S. Samuel 14 Quantitative Imaging of Podocyte Foot Processes in the Kidney Using Confocal and STED Microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 David Unnersjö-Jess 15 Synthesis and Expression of a Targeted, Ferritin-Based Tracer for PET Imaging of Kidney Glomeruli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Edwin J. Baldelomar, Kasey C. Emoto, Maria Veronica Clavijo Jordan, Jennifer R. Charlton, David E. Reichert, Neda Parvin, Courtnie Yokono, Bradley D. Hann, and Kevin M. Bennett PART III ANALYTICAL AND FUNCTIONAL MEASUREMENTS IN THE KIDNEY 16 Multiplex In Situ Hybridization in the Study of Acute Kidney Injury. . . . . . . . . . 217 Agnieszka Masztalerz, Alex Gregorieff, Serge Lemay, and Tomoko Takano 17 Spatial Transcriptomics in Kidney Tissue. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 Arti M. Raghubar, Joanna Crawford, Kahli Jones, Pui Y. Lam, Stacey B. Andersen, Nicholas A. Matigian, Monica S. Y. Ng, Helen Healy, Andrew J. Kassianos, and Andrew J. Mallett 18 Assessment of Kidney Mitochondrial Function by High-Resolution Respirometry, Transmission Electron Microscopy, and Histological Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283 Marisol Orozco-Ibarra, Omar Emiliano Aparicio-Trejo, Alexis Paulina Jiménez-Uribe, Estefani Yaquelin Hernández-Cruz, Ana Karina Aranda-Rivera, Isabel Amador-Martı́nez, Francisca Fernández-Valverde, and José Pedraza-Chaverri 19 Transdermal Measurement of Glomerular Filtration Rate in Preclinical Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 Thomas P. Mullins, Daniel Schock-Kusch, and Linda A. Gallo 20 The BioHybrid Assay: A Novel Method for Determining Calcification Propensity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317 Armand M. G. Jaminon, Asim C. Akbulut, Nikolas Rapp, Chris P. Reutelingsperger, and Leon J. Schurgers
- 15. Contents ix 21 Quantification of Calciprotein Particles (CPPs) in Serum/Plasma Samples Using a Fluorescent Bisphosphonate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 Yutaka Miura, Hiroshi Kurosu, and Makoto Kuro-o 22 Monitoring of Rho GTPase Activity in Podocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . 343 Jun Matsuda and Tomoko Takano Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
- 16. Contributors ANGELA AGOSTINELLI • Department of Radiology, The Royal Melbourne Hospital and The University of Melbourne, Parkville, Melbourne, VIC, Australia ASIM C. AKBULUT • Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University Medical Centre, Maastricht, The Netherlands ISABEL AMADOR-MARTÍNEZ • Facultad de Quı́mica, Departamento de Biologı́a, Universidad Nacional Aut onoma de México (UNAM), Mexico City, Mexico; Posgrado en Ciencias Biol ogicas, Universidad Nacional Aut onoma de México (UNAM), Ciudad Universitaria, Mexico City, Mexico HANS-JOACHIM ANDERS • Renal Division, Department of Medicine IV, Hospital of the Ludwig Maximilians University, Munich, Germany STACEY B. ANDERSEN • Genome Innovation Hub, University of Queensland, Brisbane, QLD, Australia; UQ Sequencing Facility, Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia OMAR EMILIANO APARICIO-TREJO • Departamento de Fisiopatologı́a Cardio-Renal, Instituto Nacional de Cardiologı́a Ignacio Chávez, Mexico City, Mexico ANA KARINA ARANDA-RIVERA • Facultad de Quı́mica, Departamento de Biologı́a, Universidad Nacional Aut onoma de México (UNAM), Mexico City, Mexico; Posgrado en Ciencias Biol ogicas, Universidad Nacional Aut onoma de México (UNAM), Ciudad Universitaria, Mexico City, Mexico EDWIN J. BALDELOMAR • Mallinckrodt Institute of Radiology, Washington University in St. Louis, St. Louis, MO, USA KEVIN M. BENNETT • Mallinckrodt Institute of Radiology, Washington University in St. Louis, St. Louis, MO, USA SADMAN BHUIYAN • Cardiovascular Disease Program, Biomedicine Discovery Institute and Department of Pharmacology, Monash University, Melbourne, VIC, Australia JENNIFER R. CHARLTON • Department of Pediatrics, Division of Nephrology, University of Virginia, Charlottesville, VA, USA SYLVIE COSCOY • Institut Curie, Université PSL, Sorbonne Université, CNRS UMR168, Laboratoire Physico Chimie Curie, Paris, France JOANNA CRAWFORD • Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia ALAN J. DAVIDSON • Department of Molecular Medicine Pathology, University of Auckland, Auckland, New Zealand STÉPHANIE DESCROIX • Institut Curie, Université PSL, Sorbonne Université, CNRS UMR168, Laboratoire Physico Chimie Curie, Paris, France KASEY C. EMOTO • Department of Biology, University of Hawaii at Manoa, Honolulu, HI, USA FRANCISCA FERNÁNDEZ-VALVERDE • Laboratorio de Patologı́a Experimental, Instituto Nacional de Neurologı́a y Neurocirugı́a Manuel Velasco Suárez, Mexico City, Mexico NICHOLAS FERRELL • Department of Internal Medicine, Division of Nephrology, Ohio State University Wexner Medical Center, Columbus, OH, USA xi
- 17. xii Contributors LINDA A. GALLO • School of Biomedical Sciences, The University of Queensland, St Lucia, Brisbane, QLD, Australia ALEX GREGORIEFF • Research Institute, McGill University Health Centre, Montreal, QC, Canada; Department of Pathology, McGill University Health Centre, Montreal, QC, Canada GIACOMO GROPPLERO • Institut Curie, Université PSL, Sorbonne Université, CNRS UMR168, Laboratoire Physico Chimie Curie, Paris, France LORNA J. HALE • Murdoch Children’s Research Institute, Parkville, VIC, Australia; UCB Pharma Ltd, Slough, UK BRADLEY D. HANN • Department of Biology, University of Hawaii at Manoa, Honolulu, HI, USA HELEN HEALY • Kidney Health Service, Royal Brisbane and Women’s Hospital, Herston, QLD, Australia; Conjoint Internal Medicine Laboratory, Chemical Pathology, Pathology Queensland, Health Support Queensland, Herston, QLD, Australia; Faculty of Medicine, University of Queensland, Brisbane, QLD, Australia ESTEFANI YAQUELIN HERNÁNDEZ-CRUZ • Facultad de Quı́mica, Departamento de Biologı́a, Universidad Nacional Aut onoma de México (UNAM), Mexico City, Mexico; Posgrado en Ciencias Biol ogicas, Universidad Nacional Aut onoma de México (UNAM), Ciudad Universitaria, Mexico City, Mexico TIM D. HEWITSON • Department of Nephrology, The Royal Melbourne Hospital, Parkville, Melbourne, VIC, Australia; Department of Medicine, University of Melbourne, Parkville, Melbourne, VIC, Australia NEIL A. HUKRIEDE • Department of Developmental Biology, University of Pittsburgh, Pittsburgh, PA, USA ARMAND M. G. JAMINON • Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University Medical Centre, Maastricht, The Netherlands MICHAEL SCHOU JENSEN • Department of Clinical Medicine, Aarhus University, Aarhus, Denmark ALEXIS PAULINA JIMÉNEZ-URIBE • Facultad de Quı́mica, Departamento de Biologı́a, Universidad Nacional Aut onoma de México (UNAM), Mexico City, Mexico KAHLI JONES • Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia; School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia MARIA VERONICA CLAVIJO JORDAN • Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital/Harvard Medical School, Charlestown, MA, USA ANDREW J. KASSIANOS • Kidney Health Service, Royal Brisbane and Women’s Hospital, Herston, QLD, Australia; Conjoint Internal Medicine Laboratory, Chemical Pathology, Pathology Queensland, Health Support Queensland, Herston, QLD, Australia; Faculty of Medicine, University of Queensland, Brisbane, QLD, Australia MAKOTO KURO-O • Division of Anti-aging Medicine, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan HIROSHI KUROSU • Division of Anti-aging Medicine, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan PUI Y. LAM • Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia
- 18. Contributors xiii BRICE LAPIN • Institut Curie, Université PSL, Sorbonne Université, CNRS UMR168, Laboratoire Physico Chimie Curie, Paris, France SERGE LEMAY • Research Institute, McGill University Health Centre, Montreal, QC, Canada; Department of Medicine, Division of Nephrology, McGill University Health Centre, Montreal, QC, Canada MELISSA H. LITTLE • Murdoch Children’s Research Institute, Parkville, VIC, Australia; Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, VIC, Australia ANDREW J. MALLETT • Faculty of Medicine, University of Queensland, Brisbane, QLD, Australia; Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia; College of Medicine Dentistry, James Cook University, Townsville, QLD, Australia; Department of Renal Medicine, Townsville University Hospital, Townsville, QLD, Australia JULIAN A. MARSCHNER • Renal Division, Department of Medicine IV, Hospital of the Ludwig Maximilians University, Munich, Germany; Department of Pharmacy, Ludwig Maximilians University, Munich, Germany KYLIE MARTIN • Department of Nephrology, The Royal Melbourne Hospital, Parkville, Melbourne, VIC, Australia; Department of Medicine (RMH), University of Melbourne, Melbourne, VIC, Australia LUCAS MARTIN • Renal Division, Department of Medicine IV, Hospital of the Ludwig Maximilians University, Munich, Germany AGNIESZKA MASZTALERZ • Research Institute, McGill University Health Centre, Montreal, QC, Canada NICHOLAS A. MATIGIAN • QCIF Facility for Advanced Bioinformatics, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia JUN MATSUDA • Department of Nephrology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan MARIA ELENA MELICA • Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Florence, Italy CAMILLA MERRILD • Department of Clinical Medicine, Aarhus University, Aarhus, Denmark YUTAKA MIURA • Division of Anti-aging Medicine, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan JANINA MÜLLER-DEILE • Friedrich-Alexander Universi€ at Erlangen-Nuremberg, Erlangen, Germany THOMAS P. MULLINS • School of Biomedical Sciences, The University of Queensland, St Lucia, Brisbane, QLD, Australia HENRICUS A. M. MUTSAERS • Department of Clinical Medicine, Aarhus University, Aarhus, Denmark SARAH MYRAM • Institut Curie, Université PSL, Sorbonne Université, CNRS UMR168, Laboratoire Physico Chimie Curie, Paris, France MONICA S. Y. NG • Kidney Health Service, Royal Brisbane and Women’s Hospital, Herston, QLD, Australia; Conjoint Internal Medicine Laboratory, Chemical Pathology, Pathology Queensland, Health Support Queensland, Herston, QLD, Australia; Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia; Nephrology Department, Princess Alexandra Hospital, Woolloongabba, QLD, Australia MANH-LOUIS NGUYEN • Institut Curie, Université PSL, Sorbonne Université, CNRS UMR168, Laboratoire Physico Chimie Curie, Paris, France
- 19. xiv Contributors RIKKE NØRREGAARD • Department of Clinical Medicine, Aarhus University, Aarhus, Denmark PETER OLINGA • Department of Pharmaceutical Technology and Biopharmacy, University of Groningen, Groningen, The Netherlands MARISOL OROZCO-IBARRA • Departamento de Bioquı́mica, Instituto Nacional de Cardiologı́a Ignacio Chávez, Mexico City, Mexico NEDA PARVIN • Mallinckrodt Institute of Radiology, Washington University in St. Louis, St. Louis, MO, USA JOSÉ PEDRAZA-CHAVERRI • Facultad de Quı́mica, Departamento de Biologı́a, Universidad Nacional Aut onoma de México (UNAM), Mexico City, Mexico ANETA PRZEPIORSKI • Department of Developmental Biology, University of Pittsburgh, Pittsburgh, PA, USA ARTI M. RAGHUBAR • Kidney Health Service, Royal Brisbane and Women’s Hospital, Herston, QLD, Australia; Conjoint Internal Medicine Laboratory, Chemical Pathology, Pathology Queensland, Health Support Queensland, Herston, QLD, Australia; Faculty of Medicine, University of Queensland, Brisbane, QLD, Australia; Anatomical Pathology, Pathology Queensland, Health Support Queensland, Herston, QLD, Australia; Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia GOPALA K. RANGAN • Michael Stern Laboratory for Polycystic Kidney Disease, Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW, Australia; Department of Renal Medicine, Westmead Hospital, Western Sydney Local Health District, Westmead, NSW, Australia; Westmead Clinical School, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia NIKOLAS RAPP • Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University Medical Centre, Maastricht, The Netherlands DAVID E. REICHERT • Mallinckrodt Institute of Radiology, Washington University in St. Louis, St. Louis, MO, USA CHRIS P. REUTELINGSPERGER • Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University Medical Centre, Maastricht, The Netherlands PATSY ROBERTSON • Department of Radiology, The Royal Melbourne Hospital and The University of Melbourne, Parkville, Melbourne, VIC, Australia CHRISHAN S. SAMUEL • Cardiovascular Disease Program, Biomedicine Discovery Institute and Department of Pharmacology, Monash University, Melbourne, VIC, Australia; Department of Biochemistry and Pharmacology, The University of Melbourne, Melbourne, VIC, Australia VERONIKA SANDER • Department of Molecular Medicine Pathology, University of Auckland, Auckland, New Zealand SAYANTHOORAN SARAVANABAVAN • Michael Stern Laboratory for Polycystic Kidney Disease, Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW, Australia; Department of Renal Medicine, Westmead Hospital, Western Sydney Local Health District, Westmead, NSW, Australia DANIEL SCHOCK-KUSCH • Institute of Process Control and Innovative Energy Conversion, Mannheim University of Applied Sciences, Mannheim, Germany LEON J. SCHURGERS • Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University Medical Centre, Maastricht, The Netherlands
- 20. Contributors xv EDWARD R. SMITH • Department of Nephrology, The Royal Melbourne Hospital, Parkville, Melbourne, VIC, Australia; Department of Medicine, University of Melbourne, Parkville, Melbourne, VIC, Australia NINA SOPEL • Department of Medicine 4 – Nephrology and Hypertension, Universit€ atsklinikum Erlangen, Erlangen, Germany; Friedrich-Alexander Universi€ at Erlangen-Nuremberg, Erlangen, Germany DANIEL ST€ AB • MR Research Collaborations, Siemens Healthcare Pty Limited, Melbourne, VIC, Australia TOMOKO TAKANO • Research Institute, McGill University Health Centre, Montreal, QC, Canada; Department of Medicine, Division of Nephrology, McGill University Health Centre, Montreal, QC, Canada SVEN-JEAN TAN • Department of Nephrology, The Royal Melbourne Hospital, Parkville, Melbourne, VIC, Australia; Department of Medicine (RMH), University of Melbourne, Melbourne, VIC, Australia BRADEN THAI • Department of Radiology, The Royal Melbourne Hospital and The University of Melbourne, Parkville, Melbourne, VIC, Australia NIGEL D. TOUSSAINT • Department of Nephrology, The Royal Melbourne Hospital, Parkville, Melbourne, VIC, Australia; Department of Medicine (RMH), University of Melbourne, Parkville, Melbourne, VIC, Australia DAVID UNNERSJÖ-JESS • Department II of Internal Medicine and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany; MedTechLabs, Karolinska University Hospital, Solna, Sweden VIJAY VENKATRAMAN • Department of Radiology, The Royal Melbourne Hospital and The University of Melbourne, Parkville, Melbourne, VIC, Australia DAN WANG • Department of Internal Medicine, Division of Nephrology, Ohio State University Wexner Medical Center, Columbus, OH, USA ROBERT E. WIDDOP • Cardiovascular Disease Program, Biomedicine Discovery Institute and Department of Pharmacology, Monash University, Melbourne, VIC, Australia GREGOR WILKEN • Renal Division, Department of Medicine IV, Hospital of the Ludwig Maximilians University, Munich, Germany EISHIN YAOITA • Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan COURTNIE YOKONO • Department of Biology, University of Hawaii at Manoa, Honolulu, HI, USA
- 21. Part I In Vitro, In Vivo, and Ex Vivo Models of Kidney Disease
- 22. Chapter 1 Restoration of Podocyte Phenotype in Culture Eishin Yaoita Abstract The most distinctive characteristic of podocytes in the kidney is the presence of interdigitating cell processes with nephrin and podocin that are concentrated at sites of cell–cell contact. Unfortunately, these defining features are easily lost in culture. We previously reported culture conditions that can restore the differ- entiated phenotypes in primary cultures of rat podocyte. Since then, some of the materials used have been discontinued or improved. In this chapter, we therefore provide our most recent protocol for the restora- tion of the podocyte phenotype in culture. Key words Podocyte, Glomerulus, Primary culture, Rat, Interdigitation, Dextran sulfate, All-trans- retinoic acid, Laminin 1 Introduction Podocytes in the kidney exhibit a unique 3D morphology consist- ing of interdigitating primary processes and foot processes with slit diaphragms. These cells play a critical role in maintaining the glo- merular filtration barrier, with podocyte injury being a defining cause of glomerulosclerosis [1]. Therefore, podocytes have attracted much interest, with many studies having been performed using various culture systems. Podocyte cell lines are most com- monly used [2–4]. These cell lines, however, express low levels of podocyte-specific genes and show a polygonal morphology without interdigitating processes when at confluence [5]. No one has suc- ceeded in inducing the interdigitation of cell processes using immortalized cells. In addition, the podocyte origin of these cell lines is controversial as cultured parietal epithelial cells of the Bow- man’s capsule (PECs) express many of the same proteins used to define podocytes [6]. Conversely, primary cultured podocytes, especially those derived from isolated glomeruli, are often assumed to retain their phenotypes in vivo because of the short-term culture. Although glomeruli are conventionally isolated by serial sieving, podocytes Tim D. Hewitson et al. (eds.), Kidney Research: Experimental Protocols, Methods in Molecular Biology, vol. 2664, https://doi.org/10.1007/978-1-0716-3179-9_1, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023 3
- 23. are damaged by this process [7], meaning that most of the cell outgrowths are derived from PECs [8, 9]. To address this, Takemoto et al. devised a new way of isolating glomeruli to mini- mize podocyte damage and PEC contamination, a process involv- ing the perfusion of kidneys with magnetic particles followed by collagenase digestion [10]. Using glomeruli isolated by this meth- odology, we were able to generate primary podocyte cultures [11] and succeeded in establishing culture conditions that restore podo- cytes to a physiological morphology with interdigitating cell pro- cesses and high expression levels of podocyte-specific markers [12]. The recapitulation of in vivo phenotype is in itself a robust validation of podocyte phenotype in these cells. The established culture system has also been shown to reproduce well the patho- logical changes of podocytes seen in vivo [13]. In this chapter, our current protocol for podocyte culture is provided. 4 Eishin Yaoita 2 Materials Use sterile materials. Store reagents at 4 °C (unless indicated otherwise). 2.1 Animals Male rats 8–10 weeks old (see Note 1). 2.2 Perfusion of Kidneys 1. Anesthetic agent, dissecting instruments. 2. Phosphate-buffered saline (PBS): Sterilize by autoclave. 3. Carbonyl iron powder (CS grade, φ6.0–7.0 μm) (BASF SE, Ludwigshafen, Germany): 20 mg/mL PBS. Sterilize by autoclaving. 4. 10 mL syringe. 5. 21G or 23G butterfly needle. 6. 90 mm petri dish. 2.3 Isolation of Glomeruli 1. Hanks’ balanced salt solution with calcium and magnesium (HBSS). 2. Collagenase A: 100 mg/5 mL HBSS. Subdivide into small portions, and store at -20 °C. 3. Deoxyribonuclease I (DNase I): 100 mg/10 mL HBSS. Sub- divide into small portions, and store at -20 °C. 4. Magnetic particle concentrator. 5. 100 μm cell strainer, 70 μm cell strainer. 6. 90 mm petri dish, 14 mL round bottom tube, 50 mL tube. 7. Syringe plunger with a flat tip.
- 24. Podocytes in Culture 5 2.4 Culture of Glomeruli 1. 60 mm culture dish without ECM coating. 2. Dulbecco’s modified Eagle medium/Nutrient Mixture F-12 (DMEM/F12). 3. Insulin-transferrin-selenite media supplement (ITS). 4. 10,000 U/mL penicillin, 10 mg/mL streptomycin. Subdivide into small portions, and store at -20 °C. 5. Fetal bovine serum (FBS): Heat for 30 min at 56 °C with mixing to inactivate complement proteins. Subdivide into small portions, and store at -80 °C. 6. Medium (A): DMEM/F12 supplemented with 5% FBS, 0.5% ITS, 100 U/mL penicillin, and 100 μg/mL streptomycin. 2.5 Subculture of Podocytes 1. Non-enzymatic cell dissociation solution (CDS) (Sigma- Aldrich, Saint Louis, MO). 2. 40 μm cell strainer. 3. Laminin-521: 100 μg/mL. Subdivide into small portions, and store at -80 °C. 4. Glass slides printed with a highly water-repellent mark with 5 mm diameter wells (TF1205M, Matsunami Glass Ind., Ltd., Osaka, Japan): Sterilize by dry heat sterilization at 180 ° C for 1 h (see Note 2). 5. All-trans-retinoic acid (ATRA): 16 mM in DMSO. Subdivide into small portions, and store at -80 °C. 6. Dextran sulfate (DS): 50 mg/mL ddH20. Sterilize by passing through a 0.4 μm filter. 7. Medium (B): DMEM/F12 supplemented with 0.5% FBS, 0.5% ITS, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.2 mg/ mL DS, and 0.2 μM ATRA. 8. Medium (C): DMEM/F12 supplemented with 0.5% ITS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.2 μM ATRA. 9. Hemocytometer. 10. 90 mm petri dish, 50 mL tube. 3 Methods 3.1 Perfusion of Kidneys (Fig. 1) 1. Add 0.6 mL of carbonyl iron powder solution to 10 mL of PBS. Fill a 10 mL syringe of the solution, and connect the syringe to a butterfly needle. 2. After induction of anesthesia, open the abdomen without dam- aging the diaphragm. Move the internal organs such as the stomach and intestines to the right, and approach from the left.
- 25. 6 Eishin Yaoita Fig. 1 Perfusion of the kidney with iron powder solution (A–F) 3. Exteriorize the abdominal aorta, inferior vena cava, and left renal vein, being careful not to cause bleeding. 4. Insert tweezers under the aorta and vena cava to keep them taut (A). 5. Insert the butterfly needle into the abdominal aorta as horizon- tally as possible to avoid penetrating the aorta (B) (see Note 3). 6. Ligate the abdominal aorta at the site of needle insertion (C) and cephalad to renal arteries (D), and cut a hole in the vena cava (E). 7. Perfuse kidneys slowly while tilting the syringe so that all the iron powder flows out (F), and take out the left and right kidneys (see Note 4). 3.2 Isolation and Culture of Glomeruli (Fig. 2) Carry out the following procedures on a clean bench: 1. After removing the renal capsule, dissect the kidney cortices, and cut them gently into small pieces (1–2 mm cubes) with a surgical blade in 12 mL of HBSS on a 90 mm petri dish on ice. 2. Transfer the minced sample to a 14 mL round bottom tube, and centrifuge for 5 min at 50 × g. 3. Remove the supernatant to adjust the volume to 6 mL.
- 26. Other documents randomly have different content
- 27. “A clever novel. The incidents are exciting, and the interest is maintained to the close. It may be doubted if Sir Walter Scott himself ever painted a Scotch fireside with more truth than Dr. Mac Donald.”—Morning Post. “David Elginbrod is the finest character we have met in fiction for many a day. The descriptions of natural scenery are vivid, truthful, and artistic; the general reflections are those of a refined, thoughtful, and poetical philosopher, and the whole moral atmosphere of the book is lofty, pure, and invigorating.”—Globe. SIR GIBBIE. “‘Sir Gibbie’ is a book of genius.”—Pall Mall Gazette. “This book has power, pathos, and humour. There is not a character which is not lifelike. There are many powerful scenes, and the portraits will stay long in our memory.”—Athenæum. “‘Sir Gibbie’ is unquestionably a book of genius. It abounds in humour, pathos, insight into character, and happy touches of description.”—Graphic. “‘Sir Gibbie’ contains some of the most charming writing the author has yet produced.”—Scotsman. “‘Sir Gibbie’ is one of the most touching and beautiful stories that has been written for many years. It is not a novel to be idly read and laid aside; it is a grand work, to be kept near at hand, and studied and thought over.”—Morning Post. LONDON: HURST AND BLACKETT, PUBLISHERS.
- 28. WORKS by the AUTHOR of ‘JOHN HALIFAX.’ Each in a Single Volume, with Frontispiece, price 5s. JOHN HALIFAX, GENTLEMAN. A WOMAN’S THOUGHTS ABOUT WOMEN. A LIFE FOR A LIFE. NOTHING NEW. MISTRESS AND MAID. THE WOMAN’S KINGDOM. CHRISTIAN’S MISTAKE. A NOBLE LIFE. HANNAH. THE UNKIND WORD. A BRAVE LADY. STUDIES FROM LIFE. YOUNG MRS. JARDINE. WORKS by GEORGE MAC DONALD, LL.D. Each in a Single Volume, with Frontispiece, price 5s. DAVID ELGINBROD. ROBERT FALCONER. ALEC FORBES. SIR GIBBIE.
- 29. WORKS by MRS. OLIPHANT. Each in a Single Volume, with Frontispiece, price 5s. IT WAS A LOVER AND HIS LASS. THE LAIRD OF NORLAW. A ROSE IN JUNE. ADAM GRAEME OF MOSSGRAY. PHŒBE, JUNIOR. AGNES. THE LIFE OF THE REV. EDWARD IRVING. WORKS by the AUTHOR of ‘SAM SLICK.’ Each in a Single Volume, with Frontispiece, price 5s. NATURE AND HUMAN NATURE. WISE SAWS AND MODERN INSTANCES. THE OLD JUDGE; OR, LIFE IN A COLONY. TRAITS OF AMERICAN HUMOUR. THE AMERICANS AT HOME. LONDON: HURST AND BLACKETT.
- 30. FOOTNOTES: 1 It may be objected that this story pertains more to the seventeenth than the eighteenth century; but, as the man Roderick was alive in the last century, I claim him as belonging to it. 2 ‘The History of St. Kilda,’ etc. By the Rev. Mr. Kenneth Macaulay. London, 1764. 3 ‘Description of the Western Isles of Scotland, called Hebrides,’ etc. 4 Harris. 5 Scottice, are without. 6 ‘A Late Voyage to St. Kilda, the Remotest of all the Hebrides,’ etc., London, 1698. 7 Head-dress. 8 Venus, her lap dog. 9 A game at cards introduced into France by Signor Justiniani, Ambassador of Venice in 1674. The players are the dealer or banker, his assistant, who looks after
- 31. the losing cards—a croupier, in fact—and the punters, or anyone who plays against the banker. 10 To understand the numerous allusions to the game of cards called Quadrill, it is necessary that the principles of the game should be given. It was played by four persons, each having ten cards dealt to them. The general laws of this game are, 1. It is not permitted to deal the cards otherwise than four by three, the dealer being at liberty to begin with which of those numbers he pleases. 2. If he who plays either sans prendre, or calling a king, names a trump of a different suit from that his game is in, or names two several suits, that which he first named must be the trump. 3. He who plays must name the trump by its proper name, as he likewise must the king he calls. 4. He who has said ‘I pass,’ must not be again admitted to play, except he plays by force, upon account of his having Spadille. 5. He who has asked the question, and has leave given him to play, is obliged to do it: but he must not play sans prendre except he is forced to do it. 6. He who has the four kings may call the queen of either of his kings. 7. Neither the king nor queen of the suit which is trumps must be called. 8. He who has one or several kings may call any king he has in his hand; in such case, if he wins, he alone must make six tricks; if he wins, it is all his own, and if he loses, he pays all by himself. 9. Everyone ought to play in his turn, but for having done otherwise, no one must be beasted. 10. He, however, whose turn is not to play, having in his hand the king the ombre has called, and who shall tramp about with either spadille, manille, or basto, or shall even play down the king that was called, to give notice of his being the friend, must not pretend to undertake the vole; nay, he must be condemned to be beasted if it appears that he did it
- 32. with any fraudulent design. 11. He who has drawn a card from his game, and presented it openly in order to play it, is obliged so to do, if his retaining it may be either prejudicial to his game, or give any information to his friend, especially if the card is a matadore; but he who plays sans prendre, or calls upon his own king, is not subject to this law. 12. None ought to look upon the tricks, nor to count aloud what has been played, except when it is his turn to play, but to let everyone reckon for himself. 13. He who, instead of turning up the tricks before any one of his players, shall turn up and discover his game, must be equally beasted with him whose cards he has so discovered, the one paying one half, and the other the like. 14. He who renounces must be beasted, as many times as he has so done, but, if the cards are mixed, he is to pay but one beast. 15. If the renounce prejudices the game, and the deal is not played out, everyone may take up his cards, beginning at the trick where the renounce was made, and play them over again. 16. He who shows the game before the deal is out must be beasted, except he plays sans prendre. 17. None of the three matadores can be commanded down by an inferior trump. 18. If he who plays sans prendre with the matadores in his hand, demands only one of them, he must receive only that he mentioned. 19. He who, instead of sans prendre, shall demand matadores, not having them, or he who shall demand sans prendre instead of matadores, cannot compel the players to pay him what is really his due. 20. Matadores are only paid when they are in the hands of the ombre, or of the king his ally, whether all in one hand, or separately in both. 21. He who undertakes the vole, and does not make it, must pay as much as he would have received had he won it. 22. He who plays and does not make three tricks is to be beasted alone, and must pay all that is to be paid; and, if he makes no
- 33. tricks at all, he must also pay to his two adversaries the vole, but not to his friend.’—The Oxford Encyclopædia, 1828. 11 Dressing-gown. 12 Entendres. 13 Wonders. 14 These leaden combs were used for darkening the hair. 15 Pulled down 1885. 16 Forsitan et nostros ducat de marmore vultus Nectens aut Paphia myrti aut Parnasside lauri Fronde comas—At ego secura pace quiescam. Milton in Manso. 17 John Speed, the historian, died 1629, and was buried in the church of St. Giles’, Cripplegate. 18 The few hairs of a lighter colour, are supposed to have been such as had grown on the sides of the cheeks after the corpse had been interred. 19 ‘MDCLV. May vi, died my (now) only and eldest son, John Smith (Proh Dolor, beloved of all men!) at Mitcham in Surrey. Buried May ix in St. Giles, Cripplegate.’ 20 Edward Philips or Phillips, in his life of Milton, attached to ‘Letters of State, written by Mr. John Milton,’ c., London, 1694, (p. 43), says: ‘He is said to have dyed worth £1,500 in Money (a considerable Estate, all things considered), besides Household Goods; for he sustained such losses as might well have broke any person less
- 34. frugal and temperate than himself; no less than £2,000 which he had put for Security and Improvement into the Excise Office, but, neglecting to recal it in time, could never after get it out, with all the Power and Interest he had in the Great ones of those Times; besides another great Sum by mismanagement and for want of good advice.’ 21 Thomas Newton, Bishop of Bristol, thus writes in his life of Milton, prefixed to his edition of ‘Paradise Lost,’ London, 1749: ‘His body was decently interred near that of his father (who had died very aged about the year 1647) in the chancel of the church of St. Giles, Cripplegate; and all his great and learned friends in London, not without a friendly concourse of the common people, paid their last respects in attending it to the grave. Mr. Fenton, in his short but elegant account of the life of Milton, speaking of our author’s having no monument, says that “he desired a friend to inquire at St. Giles’s Church, where the sexton showed him a small monument, which he said was supposed to be Milton’s; but the inscription had never been legible since he was employed in that office, which he has possessed about forty years. This sure could never have happened in so short a space of time, unless the epitaph had been industriously erased; and that supposition, says Mr. Fenton, carries with it so much inhumanity that I think we ought to believe it was not erected to his memory.” It is evident that it was not erected to his memory, and that the sexton was mistaken. For Mr. Toland, in his account of the life of Milton, says that he was buried in the chancel of St. Giles’s Church, “where the piety of his admirers will shortly erect a monument becoming his worth, and the encouragement of letters in King William’s reign.” This plainly implies that no monument was erected to him at that time, and this was written in
- 35. 1698, and Mr. Fenton’s account was first published, I think, in 1725; so that not above twenty-seven years intervened from the one account to the other; and consequently the sexton, who it is said was possessed of his office about forty years, must have been mistaken, and the monument must have been designed for some other person, and not for Milton.’ 22 Between the creditable trades of pawnbroker and dram- seller there is a strict alliance. As Hogarth observes, the money lent by Mr. Gripe is immediately conveyed to the shop of Mr. Killman, who, in return for the produce of rags, distributes poison under the specious name of cordials. See Hogarth’s celebrated print called Gin Lane. 23 Probably in the month of September, as the entry of his baptism in the registry of the chapelry of Middlesmoor, in Netherdale, says ‘Eugenius Aram, son of Peter Aram, baptized the 2nd of October.’ 24 Though no warrants were issued against them, Aram was arrested for debt, in order to keep him; yet he immediately discharged this debt—not only so, he paid off a mortgage on his property at Bondgate. Suspicious facts, considering he was, notably, a poor man. 25 Finding. 26 The esne was a man of the servile class, a poor mercenary, serving for hire, or for his land, but was not of so low a rank as the other classes. 27 An Act relative to German and Swiss redemptioners. 28 Bedlam was then in Moorfields. 29 A large wickerwork receptacle behind the mail-coach.
- 36. 30 Palmer invented the mail-coach, and supplied horses to the Post-Office. 31 Lunardi made the first balloon ascent in England, Sept. 21, 1784. 32 Birmingham halfpence, struck by Boulton and Watts at their works at Soho, Birmingham. 33 Kew Bridge was opened to the public, September, 1789. 34 Some idea of the duelling that went on in Ireland in the latter part of last century may be gathered from the following extract from Sir Jonah’s book (vol. ii, p. 3): ‘I think I may challenge any country in Europe to show such an assemblage of gallant judicial and official antagonists at fire and sword as is exhibited even in the following list: The Lord Chancellor of Ireland, Earl Clare, fought the Master of the Rolls, Curran. The Chief Justice, K.B. Lord Clonmell, fought Lord Tyrawley (a privy counsellor), Lord Llandaff, and two others. The judge of the county of Dublin, Egan, fought the Master of the Rolls, Roger Barrett, and three others. The Chancellor of the Exchequer, the Right Hon. Isaac Corry, fought the Right Hon. Henry Grattan (a privy counsellor), and another. A Baron of the Exchequer, Baron Medge, fought his brother-in-law and two others.
- 37. The Chief Justice, C. P. Lord Norbury, fought Fire-eater Fitzgerald and two other gentlemen, and frightened Napper Tandy, and several besides: one hit only. The judge of the Prerogative Court, Dr. Dingenan, fought one barrister and frightened another on the ground. N.B.—The latter case a curious one. The Chief Counsel to the Revenue, Henry Deane Grady, fought Counsellor O’Mahon, Counsellor Campbell, and others: all hits. The Master of the Rolls fought Lord Buckinghamshire, the Chief Secretary, c. The provost of the University of Dublin, the Right Hon. Hely Hutchinson, fought Mr. Doyle, Master in Chancery, and some others. The Chief Justice C. P. Patterson, fought three country gentlemen, one of them with swords, another with guns, and wounded all of them. The Right Hon. George Ogle (a privy counsellor) fought Barney Coyle, a distiller, because he was a Papist. They fired eight shots, and no hit; but the second broke his own arm. Thomas Wallace, K.C., fought Mr. O’Gorman, the Catholic Secretary. Counsellor O’Connell fought the Orange chieftain; fatal to the champion of Protestant ascendency. The collector of the customs of Dublin, the Hon. Francis Hutchinson, fought the Right Hon. Lord Mountmorris.
- 38. Two hundred and twenty-seven memorable and official duels have actually been fought during my grand climacteric. 35 ‘The Female Soldier; or, The Surprising Life and Adventures of Hannah Snell,’ c. London, 1750. 36 A farmer of repute. 37 For a pension. 38 The action off Cape St. Vincent, when Sir John Jervis, with fifteen sail of the line, attacked and defeated the Spanish fleet, consisting of twenty-seven sail of the line. 39 ‘The case of Mr. John Walter, of London, Merchant.’ London, 1781. 40 Then in Lombard Street. 41 Lord North resigned, and Lord Rockingham succeeded as Premier, 1782. 42 Logotypes—or printing types in which words, etc., were cast, instead of single letters. 43 The centenary of the Times was improperly celebrated in that paper on the 1st of January, 1885. 44 i.e., in the liberty or Rules of the Fleet. 45 A foot-lock or hobble. 46 From the link-boy’s natural hatred of ‘the Parish Lantern,’ which would deprive him of his livelihood. 47 In throwing dice a corruption of the French numerals is used, as ace (one), deuce (two), tray (three), c.
- 39. 48 I.e., That sentence of death, owing to his pleading benefit of clergy, or ability to read, was commuted to imprisonment, and branding on the face with a red-hot iron. By degrees, however, the iron got colder, until, at last, it was barely warm. 49 Mews, or horse-pond. 50 ‘The Humours of the Fleet.’ A Poem, by W. Paget, Comedian, c. Birmingham. 51 Where the Fleet Market is now, there was, a few Years since, a Ditch, with a muddy Channel of Water. The Market was built at the Expense of the Lord Mayor and Court of Aldermen, who receive the Rent for it. 52 The Door-keeper, or he who opens and shuts the Jigg, is call’d the Jigger. 53 Billiards is a very common game here. 54 Fine Ale drank in the Coffee-room, call’d the ‘Alderman,’ because brew’d by Alderman Parsons. 55 A Runner is a Fellow that goes abroad of Errands for the Prisoners. 56 Begs. 57 Persons who give any Considerable offence are often try’d, and undergo the Discipline of the Pump. The Author was one of these in a drunken Frolick, for which he condemns himself. 58 A Spacious place, where there are all sorts of Exercises, but especially Fives.
- 40. 59 A Publick Place, free for all Prisoners. 60 Where those lie who can’t pay their Master’s Fee. 61 There are several of these Jiggers, or Door-keepers, who relieve one another, and, when a Prisoner comes first in, they take a nice Observation of him, for fear of his escaping. 62 A cant Word for giving some Money in order to show a Lodging. 63 Which is One Pound, Six, and Eightpence, and then you are entitled to a bed on the Master’s-side, for which you pay so much per Week. 64 Mount-scoundrel, so-call’d from its being highly situated, and belonging, once, to the Common-side, tho’ lately added to the Master’s; if there be room in the House, this Place is first empty, and the Chamberlain commonly shows this to raise his Price upon you for a better. 65 Half-a-guinea. 66 A Bed-fellow so call’d. 67 When you have a Chum, you pay but fifteen Pence per Week each, and, indeed, that is the Rent of a whole Room, if you find Furniture. 68 The Upper Floors are accounted best here, for the same Reason as they are at Edinburgh, which, I suppose, every Body knows. 69 It is common to mention the Fleet by the name of the Place, and I suppose it is call’d the Place by way of Eminence, because there is not such another.
- 41. 70 A Cant Word for a Dram of Geneva. 71 A Chew of Tobacco—supposed to be given him. 72 When there are Holes above Heel, or the Feet are so bad in a Stocking that you are forced to pull them to hide the Holes, or cover the Toes, it is call’d Coaxing. 73 As the Prison is often called the College, so it is common to call a Prisoner a Collegian; and this Character is taken from a Man who had been many Years in the Place, and like to continue his Life. 74 The Name of the Cook of the Kitchen. 75 A place in the Cellar call’d Bartholomew Fair. 76 Who goes out? is repeated by Watchmen Prisoners from half-an-hour after nine till St. Paul’s Clock strikes Ten, to give Visitors Notice to depart. 77 While St. Paul’s is striking Ten, the Watchman don’t call Who goes out? but when the last stroke is given they cry All told! at which time the Gates are lock’d and nobody suffer’d to go out upon any Account. 78 A werst is one thousand and sixty-seven metres. 79 Then valued at four shillings each, or eight pounds in all. 80 Gay, in his ‘Trivia,’ book i, says, ‘Let Persian Dames th’Umbrella’s Ribs display, To guard their Beauties from the Sunny Ray.’ 81 ‘A Review of the proposed Naturalization of the Jews.’ 82 Among other Bills which then received the Royal Assent was one for purchasing Sloane Museum and the
- 42. Harleian MSS., and for providing a general repository for the same—by means of a lottery—the commencement of the British Museum. 83 ‘Parliamentary History,’ Hansard, vol. xv, p. 154. 84 ‘Eight Letters to his Grace—Duke of Newcastle—on the custom of Vails-giving in England, c.,’ 1760, p. 20. 85 ‘The East Neuk of Fife,’ by Rev. Walter Wood. Edinburgh, 1862, p. 208. 86 Tickled the palms of their hands. 87 ‘The English Treasury of Wit and Language,’ etc., ed. 1655, pp. 223, 224. 88 Or surfel—to wash the cheeks with mercurial or sulphur water. 89 Face-washes and ointments. 90 Edition 1699, p. 19. The poem had reference to the College of Physicians, establishing a dispensary of their own, owing to the excessive charges of the apothecaries. The institution did not last very long. 91 Gold. 92 ‘The Female Physician, c.,’ by John Ball, M.D.—London, 1770, pp. 76, 77. 93 This water, as its name implies, was supposed to be a sovereign remedy for gunshot wounds. It was also called aqua vulneraria, aqua sclopetaria, and aqua catapultarum. 94 Now called an entire horse, or stallion.
- 43. 95 ‘The London Spy,’ ed. 1703, p. 124. 96 An allusion to the dispensary which the College of Physicians set up in the latter part of the seventeenth century, and which was the subject of Sir S. Garth’s satirical poem, called ‘The Dispensary.’ 97 A seventh son of a seventh son is supposed to be endowed with extraordinary faculties of healing, and many of these quacks pretended to such a descent. 98 ‘The London Spy,’ ed. 1703, p. 64. 99 A covering, or gaiter, to protect the legs from dirt or wet. 100 ‘The Liturgy and other Divine Offices of the Church.’ London, Bosworth, 1880, p. 638. 101 ‘The Liturgy and other Divine Offices of the Church,’ p. 584. 102 General Advertiser, March 26, 1782. 103 General Advertiser, May 1, 1783. 104 General Advertiser, February 13, 1784. 105 Gentleman’s Magazine, 1736, pp. 617-618. 106 By Dr. Zachary Pearce, Bishop of Rochester. 107 A pickle herring was a Merry-Andrew or clown, and this means that the quack was too poor to afford either horse or attendant. 108 A false witness—one who would swear to anything for a trifle.
- 44. 109 I have before me now twelve lives of him, and that is by no means an exhaustive list. 110 ‘Memoire pour le Comte de Cagliostro, accusé: contre Monsieur le Procureur-General, accusateur; en presence de Monsieur le Cardinal de Rohan, de la Comtesse de la Motte, et autres co-accusés.’ Paris, 1786, 4to. 111 Of this work there was a French translation published in 1791 at Paris and Strasbourg, under the title of ‘Vie de Joseph Balsamo, connu sous le nom de Comte Cagliostro,’ c. 2nd edition. 112 Editor of the Morning Chronicle, 1772-89. 113 Locusta, or, more correctly, Lucusta, was a celebrated poisoner. She was employed by Aggripina to poison the Emperor Claudius, and by Nero to kill Britannicus. For this she was most handsomely rewarded by Nero; but was executed for her crimes by Galba. 114 i.e., to serve on the convict hulks there, to dredge the Thames. The treatment on board was based on good principles; those convicts who were well-behaved had remission of sentence, those who were recalcitrant had unmerciful punishment. Transcriber’s Note:
- 45. Inconsistent spelling and hyphenation are as in the original.
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