Investigating the mechanisms of renal fibrosis following ischaemia and reperfusion injury

I Acknowledgements . II List of Abbreviations . III Table of contents . V List of Figures . XI List of Tables . XIV Chapter 1: Introduction . 1 1.1 The kidney and its function . 1 1.1.1 Anatomy of Kidney . 1 1.1.2 The Functional Unit of Kidney: The Nephron . 1 1.1.3 Function of the Kidney . 2 1.2 Acute and Chronic Kidney Disease . 2 1.2.1 Epidemiology of Acute Kidney Injury . 2 1.2.2 Aetiology . 3 1.2.3 Pathophysiology of Acute Kidney Injury . 4 1.2.4 Early consequences of Acute Kidney Injury . 5 1.2.5 AKI leading to Chronic Kidney disease . 5 1.2. 6 Chronic Kidney Disease . 7 1.2. 7 Classification of stages of CKD . 7 1.2. 8 Management of CKD . 8 1.3 Ischaemia and Reperfusion Injury (IRI). 9 1.3.1 Ischaemia . 9 1.3.1.1 Hypoxia Inducible factor (HIF) . 10 1.3.1.1.2 Downstream effects of HIF-1 signalling . 11 1.3.2 Reperfusion . 12 1.3.2.1 Oxidative stress . 13 1.3.2.2 Mitochondrial dysfunction . 13 1.3.2.3 Nitric oxide . 13 1.3. 3 Cellular and metabolic consequences of tubular injury . 14 1.4 Tissue Repair after IRI . 15 1.4. 1 Extracellular matrix synthesis . 15 1.4. 1.1 Collagen . 15 1.4. 1.2 Glycoproteins . 15 VI 1.4. 1.3 Metalloproteinases (MMPs) . 16 1.4. 2 Growth factors contributing to the extracellular matrix deposition . 16 1.5 Transforming growth factor . 17 1.5. 1 Latent TGF-β and its activation . 17 1.5. 2 Activation of TGF-β . 18 1.5. 2.1 Activation by Plasmin . 18 1.5. 2.2 Activation by Thrombospondin-1 . 18 1.5. 2.3 Activation by Reactive oxygen species (ROS) . 19 1.5. 2.4 Activation by pH . 19 1.5. 2.5 Activation by Heat . 19 1.5. 3 Structure of TGF-β receptors for TGF-β family . 19 1.5. 4 TGF-β/SMAD signalling pathway . 21 1.5. 5 SMAD independent signalling . 21 1.5. 6 TGF- in kidney diseases . 23 1.5. 6.1 Cell culture models of Renal Fibrosis . 23 1.5. 6.2 Animal and preclinical models . 23 1.5. 6.3 Inhibition of TGF- . 24 1.6 Role of Integrins in kidney injury . 25 1.6.1 Integrin structure, expression and function . 25 1.6.2 Role of integrins in TGF-β activation . 27 1.6.2.1 Integrin expression in kidney . 27 1.7 Hypothesis . 29 1.8 Specific Aims . 29 Chapter 2: Material and Methods . 30 2.1 General Practise . 30 2.2 Materials . 30 2.2.1 DNA Buffers . 30 2.2.2 Western Blot Buffer solutions . 31 2.2.3 Flow Cytometry Buffer solutions . 31 2.2.4 Immunofluorescence Buffer solutions . 31 2.2.5 Bacterial Transformation . 32 2.2. 6 ELISA (Enzyme Linked Immunosorbent Assay) . 32 2.2. 7 Collagen co-culture system . 33 2.2. 8 ALK5 reconstitution . 33 VII 2.2. 9 CoCl2 preparation . 33 2.2.10 H2O2 solution . 33 2.3 Cell methodology . 34 2.3.1 Cell culture . 34 2.3.2 Culture Media . 34 2.3.2.1 DMEM-HAM F-12 media . 34 2.3.1.2 DMEM media . 35 2.3. 3 Cell lines . 35 2.3. 3.1 Human kidney epithelial cell lines (HKC8 and HK-2) . 35 2.3. 3.2 Human lung fibroblast cell line (MRC-5) . 36 2.3. 4 Cell viability . 36 2.3. 5 Cell Storage . 36 2.3. 6 Mycoplasma testing. 37 2.3. 7 WST-1 cell proliferation assay . 37 2.3. 8 Establishing co-culture system . 38 2.3. 8.1 Collagen concentration . 38 2.3. 8.2 Cell seeding density . 39 2.3. 9 Bacterial transformation . 40 2.3. 9.1 Bacterial culture . 41 2.4 Protein expression . 42 2.4. 1 Immunofluorescent cell staining . 42 2.4. 1.1 Quantification of Immunofluorescence per Cell (IF/Cell) . 44 2.4. 2 Immunohistochemistry (IHC) . 44 2.4. 3 Flow cytometry . 45 2.4. 4 Measurement of protein concentration .