THE BIOLOGY OF IMMUNOGLOBULIN FREE LIGHT CHAINS IN KIDNEY DISEASE: A STUDY OF MONOCLONAL AND POLYCLONAL LIGHT CHAINS BY KOLITHA INDIKA BASNAYAKE A thesis submitted to The University of Birmingham For the degree of DOCTOR OF PHILOSOPHY School of Immunity and Infection College of Medical and Dental Sciences The University of Birmingham January 2011
THE BIOLOGY OF IMMUNOGLOBULIN FREE LIGHT CHAINS IN KIDNEY DISEASE: A STUDY OF MONOCLONAL AND POLYCLONAL LIGHT CHAINS
Jan 12, 2023
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THE BIOLOGY OF IMMUNOGLOBULIN FREE LIGHT CHAINS IN KIDNEY DISEASE:
CHAINS
BY
College of Medical and Dental Sciences
The University of Birmingham
University of Birmingham Research Archive
e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
ABSTRACT
Monoclonal immunoglobulin free light chains (FLCs) cause a range of disorders in the
kidney. In multiple myeloma, FLCs can activate the proximal tubule to release MCP-1,
an important cytokine in renal fibrosis. Distal tubular cast formation can also occur
when FLCs co-precipitate with uromodulin. However a pathogenic role for the elevated
polyclonal FLC concentrations seen in chronic kidney disease has not been assessed to
date. This thesis explores the biology of monoclonal FLCs as well as polyclonal FLCs.
Detailed histological analyses demonstrated that in multiple myeloma, interstitial
fibrosis can progress rapidly in situ and indicated that intratubular cast numbers might
be linked to potential for renal recovery. The functional basis of this fibrosis was
explored by in vitro studies, which showed that upon endocytosis of FLCs, oxidative
stress activated redox signalling, resulting in MCP-1 production. Further in situ
analyses showed that in chronic kidney disease, polyclonal FLCs co-localised with
uromodulin in distal tubular casts. Relationships between these casts and markers of
progression of chronic kidney disease were demonstrated. In vitro analyses then showed
that polyclonal FLCs bind to uromodulin and promote aggregation. These findings:
(i) further delineate the pathways for proximal tubular injury in myeloma and (ii)
indicate a potential pathogenic role for polyclonal FLCs in the distal nephron.
DEDICATION
ACKNOWLEDGMENTS
The work presented in this thesis would not have been possible without the
contributions of many people. First and foremost, I must thank Dr Paul Cockwell, who
invested a huge amount of time and effort, in the roles of supervisor, colleague and
friend. I thank Prof Arthur Bradwell for all the encouragement and the tremendous
support he has given. I thank Prof Mark Drayson for his help. Prof Paul Sanders
deserves special mention – thank you for all your support, I will always remember
Alabama fondly. I thank Dr Gregg Wallis for his expert guidance. A special thank you
to Miss for her assistance. I would also like to thank Ltd, the National Institutes for
Health and the Department of Veterans Affairs. In addition, the individuals listed below
have all helped me at various stages, for which I am very grateful.
Miss Kristal Aaron Dr Matthew Morgan
Mr Peterson Anand Dr Timothy Plant
Mr Richard Barber Dr Paul Showell
Mr Simon Blackmore Dr Helen Smith
Dr Alastair Ferraro Mrs Norma Stewart
Dr Tarek Ghonemy Dr Stephanie Stringer
Mr John Gregory Dr Phillip Stubbs
Dr Richard Hampton Mr Phillip Walsh
Miss Lisa Hasty Dr Betsy Wang
Dr Colin Hutchison Mrs Ellie White
Dr Laura Ismail Dr Wei Zhong Ying
Dr Graham Mead Miss Alice Zhou
CONTENTS
1.1 Introduction ........................................................................................................ 1
1.3 Renal Handling of FLCs .................................................................................... 8
1.4 Why do Some Clonal FLCs Cause Kidney Injury? ...................................... 10
1.5 Resident Renal Cells are Differentially Predisposed to Injury by FLC ...... 10
1.6 Disease Specific Considerations for Mesangial Cells and Glomerular
Pathology .................................................................................................................... 11
1.6.4 Light Chain Deposition Disease.................................................................. 16
1.7 Cell Specific Processing and Activation Leads to the Differential Patterns
that are seen in FLC Disease ................................................................................... 17
1.8 The Differential Role of TGF Beta in Amyloid and LCDD ......................... 19
1.9 Matrix Metalloproteinases .............................................................................. 20
Tubule ........................................................................................................................ 21
1.10.3 Proximal Tubular Toxicity ........................................................................ 23
1.11 Distal Tubular Cast Formation..................................................................... 30
1.12 Polyclonal Elevations in Light Chain Levels ............................................... 36
1.13 Scope of this Thesis and Hypothesis ............................................................. 40
2. MATERIALS AND METHODS .......................................................................... 52
2.1 Introduction ...................................................................................................... 52
2.2.1 Background ................................................................................................. 52
2.2.5 Detection Systems ....................................................................................... 54
2.2.6 Chromogenic Detection .............................................................................. 54
2.2.7 Fluorescence Detection ............................................................................... 55
2.2.9 Tissue Fixation and Embedding .................................................................. 57
2.2.10 Tissue Sectioning ...................................................................................... 58
2.2.16 Quenching and Blocking Steps – Prevention of Background Staining..... 60
2.2.17 Endogenous Peroxidase Quenching with Hydrogen Peroxide ................. 60
2.2.18 Endogenous Biotin Block with Avidin and Biotin ................................... 61
2.2.19 Fc Receptor Block with Serum ................................................................. 61
2.2.20 Primary Antibody ...................................................................................... 61
2.2.21 Isotype Control .......................................................................................... 62
2.2.23 Protocol for Quenching of Autofluorescence ........................................... 63
2.2.24 Protocol for Multiple Immunofluorescent Staining of FLC in Kidneys ... 64
2.2.25 Protocol for multiple IF staining - controls ............................................... 65
2.2.26 Visualisation of Immunofluorescent Staining and Image Acquisition ..... 65
2.2.27 Protocol for Immunohistochemical Staining of Macrophages and
Interstitial Capillaries .............................................................................................. 66
2.3.1 Patients ........................................................................................................ 68
Capillary Density .................................................................................................... 68
2.3.4 Cast Counting .............................................................................................. 70
2.4.1 Culture and Propagation of HK-2 Cells ...................................................... 70
2.4.2 Containers ................................................................................................... 71
2.4.6 Enumeration of Cells using a Haemocytometer.......................................... 73
2.4.7 Cryopreservation ......................................................................................... 74
2.4.8 Preparation of Polyclonal FLC Stock Solution for In Vitro use ................. 74
2.4.9 Protocol for Incubation of HK-2 Cells with FLC ....................................... 74
2.4.10 Protocol for Cell Lysis ............................................................................. 75
2.4.11 Hydrogen Peroxide Assay ........................................................................ 75
2.4.12 MCP-1 Assay ........................................................................................... 76
2.4.14 Silencing of Gene Expression with siRNA .............................................. 78
2.4.15 Immunoblotting of Cell Culture Lysates................................................... 79
2.4.15.1 Western Blotting - c-Src Phosphorylation ............................................. 79
2.4.15.2 Western Blotting - Megalin and Cubilin ................................................ 80
2.4.15.3 Densitometry .......................................................................................... 80
2.4.17 Removal of Extracellular and Intracellular H2O2 ..................................... 82
2.5 Purification of Polyclonal FLCs and Protein Chemistry.............................. 83
2.5.1 Total Soluble Protein (TSP) Quantification ................................................ 83
2.5.1.1 Ultraviolet Absorbance at 280 nm (A280) ............................................... 83
2.5.1.2 Bicinchoninic Acid (BCA) Assay ............................................................ 84
2.5.2 Free Light Chain Quantification ................................................................. 84
2.5.3 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis ................ 85
2.5.3.1 Coomassie Brilliant Blue ......................................................................... 85
2.5.3.2 Silver Stain ............................................................................................... 85
2.5.4.2 Dot Blotting .............................................................................................. 86
2.5.5 Endotoxin Assays ........................................................................................ 87
2.5.6 Endotoxin Removal ..................................................................................... 88
2.5.8.1 Initial Steps .............................................................................................. 89
2.5.8.3 Affinity Chromatography ......................................................................... 90
2.5.8.4 Size-Exclusion Chromatography ............................................................. 91
2.5.9 Lyophilisation of Polyclonal FLC for Storage and Transportation ............ 92
2.6 Uromodulin ....................................................................................................... 92
2.6.2 Binding of Uromodulin to Polyclonal FLC ................................................ 93
2.6.2.1 Indirect ELISA ......................................................................................... 93
2.6.2.2 Sandwich ELISA ...................................................................................... 94
2.6.2.3 Dot Blot .................................................................................................... 94
2.7.2 Normalisation of Data ................................................................................. 96
2.7.3 Correlations ................................................................................................. 96
2.7.4 ANOVA ...................................................................................................... 96
2.7.5 Test of Intra- and Inter-observer Variability of Image Analysis Data ........ 96
2.7.6 Assessment of Distribution of Casts ........................................................... 97
3. RENAL INFLAMMATION AND FIBROSIS IN MONOCLONAL DISEASE:
IN SITU STUDIES ........................................................................................................ 98
3.2.3 Index of Chronic Damage ......................................................................... 103
3.2.4 Number of Tubules with Casts .................................................................. 103
3.2.5 Interstitial Infiltrate ................................................................................... 106
IN VITRO STUDIES .................................................................................................. 111
4.2.2 DMTU Inhibits c-Src Activation .............................................................. 114
4.2.3 Inhibition of c-Src Suppresses MCP-1 Production but does not Suppress
H2O2 Production .................................................................................................... 116
4.2.4 Removal of Extracellular H2O2 by Catalase has no Impact on MCP-1
Production ............................................................................................................. 118
4.2.5 Silencing of c-Src Expression Suppresses MCP-1 Production in Response
to Light Chain Exposure ....................................................................................... 118
4.2.6 c-Src is Oxidised Following Light Chain Treatment ................................ 118
4.2.7 Silencing of Megalin and Cubilin Suppresses MCP-1 Production ........... 120
4.3 Discussion ........................................................................................................ 121
5.1 Introduction .................................................................................................... 128
5.2 Choice of Source of Polyclonal Free Light Chains ...................................... 128
5.3 Analysis of Resuspended Sera ....................................................................... 130
5.4 Extraction of Proteins Containing Light Chains from Starting Sample .. 133
5.4.1 Anti- Light Chain Matrix – Assessment of Suitability ........................... 133
5.4.2 Anti- Light Chain Matrix – Assessment of Suitability ........................... 136
5.4.3 Extraction of -Light Chain Containing Proteins From Starting Sample . 140
5.4.4 Anti- Light Chain Matrix Manufacture................................................... 142
5.4.5 Anti- Light Chain Column – Assessment of Capacity ............................ 143
5.4.6 Extraction of -Light Chain Containing Proteins From the Anti- Unbound
Fraction ................................................................................................................. 145
Contaminants .......................................................................................................... 148
5.5.3 Size Exclusion Chromatography ............................................................... 154
5.6 Assessment of Polyclonal FLC Purity .......................................................... 154
5.7 FLC – Removal of IgG, Human Serum Albumin and Transferrin ....... 158
5.8 Detection and Removal of Endotoxin ........................................................... 160
5.9 Assessment of Solubility After Lyophilisation ............................................. 161
Table 5.4. FLC recovery after lyophilisation..................................................... 162
5.10 Discussion ...................................................................................................... 163
CHRONIC KIDNEY DISEASE: IN SITU STUDIES ............................................. 166
6.1 Introduction .................................................................................................... 166
6.2 Patients ............................................................................................................ 168
6.3 Results ............................................................................................................. 169
6.3.1 Immunofluorescence ................................................................................. 169
6.3.3 Polyclonal FLC Co-localise with Uromodulin in Distal Tubules ............. 170
6.4 Measurement of Cast Numbers, Index of Chronic Damage, Interstitial
Capillary Density and Macrophage Numbers ...................................................... 175
6.4.1 Test of Normality of Data ......................................................................... 175
6.4.2 Assessment of Validity of Quantification Methods .................................. 180
6.4.3 Casts in CKD are Situated in Areas of Established Chronic Damage ...... 180
6.4.4 Capillary Density Correlates with the Index of Chronic Damage and
Macrophage Numbers ........................................................................................... 182
6.4.5 Macrophage Numbers Correlate with Index of Chronic Damage ............ 185
6.4.6 Cast Numbers Correlate with Index of Chronic Damage, Capillary Density
and Macrophage Numbers .................................................................................... 185
6.5 Discussion ........................................................................................................ 188
VITRO STUDIES ....................................................................................................... 193
7.1 Introduction .................................................................................................... 193
7.2 PTEC Culture ................................................................................................. 194
7.2.1 Effect of Polyclonal Free Light Chains on Inflammatory Signalling ....... 194
7.2.2 Cytotoxic Effects of Polyclonal Free Light Chains on Proximal Tubule
Epithelial Cells ...................................................................................................... 195
7.3 Uromodulin ..................................................................................................... 196
7.3.1 Uromodulin is Highly Aggregated in High Salt Solutions ....................... 196
7.3.2 Uromodulin Aggregation is Reduced in Water and by Alkaline pH ........ 197
7.4 Polyclonal Free Light Chains Interact with Uromodulin: Dot Blotting ... 200
7.5 Polyclonal Free Light Chains Interact with Uromodulin: ELISA ............ 205
7.5.1 Plate Coated with Uromodulin .................................................................. 205
7.5.2 Plate Coated with Polyclonal Free Light Chains ...................................... 206
7.5.3 Sandwich ELISA ....................................................................................... 209
7.6 Polyclonal Free Light Chains Interact with Uromodulin: Nephelometry 209
7.6.1 Experiments in Buffer Containing 50 mM NaCl ...................................... 212
7.6.2 Experiments in Buffer Containing 100 mM NaCl .................................... 212
7.6.3 Experiments in Buffer Containing 150 mM NaCl .................................... 216
7.7 Discussion ........................................................................................................ 217
AND THERAPEUTIC STRATEGIES ..................................................................... 226
8.3 Histological Markers of Renal Outcome in Cast Nephropathy ................. 228
8.4 Proximal Tubular Damage in Cast Nephropathy ....................................... 229
8.4.1 The Role of c-Src in Signal Transduction ................................................. 230
8.5 Distal Tubular Damage in Cast Nephropathy ............................................. 231
8.6 Potential Therapeutic Approaches to Cast Nephropathy .......................... 232
8.6.1 Reduction of FLC Load Delivered to Nephrons ....................................... 232
8.6.2 Prevention of PTEC Damage .................................................................... 232
8.6.2.1 Prevention of Endocytosis ..................................................................... 232
8.6.2.2 Reduction of Intracellular Oxidative Stress ........................................... 234
8.6.2.3 c-Src Inhibition....................................................................................... 234
8.7 The Inflammatory Role of Filtered Proteins in CKD ................................. 238
8.8 The Role of Polyclonal FLCs in CKD .......................................................... 238
8.8.1 The Effects of Polyclonal FLCs on PTECs .............................................. 238
8.8.2 The Interaction of Uromodulin and Polyclonal FLCs in CKD ................. 240
8.9 Conclusion ....................................................................................................... 242
9.1 Papers .............................................................................................................. 244
9.2 Abstracts ......................................................................................................... 244
10. APPENDIX ......................................................................................................... 246
A.3 SDS-PAGE - Gel Recipes and Calculation of Measures ............................ 252
A.4 Useful Common Protein Extinction Coefficients for A280 Measurements 253
A.5 BCA Assay Standards, Working Range 20 – 2000 µg/ml .......................... 253
A.6 Useful Numbers for Cell Culture ................................................................ 254
A.7 Amino Acids ............................................................................................... 254
A.9 Normal HK-2 Cells ..................................................................................... 257
A.10 A Neubauer Haemocytometer ................................................................... 257
A.11 siRNA Mode of Action ............................................................................. 258
A.12 Chromatography Apparatus ...................................................................... 259
Figure 1.2.Interactions of FLCs with mesangial cells..................................................... 12
Figure 1.3. Cast nephropathy and accelerated in situ progression of interstitial fibrosis in
a patient with multiple myeloma. ............................................................................ 24
Figure 1.4. Interactions of FLCs with proximal tubule epithelial cells. ......................... 25
Figure 1.5. Light chain interactions in the distal nephron............................................... 32
Figure 3.1. Renal biopsies from patients A-D............................................................... 104
Figure 3.2. Changes in the index of chronic damage and cast numbers over six weeks.
............................................................................................................................... 105
Figure 4.3. DMTU inhibits c-Src activation. ................................................................ 116
Figure 4.4. Inhibition of c-Src suppresses MCP-1 production but not H2O2 production.
............................................................................................................................... 117
Figure 4.5. Silencing of c-Src expression suppresses MCP-1 production in response to
light chain exposure. ............................................................................................. 119
Figure 4.6. c-Src in oxidised following light chain treatment....................................... 121
Figure 4.7. Silencing of megalin and cubilin suppress MCP-1 production. ................. 122
Figure 5.1. Electrophoretic (SDS-PAGE) analysis of the FLC extract after passage
through the protein A column. .............................................................................. 132
Figure 5.2. Polyclonal FLC purification protocol. ........................................................ 134
Figure 5.3. Chromatogram of assessment of suitability of anti- light chain matrix. .. 135
Figure 5.4. Dot blot demonstrating anti- light chain matrix capacity. ........................ 136
Figure 5.5. SDS-PAGE analysis of pooled elutions from the anti- column. .............. 137
Figure 5.6. Chromatogram of assessment of suitability of anti- light chain matrix. .. 138
Figure 5.7. Dot blot demonstrating anti- light chain matrix capacity. ........................ 139
Figure 5.8. SDS-PAGE analysis of pooled elutions from the anti- column. .............. 140
Figure 5.9. Chromatograms showing extraction of -light chain containing proteins
from the starting sample. ....................................................................................... 141
Figure 5.10. Western blots of anti- elution pool. ........................................................ 143
Figure 5.11. Chromatogram of assessment of suitability of manufactured anti- matrix.
............................................................................................................................... 144
Figure 5.12, Dot blot demonstrating capacity of the manufactured anti- light chain
matrix. ................................................................................................................... 145
from the anti- unbound fraction. ......................................................................... 147
Figure 5.14. Western blot showing enrichment of -light chain containing proteins in
the anti-total elutions.......................................................................................... 148
Figure 5.15. Chromatograms showing anti- and anti- elutions applied to protein G
column. .................................................................................................................. 151
Figure 5.16. Chromatogram showing the -FLC sample applied to anti-IgA and anti-
IgM columns. ........................................................................................................ 152
Figure 5.17. Chromatograms showing the -FLC sample applied to anti-IgA and anti-
IgM columns. ........................................................................................................ 153
Figure 5.18. Chromatograms showing removal of impurities from and FLC samples
by size-exclusion chromatography. ....................................................................... 155
Figure 5.19. SDS-PAGE analysis of the purity of and FLC samples. .................... 159
Figure 5.20. Western blots showing the presence of albumin and transferrin
contaminants in the -FLC sample. ...................................................................... 160
Figure 5.21. SDS_PAGE analysis of unbound fractions after incubation with anti-HSA,
anti-HSA+anti-TF and protein G. ......................................................................... 161
Figure 5.22. SDS-PAGE analysis of -FLC sample, after incubation with anti-
HSA+anti-TF and protein G. ................................................................................ 162
Figure 5.23. SDS-PAGE analysis of FLC recovery after lyophilisation. .................. 163
Figure 6.1. A proteinaceous cast in a renal biopsy from a patient with CKD. ............. 167
Figure 6.2. Uromodulin is produced in the distal nephron and is present in casts........ 168
Figure 6.3. Free light chains in the proximal tubule in CKD. ....................................... 171
Figure 6.4. Confocal image of proximal and distal tubules showing distribution of -
FLC, -FLC and uromodulin. ............................................................................... 172
Figure 6.5. Confocal image of casts in a renal biopsy from a patient with CKD. ........ 173
Figure 6.6. High-power confocal image of a cast in a renal biopsy from a patient with
CKD. ..................................................................................................................... 174
Figure 6.7. Quantification of the index of chronic damage. ......................................... 176
Figure 6.8. Quantification of cast numbers. .................................................................. 177
Figure 6.9. Quantification of interstitial capillary density. ........................................... 178
Figure 6.10. Quantification of macrophage numbers.................................................... 179
Figure 6.11. Comparison of cast numbers between areas with or without chronic
damage. ................................................................................................................. 182
Figure 6.12. Correlations of capillary density with index of chronic damage and
macrophage numbers. ........................................................................................... 184
Figure 6.13. Correlation of macrophage numbers with index of chronic damage. ....... 185
Figure 6.14. Correlations of cast numbers with index of chronic damage, capillary
density and macrophage numbers. ........................................................................ 186
Figure 7.1. Effect of polyclonal free light chains on inflammatory signalling. ............ 196
Figure 7.2. Cytotoxic effects of polyclonal free light chains on proximal tubule cells.197
Figure 7.3. Uromodulin from healthy volunteer urines prior to dialysis. ..................... 198
Figure 7.4. Uromodulin aggregation is reduced by dialysis into water and by alkaline
pH. ......................................................................................................................... 199
Figure 7.5. Dot blot showing binding of polyclonal FLCs to uromodulin (polyclonal
FLC dots). ............................................................................................................. 202
Figure 7.6. Dot blot demonstrating binding of polyclonal FLC to uromodulin
(uromodulin dots). ................................................................................................. 203
Figure 7.7. Dot blot (repeated) demonstrating binding of polyclonal FLC to uromodulin
(FLC dots). ............................................................................................................ 204
Figure 7.8. Assessment of binding of polyclonal FLC to uromodulin by ELISA: Plate
coated with uromodulin......................................................................................... 207
Figure 7.9. Assessment of binding of polyclonal FLC to uromodulin by ELISA: Plate
coated with FLC. ................................................................................................... 208
Figure 7.10. Assessment of binding of polyclonal FLC to uromodulin by ELISA:
Sandwich ELISA. .................................................................................................. 211
Figure 7.11. Nephelometric assessment of the formation of higher molecular weight
aggregates with uromodulin; comparison of polyclonal and monoclonal FLCs; 50
mM NaCl. .............................................................................................................. 213
Figure 7.12. Nephelometric assessment of the formation of higher molecular weight
aggregates with uromodulin; comparison of polyclonal FLCs with HSA and
uromodulin; 50 mM NaCl. .................................................................................... 214
Figure 7.13. Nephelometric assessment of the formation of higher molecular weight
aggregates with uromodulin; comparison of monoclonal and polyclonal FLCs; 100
mM NaCl. .............................................................................................................. 215
Figure 7.14. Nephelometric assessment of the formation of higher molecular weight
aggregates with uromodulin; comparison of polyclonal FLCs with HSA or
uromodulin alone; 100 mM NaCl. ........................................................................ 216
Figure 7.15. Nephelometric assessment of the formation of higher molecular weight
aggregates with uromodulin; comparison of monolconal and polyclonal FLCs; 150
mM NaCl. .............................................................................................................. 220
Figure 7.16. Nephelometric assessment of the formation of higher molecular weight
aggregates with uromodulin; comparison of polyclonal FLCs with HSA or
uromodulin alone; 150 mM NaCl. ........................................................................ 221
TABLES
Table 1.1 Renal manifestations of plasma cell dyscrasias. ............................................... 2
Table 1.2. Renal manifestations of plasma cell dyscrasias, site and composition of
deposits and summary of clinical and histological features. ................................... 44
Table 3.1. Summary of patient demographics, histological findings and biochemical
data. ....................................................................................................................... 102
Table 5.1. Batches of resuspended sera received after filtration and passage through
protein A column................................................................................................... 131
Table 5.2. Nephelometric analysis of immunoglobulin content of fraction unbound to
protein G. .............................................................................................................. 152
Table 5.3. Nephelometric analysis of FLC content of and samples after SEC. ..... 156
Table 5.4. FLC recovery after lyophilisation. ............................................................ 162
Table 6.1. Assessment of agreement between two observers by the Bland-Altman
method. .................................................................................................................. 181
Table 6.2. Univariate analyses of cast numbers, index of chronic damage, capillary
density and macrophage numbers. ........................................................................ 183
Table 6.3. Multivariate analysis of correlations between index of chronic damage,
capillary density, macrophage numbers and cast numbers. .................................. 187
ABBREVIATIONS
ACR Albumin/Creatinine Ratio
DMTU Dimethyl Thiourea
ECM Extracellular Matrix
EMT Epithelial-to-Mesenchymal Transition
LDH Lactate Dehydrogenase
MC Mesangial Cell
MMP Matrix Metalloproteinase
MW Molecular Weight
NF-B Nuclear Factor kappa- light-chain-enhancer of activated B cells
PACAP38 Pituitary Adenylate Cyclase Activating Polypeptide with 38 residues
PAMS Periodic Acid-Methenamine Silver
PCD Plasma cell dyscrasia
PP2 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine
RAP Receptor Associate Protein
ROS Reactive Oxygen Species
SAP Serum Amyloid Protein
1.1 Introduction
Plasma cell dyscrasias (PCD) are relatively common disorders, the prevalence of
monoclonal gammopathy of undetermined significance (MGUS) in the over-50’s is
around 3.2%, and multiple myeloma accounting for 10% of all haematological
malignancies.(Kyle and Rajkumar 2004; Kyle et al. 2006) Plasma cell dyscrasias are
characterised by the proliferation of a clone of B-cell lineage. There is associated
production of clonal immunoglobulin (Ig) which frequently includes a variable quantity
of clonal immunoglobulin free light chain (FLC). Each clone of FLC has distinct
physico-chemical properties which may lead to differential injury at tissue sites. As
FLCs are primarily cleared from the circulation by the kidneys,(Abraham and
Waterhouse 1974; Wochner et al. 1967; Maack et al. 1979) this organ is often damaged
in the setting of PCD (Table 1.1). The clinical features of this are wide, ranging from
slowly progressive chronic kidney disease (CKD) often associated with heavy
proteinuria, to life-threatening acute kidney injury (AKI). In multiple myeloma alone,
up to 50% of patients can have renal impairment at diagnosis, 20% may have AKI and
up to 10% require dialysis.(Kyle et al. 2003; Knudsen et al. 2000; Gertz 2005; Blade et
al. 1998)
in disease-dependent patterns.(Sanders et al. 1991) For example: primary (AL)
amyloidosis can affect all compartments of the kidney, but predominantly involves the
glomeruli; in myeloma kidney (cast nephropathy), there is proximal tubular
2
Plasma Cell Dyscrasia Renal Manifestation
MGUS None…
CHAINS
BY
College of Medical and Dental Sciences
The University of Birmingham
University of Birmingham Research Archive
e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
ABSTRACT
Monoclonal immunoglobulin free light chains (FLCs) cause a range of disorders in the
kidney. In multiple myeloma, FLCs can activate the proximal tubule to release MCP-1,
an important cytokine in renal fibrosis. Distal tubular cast formation can also occur
when FLCs co-precipitate with uromodulin. However a pathogenic role for the elevated
polyclonal FLC concentrations seen in chronic kidney disease has not been assessed to
date. This thesis explores the biology of monoclonal FLCs as well as polyclonal FLCs.
Detailed histological analyses demonstrated that in multiple myeloma, interstitial
fibrosis can progress rapidly in situ and indicated that intratubular cast numbers might
be linked to potential for renal recovery. The functional basis of this fibrosis was
explored by in vitro studies, which showed that upon endocytosis of FLCs, oxidative
stress activated redox signalling, resulting in MCP-1 production. Further in situ
analyses showed that in chronic kidney disease, polyclonal FLCs co-localised with
uromodulin in distal tubular casts. Relationships between these casts and markers of
progression of chronic kidney disease were demonstrated. In vitro analyses then showed
that polyclonal FLCs bind to uromodulin and promote aggregation. These findings:
(i) further delineate the pathways for proximal tubular injury in myeloma and (ii)
indicate a potential pathogenic role for polyclonal FLCs in the distal nephron.
DEDICATION
ACKNOWLEDGMENTS
The work presented in this thesis would not have been possible without the
contributions of many people. First and foremost, I must thank Dr Paul Cockwell, who
invested a huge amount of time and effort, in the roles of supervisor, colleague and
friend. I thank Prof Arthur Bradwell for all the encouragement and the tremendous
support he has given. I thank Prof Mark Drayson for his help. Prof Paul Sanders
deserves special mention – thank you for all your support, I will always remember
Alabama fondly. I thank Dr Gregg Wallis for his expert guidance. A special thank you
to Miss for her assistance. I would also like to thank Ltd, the National Institutes for
Health and the Department of Veterans Affairs. In addition, the individuals listed below
have all helped me at various stages, for which I am very grateful.
Miss Kristal Aaron Dr Matthew Morgan
Mr Peterson Anand Dr Timothy Plant
Mr Richard Barber Dr Paul Showell
Mr Simon Blackmore Dr Helen Smith
Dr Alastair Ferraro Mrs Norma Stewart
Dr Tarek Ghonemy Dr Stephanie Stringer
Mr John Gregory Dr Phillip Stubbs
Dr Richard Hampton Mr Phillip Walsh
Miss Lisa Hasty Dr Betsy Wang
Dr Colin Hutchison Mrs Ellie White
Dr Laura Ismail Dr Wei Zhong Ying
Dr Graham Mead Miss Alice Zhou
CONTENTS
1.1 Introduction ........................................................................................................ 1
1.3 Renal Handling of FLCs .................................................................................... 8
1.4 Why do Some Clonal FLCs Cause Kidney Injury? ...................................... 10
1.5 Resident Renal Cells are Differentially Predisposed to Injury by FLC ...... 10
1.6 Disease Specific Considerations for Mesangial Cells and Glomerular
Pathology .................................................................................................................... 11
1.6.4 Light Chain Deposition Disease.................................................................. 16
1.7 Cell Specific Processing and Activation Leads to the Differential Patterns
that are seen in FLC Disease ................................................................................... 17
1.8 The Differential Role of TGF Beta in Amyloid and LCDD ......................... 19
1.9 Matrix Metalloproteinases .............................................................................. 20
Tubule ........................................................................................................................ 21
1.10.3 Proximal Tubular Toxicity ........................................................................ 23
1.11 Distal Tubular Cast Formation..................................................................... 30
1.12 Polyclonal Elevations in Light Chain Levels ............................................... 36
1.13 Scope of this Thesis and Hypothesis ............................................................. 40
2. MATERIALS AND METHODS .......................................................................... 52
2.1 Introduction ...................................................................................................... 52
2.2.1 Background ................................................................................................. 52
2.2.5 Detection Systems ....................................................................................... 54
2.2.6 Chromogenic Detection .............................................................................. 54
2.2.7 Fluorescence Detection ............................................................................... 55
2.2.9 Tissue Fixation and Embedding .................................................................. 57
2.2.10 Tissue Sectioning ...................................................................................... 58
2.2.16 Quenching and Blocking Steps – Prevention of Background Staining..... 60
2.2.17 Endogenous Peroxidase Quenching with Hydrogen Peroxide ................. 60
2.2.18 Endogenous Biotin Block with Avidin and Biotin ................................... 61
2.2.19 Fc Receptor Block with Serum ................................................................. 61
2.2.20 Primary Antibody ...................................................................................... 61
2.2.21 Isotype Control .......................................................................................... 62
2.2.23 Protocol for Quenching of Autofluorescence ........................................... 63
2.2.24 Protocol for Multiple Immunofluorescent Staining of FLC in Kidneys ... 64
2.2.25 Protocol for multiple IF staining - controls ............................................... 65
2.2.26 Visualisation of Immunofluorescent Staining and Image Acquisition ..... 65
2.2.27 Protocol for Immunohistochemical Staining of Macrophages and
Interstitial Capillaries .............................................................................................. 66
2.3.1 Patients ........................................................................................................ 68
Capillary Density .................................................................................................... 68
2.3.4 Cast Counting .............................................................................................. 70
2.4.1 Culture and Propagation of HK-2 Cells ...................................................... 70
2.4.2 Containers ................................................................................................... 71
2.4.6 Enumeration of Cells using a Haemocytometer.......................................... 73
2.4.7 Cryopreservation ......................................................................................... 74
2.4.8 Preparation of Polyclonal FLC Stock Solution for In Vitro use ................. 74
2.4.9 Protocol for Incubation of HK-2 Cells with FLC ....................................... 74
2.4.10 Protocol for Cell Lysis ............................................................................. 75
2.4.11 Hydrogen Peroxide Assay ........................................................................ 75
2.4.12 MCP-1 Assay ........................................................................................... 76
2.4.14 Silencing of Gene Expression with siRNA .............................................. 78
2.4.15 Immunoblotting of Cell Culture Lysates................................................... 79
2.4.15.1 Western Blotting - c-Src Phosphorylation ............................................. 79
2.4.15.2 Western Blotting - Megalin and Cubilin ................................................ 80
2.4.15.3 Densitometry .......................................................................................... 80
2.4.17 Removal of Extracellular and Intracellular H2O2 ..................................... 82
2.5 Purification of Polyclonal FLCs and Protein Chemistry.............................. 83
2.5.1 Total Soluble Protein (TSP) Quantification ................................................ 83
2.5.1.1 Ultraviolet Absorbance at 280 nm (A280) ............................................... 83
2.5.1.2 Bicinchoninic Acid (BCA) Assay ............................................................ 84
2.5.2 Free Light Chain Quantification ................................................................. 84
2.5.3 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis ................ 85
2.5.3.1 Coomassie Brilliant Blue ......................................................................... 85
2.5.3.2 Silver Stain ............................................................................................... 85
2.5.4.2 Dot Blotting .............................................................................................. 86
2.5.5 Endotoxin Assays ........................................................................................ 87
2.5.6 Endotoxin Removal ..................................................................................... 88
2.5.8.1 Initial Steps .............................................................................................. 89
2.5.8.3 Affinity Chromatography ......................................................................... 90
2.5.8.4 Size-Exclusion Chromatography ............................................................. 91
2.5.9 Lyophilisation of Polyclonal FLC for Storage and Transportation ............ 92
2.6 Uromodulin ....................................................................................................... 92
2.6.2 Binding of Uromodulin to Polyclonal FLC ................................................ 93
2.6.2.1 Indirect ELISA ......................................................................................... 93
2.6.2.2 Sandwich ELISA ...................................................................................... 94
2.6.2.3 Dot Blot .................................................................................................... 94
2.7.2 Normalisation of Data ................................................................................. 96
2.7.3 Correlations ................................................................................................. 96
2.7.4 ANOVA ...................................................................................................... 96
2.7.5 Test of Intra- and Inter-observer Variability of Image Analysis Data ........ 96
2.7.6 Assessment of Distribution of Casts ........................................................... 97
3. RENAL INFLAMMATION AND FIBROSIS IN MONOCLONAL DISEASE:
IN SITU STUDIES ........................................................................................................ 98
3.2.3 Index of Chronic Damage ......................................................................... 103
3.2.4 Number of Tubules with Casts .................................................................. 103
3.2.5 Interstitial Infiltrate ................................................................................... 106
IN VITRO STUDIES .................................................................................................. 111
4.2.2 DMTU Inhibits c-Src Activation .............................................................. 114
4.2.3 Inhibition of c-Src Suppresses MCP-1 Production but does not Suppress
H2O2 Production .................................................................................................... 116
4.2.4 Removal of Extracellular H2O2 by Catalase has no Impact on MCP-1
Production ............................................................................................................. 118
4.2.5 Silencing of c-Src Expression Suppresses MCP-1 Production in Response
to Light Chain Exposure ....................................................................................... 118
4.2.6 c-Src is Oxidised Following Light Chain Treatment ................................ 118
4.2.7 Silencing of Megalin and Cubilin Suppresses MCP-1 Production ........... 120
4.3 Discussion ........................................................................................................ 121
5.1 Introduction .................................................................................................... 128
5.2 Choice of Source of Polyclonal Free Light Chains ...................................... 128
5.3 Analysis of Resuspended Sera ....................................................................... 130
5.4 Extraction of Proteins Containing Light Chains from Starting Sample .. 133
5.4.1 Anti- Light Chain Matrix – Assessment of Suitability ........................... 133
5.4.2 Anti- Light Chain Matrix – Assessment of Suitability ........................... 136
5.4.3 Extraction of -Light Chain Containing Proteins From Starting Sample . 140
5.4.4 Anti- Light Chain Matrix Manufacture................................................... 142
5.4.5 Anti- Light Chain Column – Assessment of Capacity ............................ 143
5.4.6 Extraction of -Light Chain Containing Proteins From the Anti- Unbound
Fraction ................................................................................................................. 145
Contaminants .......................................................................................................... 148
5.5.3 Size Exclusion Chromatography ............................................................... 154
5.6 Assessment of Polyclonal FLC Purity .......................................................... 154
5.7 FLC – Removal of IgG, Human Serum Albumin and Transferrin ....... 158
5.8 Detection and Removal of Endotoxin ........................................................... 160
5.9 Assessment of Solubility After Lyophilisation ............................................. 161
Table 5.4. FLC recovery after lyophilisation..................................................... 162
5.10 Discussion ...................................................................................................... 163
CHRONIC KIDNEY DISEASE: IN SITU STUDIES ............................................. 166
6.1 Introduction .................................................................................................... 166
6.2 Patients ............................................................................................................ 168
6.3 Results ............................................................................................................. 169
6.3.1 Immunofluorescence ................................................................................. 169
6.3.3 Polyclonal FLC Co-localise with Uromodulin in Distal Tubules ............. 170
6.4 Measurement of Cast Numbers, Index of Chronic Damage, Interstitial
Capillary Density and Macrophage Numbers ...................................................... 175
6.4.1 Test of Normality of Data ......................................................................... 175
6.4.2 Assessment of Validity of Quantification Methods .................................. 180
6.4.3 Casts in CKD are Situated in Areas of Established Chronic Damage ...... 180
6.4.4 Capillary Density Correlates with the Index of Chronic Damage and
Macrophage Numbers ........................................................................................... 182
6.4.5 Macrophage Numbers Correlate with Index of Chronic Damage ............ 185
6.4.6 Cast Numbers Correlate with Index of Chronic Damage, Capillary Density
and Macrophage Numbers .................................................................................... 185
6.5 Discussion ........................................................................................................ 188
VITRO STUDIES ....................................................................................................... 193
7.1 Introduction .................................................................................................... 193
7.2 PTEC Culture ................................................................................................. 194
7.2.1 Effect of Polyclonal Free Light Chains on Inflammatory Signalling ....... 194
7.2.2 Cytotoxic Effects of Polyclonal Free Light Chains on Proximal Tubule
Epithelial Cells ...................................................................................................... 195
7.3 Uromodulin ..................................................................................................... 196
7.3.1 Uromodulin is Highly Aggregated in High Salt Solutions ....................... 196
7.3.2 Uromodulin Aggregation is Reduced in Water and by Alkaline pH ........ 197
7.4 Polyclonal Free Light Chains Interact with Uromodulin: Dot Blotting ... 200
7.5 Polyclonal Free Light Chains Interact with Uromodulin: ELISA ............ 205
7.5.1 Plate Coated with Uromodulin .................................................................. 205
7.5.2 Plate Coated with Polyclonal Free Light Chains ...................................... 206
7.5.3 Sandwich ELISA ....................................................................................... 209
7.6 Polyclonal Free Light Chains Interact with Uromodulin: Nephelometry 209
7.6.1 Experiments in Buffer Containing 50 mM NaCl ...................................... 212
7.6.2 Experiments in Buffer Containing 100 mM NaCl .................................... 212
7.6.3 Experiments in Buffer Containing 150 mM NaCl .................................... 216
7.7 Discussion ........................................................................................................ 217
AND THERAPEUTIC STRATEGIES ..................................................................... 226
8.3 Histological Markers of Renal Outcome in Cast Nephropathy ................. 228
8.4 Proximal Tubular Damage in Cast Nephropathy ....................................... 229
8.4.1 The Role of c-Src in Signal Transduction ................................................. 230
8.5 Distal Tubular Damage in Cast Nephropathy ............................................. 231
8.6 Potential Therapeutic Approaches to Cast Nephropathy .......................... 232
8.6.1 Reduction of FLC Load Delivered to Nephrons ....................................... 232
8.6.2 Prevention of PTEC Damage .................................................................... 232
8.6.2.1 Prevention of Endocytosis ..................................................................... 232
8.6.2.2 Reduction of Intracellular Oxidative Stress ........................................... 234
8.6.2.3 c-Src Inhibition....................................................................................... 234
8.7 The Inflammatory Role of Filtered Proteins in CKD ................................. 238
8.8 The Role of Polyclonal FLCs in CKD .......................................................... 238
8.8.1 The Effects of Polyclonal FLCs on PTECs .............................................. 238
8.8.2 The Interaction of Uromodulin and Polyclonal FLCs in CKD ................. 240
8.9 Conclusion ....................................................................................................... 242
9.1 Papers .............................................................................................................. 244
9.2 Abstracts ......................................................................................................... 244
10. APPENDIX ......................................................................................................... 246
A.3 SDS-PAGE - Gel Recipes and Calculation of Measures ............................ 252
A.4 Useful Common Protein Extinction Coefficients for A280 Measurements 253
A.5 BCA Assay Standards, Working Range 20 – 2000 µg/ml .......................... 253
A.6 Useful Numbers for Cell Culture ................................................................ 254
A.7 Amino Acids ............................................................................................... 254
A.9 Normal HK-2 Cells ..................................................................................... 257
A.10 A Neubauer Haemocytometer ................................................................... 257
A.11 siRNA Mode of Action ............................................................................. 258
A.12 Chromatography Apparatus ...................................................................... 259
Figure 1.2.Interactions of FLCs with mesangial cells..................................................... 12
Figure 1.3. Cast nephropathy and accelerated in situ progression of interstitial fibrosis in
a patient with multiple myeloma. ............................................................................ 24
Figure 1.4. Interactions of FLCs with proximal tubule epithelial cells. ......................... 25
Figure 1.5. Light chain interactions in the distal nephron............................................... 32
Figure 3.1. Renal biopsies from patients A-D............................................................... 104
Figure 3.2. Changes in the index of chronic damage and cast numbers over six weeks.
............................................................................................................................... 105
Figure 4.3. DMTU inhibits c-Src activation. ................................................................ 116
Figure 4.4. Inhibition of c-Src suppresses MCP-1 production but not H2O2 production.
............................................................................................................................... 117
Figure 4.5. Silencing of c-Src expression suppresses MCP-1 production in response to
light chain exposure. ............................................................................................. 119
Figure 4.6. c-Src in oxidised following light chain treatment....................................... 121
Figure 4.7. Silencing of megalin and cubilin suppress MCP-1 production. ................. 122
Figure 5.1. Electrophoretic (SDS-PAGE) analysis of the FLC extract after passage
through the protein A column. .............................................................................. 132
Figure 5.2. Polyclonal FLC purification protocol. ........................................................ 134
Figure 5.3. Chromatogram of assessment of suitability of anti- light chain matrix. .. 135
Figure 5.4. Dot blot demonstrating anti- light chain matrix capacity. ........................ 136
Figure 5.5. SDS-PAGE analysis of pooled elutions from the anti- column. .............. 137
Figure 5.6. Chromatogram of assessment of suitability of anti- light chain matrix. .. 138
Figure 5.7. Dot blot demonstrating anti- light chain matrix capacity. ........................ 139
Figure 5.8. SDS-PAGE analysis of pooled elutions from the anti- column. .............. 140
Figure 5.9. Chromatograms showing extraction of -light chain containing proteins
from the starting sample. ....................................................................................... 141
Figure 5.10. Western blots of anti- elution pool. ........................................................ 143
Figure 5.11. Chromatogram of assessment of suitability of manufactured anti- matrix.
............................................................................................................................... 144
Figure 5.12, Dot blot demonstrating capacity of the manufactured anti- light chain
matrix. ................................................................................................................... 145
from the anti- unbound fraction. ......................................................................... 147
Figure 5.14. Western blot showing enrichment of -light chain containing proteins in
the anti-total elutions.......................................................................................... 148
Figure 5.15. Chromatograms showing anti- and anti- elutions applied to protein G
column. .................................................................................................................. 151
Figure 5.16. Chromatogram showing the -FLC sample applied to anti-IgA and anti-
IgM columns. ........................................................................................................ 152
Figure 5.17. Chromatograms showing the -FLC sample applied to anti-IgA and anti-
IgM columns. ........................................................................................................ 153
Figure 5.18. Chromatograms showing removal of impurities from and FLC samples
by size-exclusion chromatography. ....................................................................... 155
Figure 5.19. SDS-PAGE analysis of the purity of and FLC samples. .................... 159
Figure 5.20. Western blots showing the presence of albumin and transferrin
contaminants in the -FLC sample. ...................................................................... 160
Figure 5.21. SDS_PAGE analysis of unbound fractions after incubation with anti-HSA,
anti-HSA+anti-TF and protein G. ......................................................................... 161
Figure 5.22. SDS-PAGE analysis of -FLC sample, after incubation with anti-
HSA+anti-TF and protein G. ................................................................................ 162
Figure 5.23. SDS-PAGE analysis of FLC recovery after lyophilisation. .................. 163
Figure 6.1. A proteinaceous cast in a renal biopsy from a patient with CKD. ............. 167
Figure 6.2. Uromodulin is produced in the distal nephron and is present in casts........ 168
Figure 6.3. Free light chains in the proximal tubule in CKD. ....................................... 171
Figure 6.4. Confocal image of proximal and distal tubules showing distribution of -
FLC, -FLC and uromodulin. ............................................................................... 172
Figure 6.5. Confocal image of casts in a renal biopsy from a patient with CKD. ........ 173
Figure 6.6. High-power confocal image of a cast in a renal biopsy from a patient with
CKD. ..................................................................................................................... 174
Figure 6.7. Quantification of the index of chronic damage. ......................................... 176
Figure 6.8. Quantification of cast numbers. .................................................................. 177
Figure 6.9. Quantification of interstitial capillary density. ........................................... 178
Figure 6.10. Quantification of macrophage numbers.................................................... 179
Figure 6.11. Comparison of cast numbers between areas with or without chronic
damage. ................................................................................................................. 182
Figure 6.12. Correlations of capillary density with index of chronic damage and
macrophage numbers. ........................................................................................... 184
Figure 6.13. Correlation of macrophage numbers with index of chronic damage. ....... 185
Figure 6.14. Correlations of cast numbers with index of chronic damage, capillary
density and macrophage numbers. ........................................................................ 186
Figure 7.1. Effect of polyclonal free light chains on inflammatory signalling. ............ 196
Figure 7.2. Cytotoxic effects of polyclonal free light chains on proximal tubule cells.197
Figure 7.3. Uromodulin from healthy volunteer urines prior to dialysis. ..................... 198
Figure 7.4. Uromodulin aggregation is reduced by dialysis into water and by alkaline
pH. ......................................................................................................................... 199
Figure 7.5. Dot blot showing binding of polyclonal FLCs to uromodulin (polyclonal
FLC dots). ............................................................................................................. 202
Figure 7.6. Dot blot demonstrating binding of polyclonal FLC to uromodulin
(uromodulin dots). ................................................................................................. 203
Figure 7.7. Dot blot (repeated) demonstrating binding of polyclonal FLC to uromodulin
(FLC dots). ............................................................................................................ 204
Figure 7.8. Assessment of binding of polyclonal FLC to uromodulin by ELISA: Plate
coated with uromodulin......................................................................................... 207
Figure 7.9. Assessment of binding of polyclonal FLC to uromodulin by ELISA: Plate
coated with FLC. ................................................................................................... 208
Figure 7.10. Assessment of binding of polyclonal FLC to uromodulin by ELISA:
Sandwich ELISA. .................................................................................................. 211
Figure 7.11. Nephelometric assessment of the formation of higher molecular weight
aggregates with uromodulin; comparison of polyclonal and monoclonal FLCs; 50
mM NaCl. .............................................................................................................. 213
Figure 7.12. Nephelometric assessment of the formation of higher molecular weight
aggregates with uromodulin; comparison of polyclonal FLCs with HSA and
uromodulin; 50 mM NaCl. .................................................................................... 214
Figure 7.13. Nephelometric assessment of the formation of higher molecular weight
aggregates with uromodulin; comparison of monoclonal and polyclonal FLCs; 100
mM NaCl. .............................................................................................................. 215
Figure 7.14. Nephelometric assessment of the formation of higher molecular weight
aggregates with uromodulin; comparison of polyclonal FLCs with HSA or
uromodulin alone; 100 mM NaCl. ........................................................................ 216
Figure 7.15. Nephelometric assessment of the formation of higher molecular weight
aggregates with uromodulin; comparison of monolconal and polyclonal FLCs; 150
mM NaCl. .............................................................................................................. 220
Figure 7.16. Nephelometric assessment of the formation of higher molecular weight
aggregates with uromodulin; comparison of polyclonal FLCs with HSA or
uromodulin alone; 150 mM NaCl. ........................................................................ 221
TABLES
Table 1.1 Renal manifestations of plasma cell dyscrasias. ............................................... 2
Table 1.2. Renal manifestations of plasma cell dyscrasias, site and composition of
deposits and summary of clinical and histological features. ................................... 44
Table 3.1. Summary of patient demographics, histological findings and biochemical
data. ....................................................................................................................... 102
Table 5.1. Batches of resuspended sera received after filtration and passage through
protein A column................................................................................................... 131
Table 5.2. Nephelometric analysis of immunoglobulin content of fraction unbound to
protein G. .............................................................................................................. 152
Table 5.3. Nephelometric analysis of FLC content of and samples after SEC. ..... 156
Table 5.4. FLC recovery after lyophilisation. ............................................................ 162
Table 6.1. Assessment of agreement between two observers by the Bland-Altman
method. .................................................................................................................. 181
Table 6.2. Univariate analyses of cast numbers, index of chronic damage, capillary
density and macrophage numbers. ........................................................................ 183
Table 6.3. Multivariate analysis of correlations between index of chronic damage,
capillary density, macrophage numbers and cast numbers. .................................. 187
ABBREVIATIONS
ACR Albumin/Creatinine Ratio
DMTU Dimethyl Thiourea
ECM Extracellular Matrix
EMT Epithelial-to-Mesenchymal Transition
LDH Lactate Dehydrogenase
MC Mesangial Cell
MMP Matrix Metalloproteinase
MW Molecular Weight
NF-B Nuclear Factor kappa- light-chain-enhancer of activated B cells
PACAP38 Pituitary Adenylate Cyclase Activating Polypeptide with 38 residues
PAMS Periodic Acid-Methenamine Silver
PCD Plasma cell dyscrasia
PP2 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine
RAP Receptor Associate Protein
ROS Reactive Oxygen Species
SAP Serum Amyloid Protein
1.1 Introduction
Plasma cell dyscrasias (PCD) are relatively common disorders, the prevalence of
monoclonal gammopathy of undetermined significance (MGUS) in the over-50’s is
around 3.2%, and multiple myeloma accounting for 10% of all haematological
malignancies.(Kyle and Rajkumar 2004; Kyle et al. 2006) Plasma cell dyscrasias are
characterised by the proliferation of a clone of B-cell lineage. There is associated
production of clonal immunoglobulin (Ig) which frequently includes a variable quantity
of clonal immunoglobulin free light chain (FLC). Each clone of FLC has distinct
physico-chemical properties which may lead to differential injury at tissue sites. As
FLCs are primarily cleared from the circulation by the kidneys,(Abraham and
Waterhouse 1974; Wochner et al. 1967; Maack et al. 1979) this organ is often damaged
in the setting of PCD (Table 1.1). The clinical features of this are wide, ranging from
slowly progressive chronic kidney disease (CKD) often associated with heavy
proteinuria, to life-threatening acute kidney injury (AKI). In multiple myeloma alone,
up to 50% of patients can have renal impairment at diagnosis, 20% may have AKI and
up to 10% require dialysis.(Kyle et al. 2003; Knudsen et al. 2000; Gertz 2005; Blade et
al. 1998)
in disease-dependent patterns.(Sanders et al. 1991) For example: primary (AL)
amyloidosis can affect all compartments of the kidney, but predominantly involves the
glomeruli; in myeloma kidney (cast nephropathy), there is proximal tubular
2
Plasma Cell Dyscrasia Renal Manifestation
MGUS None…
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