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Methods in
Molecular Biology 2067

Luigi Gnudi · David A. Long Editors

Diabetic
Nephropathy
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY

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
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.
 Diabetic Nephropathy

Methods and Protocols

Edited by

Luigi Gnudi
Faculty of Life Sciences & Medicine, School of Cardiovascular Medicine & Sciences, British Heart
Foundation Centre of Research Excellence, King’s College London, London, UK

David A. Long
Developmental Biology and Cancer Programme, UCL Great Ormond Street Institute of Child Health,
University College London, London, UK
Editors
Luigi Gnudi David A. Long
Faculty of Life Sciences & Medicine Developmental Biology and Cancer Programme
School of Cardiovascular Medicine UCL Great Ormond Street Institute of Child Health
& Sciences, British Heart Foundation University College London
Centre of Research Excellence London, UK
King’s College London
London, UK

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-9840-1 ISBN 978-1-4939-9841-8 (eBook)
https://doi.org/10.1007/978-1-4939-9841-8
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Chapter 20 is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/). For further details see license information in the chapter.
This work is subject to copyright. All rights are reserved 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,
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even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations
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The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to
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The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.
Preface

Diabetic nephropathy represents the leading cause of end-stage renal failure in the Western
world. New treatments to either prevent this condition or delay its relentless progression are
urgently needed. In this book, we have put together a toolkit of novel research approaches
for investigators to study diabetic nephropathy. We hope that this will foster new research
directions and inspire ideas to enhance our understanding of diabetic nephropathy and to
develop treatments for this condition.
Experimental models are critical for basic science studies in the field of diabetic nephrop-
athy, and our book describes a wide range of current models from the fly to the fish, cells in
culture, and in vivo mammalian approaches. Successful studies in the field of diabetic
nephropathy also require powerful techniques to image the kidney. This is an area where
technological advances are rapidly occurring. Our book reflects this, and we describe not
only traditional histological techniques to visualize diabetic kidneys but also innovative new
approaches in electron, confocal, and two-photon microscopy. The pathophysiology of the
diabetic kidney is complex, and we describe a range of techniques that can unravel these
processes by examining changes in the properties of cells that make up the kidney filtration
barrier, assess endoplasmic reticulum stress, apoptosis, and cell–cell interactions. The last
decade has also seen major advances in techniques which take a global approach to assessing
microRNAs, genes, proteins, and metabolites in disease conditions, and these are described
in relation to diabetic nephropathy. Finally, advances in the renal field in gene editing and
regenerative medicine are described. Although these have not been used extensively in
diabetic kidney disease to-date, they provide great promise for the future.
We are grateful to all that have contributed to this book and whose aim is to help
patients with diabetic kidney disease. We hope this book will support the reader in their
research and help researchers to develop innovative experimental approaches in the field of
diabetic nephropathy.

London, UK Luigi Gnudi

David A. Long

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

PART I INTRODUCTION
1 Diabetic Nephropathy: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Manpreet K. Sagoo and Luigi Gnudi

PART II EXPERIMENTAL MODELS TO STUDY DIABETIC NEPHROPATHY

2 Modeling Podocyte Biology Using Drosophila Nephrocytes . . . . . . . . . . . . . . . . . . 11

Paul S. Hartley and Richard J. Coward
3 A Technique for Studying Glomerular Filtration Integrity
in the Zebrafish Pronephros . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Maria Kolatsi-Joannou and Daniel Osborn
4 Modeling Human Diabetic Kidney Disease by Combining
Hyperglycemia and Hypertension in a Transgenic Rodent Model . . . . . . . . . . . . . 41
Carolynn Cairns and Bryan Conway
5 Isolating and Culturing Mouse Podocyte Cells to Study
Diabetic Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Elisavet Vasilopoulou

PART III TECHNIQUES TO IMAGE THE DIABETIC KIDNEY

6 Histological Examination of the Diabetic Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Camillo Carrara, Mauro Abbate, Sara Conti, Daniela Rottoli,
Paola Rizzo, and Gianfranco Marchetti
7 Sample Preparation and Stereological Methods for the Study
of Glomerular Ultrastructure Using Electron Microscopy. . . . . . . . . . . . . . . . . . . . 89
Kathryn E. White
8 Tissue Clearing and Deep Imaging of the Kidney Using Confocal
and Two-Photon Microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Daniyal J. Jafree, David A. Long, Peter J. Scambler,
and Dale Moulding

PART IV ASSESSING THE FUNCTION AND PATHOPHYSIOLOGICAL

PROPERTIES OF THE DIABETIC KIDNEY

9 Transcutaneous Measurement of Glomerular Filtration Rate

in Rodents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Cristina Daniele, Daniela Nardozi, Angelo Torelli,
Arif ul Maula Khan, and Norbert Gretz

vii
viii Contents

10 An In Vitro Method to Analyze Glucose Uptake in Podocytes . . . . . . . . . . . . . . . 139

Abigail C. Lay and Richard J. Coward
11 Glomerular Endothelial Cells: Assessment of Barrier Properties
In Vitro. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Raina D. Ramnath and Simon C. Satchell
12 Methods to Detect Endoplasmic Reticulum Stress and Apoptosis
in Diabetic Nephropathy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Khurrum Shahzad, Sanchita Ghosh, Akash Mathew,
and Berend Isermann
13 Isolating Urinary Extracellular Vesicles as Biomarkers
for Diabetic Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Karina Barreiro, Tobias B. Huber, and Harry Holthofer
14 Examining Cell-Cell Interactions in the Kidney Using AFM
Single-Cell Force Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Eleftherios Siamantouras, Claire E. Hills, Kuo-Kang Liu,
and Paul E. Squires

PART V TRANSCRIPTOME, METABOLIC, AND PROTEOMIC PROFILING

OF THE DIABETIC KIDNEY

15 Genetic Strategies to Understand Human Diabetic Nephropathy:

Wet-Lab Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Laura J. Smyth, Katie Kerr, Seamus Duffy, Jill Kilner,
and Amy Jayne McKnight
16 Genetic Strategies to Understand Human Diabetic Nephropathy:
In Silico Strategies for Molecular Data—Association Studies . . . . . . . . . . . . . . . . . 241
Marisa Canadas-Garre, Laura J. Smyth, Kerry Anderson,
Katie Kerr, and Amy Jayne McKnight
17 Assessment of Urinary MicroRNAs by Quantitative Polymerase
Chain Reaction in Diabetic Nephropathy Patients . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Lucy Jade Newbury, Alexa Wonnacott, Kate Simpson,
Timothy Bowen, and Donald Fraser
18 Metabolomic and Proteomic Techniques for Establishing Biomarkers
and Improving Our Understanding of Pathophysiology in Diabetic
Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
Justyna Siwy, Linda Ahonen, Pedro Magalhães, Maria Frantzi,
and Peter Rossing

PART VI NEW APPROACHES: GENE EDITING AND STEM CELLS

TO STUDY DIABETIC KIDNEY DISEASE

19 Formation of Mature Nephrons by Implantation of Human

Pluripotent Stem Cell-Derived Progenitors into Mice . . . . . . . . . . . . . . . . . . . . . . . 309
Ioannis Bantounas, Edina Silajdžić, Adrian S. Woolf,
and Susan J. Kimber
 Contents ix

20 Generating Mutant Renal Cell Lines Using CRISPR Technologies. . . . . . . . . . . . 323

Nuria Perretta-Tejedor, Grace Freke, Marian Seda,
David A. Long, and Dagan Jenkins

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
Contributors

MAURO ABBATE  Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Bergamo, Italy
LINDA AHONEN  Steno Diabetes Center Copenhagen, Gentofte, Denmark
KERRY ANDERSON  Centre for Public Health, Queen’s University Belfast, Northern Ireland,
UK
IOANNIS BANTOUNAS  Division of Cell Matrix Biology and Regenerative Medicine, Faculty
of Biology, Medicine and Health, University of Manchester, Manchester Academic Health
Science Centre, Manchester, UK
KARINA BARREIRO  Finnish Institute of Molecular Medicine, University of Helsinki, Helsinki,
Finland
TIMOTHY BOWEN  Wales Kidney Research Unit, Heath Park Campus, Cardiff, UK
CAROLYNN CAIRNS  Centre for Cardiovascular Science, University of Edinburgh, Edinburgh,
UK
MARISA CANADAS-GARRE  Centre for Public Health, Queen’s University Belfast,
Northern Ireland, UK
CAMILLO CARRARA  Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Bergamo,
Italy
SARA CONTI  Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Bergamo, Italy
BRYAN CONWAY  Centre for Cardiovascular Science, University of Edinburgh, Edinburgh,
UK
RICHARD J. COWARD  Bristol Royal Hospital for Sick Children & University of Bristol, Bristol
Medical School, Bristol, UK; Bristol Renal, Translational Health Sciences, Bristol Medical
School, University of Bristol, Bristol, UK
CRISTINA DANIELE  Medical Faculty Mannheim, Medical Research Center, University
of Heidelberg, Mannheim, Germany
SEAMUS DUFFY  Centre for Public Health, Queen’s University Belfast, Northern Ireland, UK
MARIA FRANTZI  Mosaiques Diagnostics GmbH, Hannover, Germany
DONALD FRASER  Wales Kidney Research Unit, Heath Park Campus, Cardiff, UK
GRACE FREKE  Genetics and Genomic Medicine Programmes, Great Ormond Street Institute
of Child Health, University College London, London, UK
SANCHITA GHOSH  Institute of Clinical Chemistry and Pathobiochemistry, Otto-von-
Guericke-University, Magdeburg, Germany
LUIGI GNUDI  Faculty of Life Sciences & Medicine, School of Cardiovascular Medicine &
Sciences, British Heart Foundation Centre of Research Excellence, King’s College London,
London, UK
NORBERT GRETZ  Medical Faculty Mannheim, Medical Research Center, University
of Heidelberg, Mannheim, Germany
PAUL S. HARTLEY  Department of Life and Environmental Science, Bournemouth
University, Poole, Dorset, UK
CLAIRE E. HILLS  School of Life Sciences, University of Lincoln, Lincoln, UK
HARRY HOLTHOFER  Finnish Institute of Molecular Medicine, University of Helsinki,
Helsinki, Finland; Department of Medicine, University Medical Center Hamburg-
Eppendorf, Hamburg, Germany

xi
xii Contributors

TOBIAS B. HUBER  Department of Medicine, University Medical Center Hamburg-

Eppendorf, Hamburg, Germany
BEREND ISERMANN  Institute of Clinical Chemistry and Pathobiochemistry, Otto-von-
Guericke-University, Magdeburg, Germany
DANIYAL J. JAFREE  Developmental Biology and Cancer Programme, UCL Great Ormond
Street Institute of Child Health, University College London, London, UK; MB/PhD
Programme, Faculty of Medical Sciences, University College London, London, UK
DAGAN JENKINS  Genetics and Genomic Medicine Programmes, Great Ormond Street
Institute of Child Health, University College London, London, UK
KATIE KERR  Centre for Public Health, Queen’s University Belfast, Northern Ireland, UK
ARIF UL MAULA KHAN  Medical Faculty Mannheim, Medical Research Center, University of
Heidelberg, Mannheim, Germany
JILL KILNER  Centre for Public Health, Queen’s University Belfast, Northern Ireland, UK
SUSAN J. KIMBER  Division of Cell Matrix Biology and Regenerative Medicine, Faculty of
Biology, Medicine and Health, University of Manchester, Manchester Academic Health
Science Centre, Manchester, UK
MARIA KOLATSI-JOANNOU  Developmental Biology and Cancer Programme, UCL Great
Ormond Street Institute of Child Health, London, UK
ABIGAIL C. LAY  Bristol Renal, Translational Health Sciences, Bristol Medical School,
University of Bristol, Bristol, UK
KUO-KANG LIU  School of Engineering, University of Warwick, Warwick, UK
DAVID A. LONG  Developmental Biology and Cancer Programme, UCL Great Ormond
Street Institute of Child Health, University College London, London, UK
PEDRO MAGALHÃES  Mosaiques Diagnostics GmbH, Hannover, Germany; Department of
Pediatric Nephrology, Hannover Medical School, Hannover, Germany
GIANFRANCO MARCHETTI  Unit of Nephrology, Azienda Socio-Sanitaria Territoriale Papa
Giovanni XXIII, Bergamo, Italy
AKASH MATHEW  Institute of Clinical Chemistry and Pathobiochemistry, Otto-von-Guericke-
University, Magdeburg, Germany
AMY JAYNE MCKNIGHT  Centre for Public Health, Queen’s University Belfast, Northern
Ireland, UK
DALE MOULDING  Developmental Biology and Cancer Programme, UCL Great Ormond
Street Institute of Child Health, University College London, London, UK; Light Microscopy
Core Facility, UCL Great Ormond Street Institute of Child Health, University College
London, London, UK
DANIELA NARDOZI  Medical Faculty Mannheim, Medical Research Center, University of
Heidelberg, Mannheim, Germany
LUCY JADE NEWBURY  Wales Kidney Research Unit, Heath Park Campus, Cardiff, UK
DANIEL OSBORN  Genetics Research Centre, St George’s University of London, London, UK
NURIA PERRETTA-TEJEDOR  Developmental Biology and Cancer, Great Ormond Street
Institute of Child Health, University College London, London, UK
RAINA D. RAMNATH  Bristol Renal, Translational Health Sciences, Bristol Medical School,
University of Bristol, Bristol, UK
PAOLA RIZZO  Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Bergamo, Italy
PETER ROSSING  Steno Diabetes Center Copenhagen, Gentofte, Denmark; Department of
Clinical Medicine, University of Copenhagen, Copenhagen, Denmark
DANIELA ROTTOLI  Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Bergamo, Italy
 Contributors xiii

MANPREET K. SAGOO  Faculty of Life Sciences & Medicine, School of Cardiovascular

Medicine & Sciences, British Heart Foundation Centre of Research Excellence,
King’s College London, London, UK
SIMON C. SATCHELL  Bristol Renal, Translational Health Sciences, Bristol Medical School,
University of Bristol, Bristol, UK
PETER J. SCAMBLER  Developmental Biology and Cancer Programme, UCL Great Ormond
Street Institute of Child Health, University College London, London, UK
MARIAN SEDA  Genetics and Genomic Medicine Programmes, Great Ormond Street Institute
of Child Health, University College London, London, UK
KHURRUM SHAHZAD  Institute of Clinical Chemistry and Pathobiochemistry, Otto-von-
Guericke-University, Magdeburg, Germany; Department of Biotechnology, University
of Sargodha, Sargodha, Pakistan
ELEFTHERIOS SIAMANTOURAS  School of Life Sciences, University of Lincoln, Lincoln, UK
EDINA SILAJDŽIĆ  Division of Cell Matrix Biology and Regenerative Medicine, Faculty
of Biology, Medicine and Health, University of Manchester, Manchester Academic Health
Science Centre, Manchester, UK
KATE SIMPSON  Wales Kidney Research Unit, Heath Park Campus, Cardiff, UK
JUSTYNA SIWY  Mosaiques Diagnostics GmbH, Hannover, Germany
LAURA J. SMYTH  Centre for Public Health, Queen’s University Belfast, Northern Ireland,
UK
PAUL E. SQUIRES  School of Life Sciences, University of Lincoln, Lincoln, UK
ANGELO TORELLI  Medical Faculty Mannheim, Medical Research Center, University of
Heidelberg, Mannheim, Germany; Mannheim University of Applied Sciences, Mannheim,
Germany
ELISAVET VASILOPOULOU  Medway School of Pharmacy, University of Kent, Chatham, Kent,
UK; Developmental Biology and Cancer, UCL GOS Institute of Child Health, London,
UK
KATHRYN E. WHITE  EM Research Services, Faculty of Medical Sciences, Newcastle
University, Newcastle upon Tyne, UK
ALEXA WONNACOTT  Wales Kidney Research Unit, Heath Park Campus, Cardiff, UK
ADRIAN S. WOOLF  Division of Cell Matrix Biology and Regenerative Medicine, Faculty
of Biology, Medicine and Health, University of Manchester, Manchester Academic Health
Science Centre, Manchester, UK; Royal Manchester Children’s Hospital, Manchester, UK
 Part I

Introduction
 Chapter 1

Diabetic Nephropathy: An Overview

Manpreet K. Sagoo and Luigi Gnudi

Abstract
Diabetic nephropathy (DN) is one of the most feared diabetic chronic microvascular complications and the
major cause of end-stage renal disease (ESRD). The classical presentation of DN is characterized by
hyperfiltration and albuminuria in the early phases which is then followed by a progressive renal function
decline. The presentation of diabetic kidney disease (DKD) can vary especially in patients with T2DM
where concomitant presence of other glomerular/tubular pathologies and severe peripheral vascular disease
can become important confounders. All-cause mortality in individuals with DKD is approximately 30 times
higher than that in diabetic patients without nephropathy and a great majority of patients with DKD will die
from cardiovascular disease before they reach ESRD. The management of metabolic and hemodynamic
perturbations for the prevention and for the delay of progression of DKD is very important. DKD is a
global challenge and a significant social and economic burden; research should aim at developing new ideas
to tackle this devastating condition.

Key words Diabetes mellitus, Diabetic kidney disease, Epidemiology, Management, Pathogenesis

1 Introduction to Diabetes Mellitus

Diabetes mellitus (DM) is a common and serious metabolic condi-

tion which presents a major challenge in twenty-first-century
healthcare. The global prevalence of DM has dramatically increased
from 108 million in 1980 to 451 million in 2017 and this number
continues to rapidly rise [1]. Estimated figures from the Interna-
tional Diabetes Federation predict that 693 million people will be
living with diabetes worldwide by 2045 [2] (http://www.
diabetesatlas.org). At present, diabetes is the seventh leading
cause of mortality worldwide and thus the diabetes epidemic
requires immediate action [3].
Diabetes is characterized by chronic hyperglycemia and glucose
intolerance due to impaired insulin action and/or secretion. Micro-
and macro-diabetic chronic vascular complications affect the major-
ity of patients with diabetes in both developed and developing
countries. The microvascular complications include diabetic
nephropathy, retinopathy, and neuropathy, and are responsible for

Luigi Gnudi and David A. Long (eds.), Diabetic Nephropathy: Methods and Protocols, Methods in Molecular Biology, vol. 2067,
https://doi.org/10.1007/978-1-4939-9841-8_1, © Springer Science+Business Media, LLC, part of Springer Nature 2020

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