Kidney Research: Experimental Protocols (Methods in Molecular Biology, 2664)

Preface
Contents
Contributors
Part I: In Vitro, In Vivo, and Ex Vivo Models of Kidney Disease
	Chapter 1: Restoration of Podocyte Phenotype in Culture
		1 Introduction
		2 Materials
			2.1 Animals
			2.2 Perfusion of Kidneys
			2.3 Isolation of Glomeruli
			2.4 Culture of Glomeruli
			2.5 Subculture of Podocytes
		3 Methods
			3.1 Perfusion of Kidneys (Fig. 1)
			3.2 Isolation and Culture of Glomeruli (Fig. 2)
			3.3 Subculture of Podocytes
		4 Notes
		References
	Chapter 2: Propagation, Culture, and Characterization of Renal Fibroblasts
		1 Introduction
		2 Materials
			2.1 General Sterile Cell Culture Materials
			2.2 Specialist Cell Culture Equipment
			2.3 Cell Culture Reagents
			2.4 Immunoperoxidase Cytochemistry
				2.4.1 General Materials
				2.4.2 Immunoperoxidase Cytochemistry
			2.5 Immunofluorescence
				2.5.1 Cell Culture
				2.5.2 Preparation of Cultured Cells
				2.5.3 Immunostaining of Fixed Cells
			2.6 Flow Cytometry
		3 Methods
			3.1 Gelatin Coating of Petri Dishes
			3.2 Propagation of Renal Interstitial Cultures from Kidney Explants
			3.3 Subculture of Primary Human Cell Cultures
				3.3.1 Lifting Explant Monolayer Cultures
				3.3.2 Maintenance of Primary Renal Fibroblast Subcultures
			3.4 Characterization of Primary Renal Fibroblasts
				3.4.1 Growth Characteristics
				3.4.2 Immunocytochemistry/Immunoperoxidase Staining
				3.4.3 Immunofluorescence
				3.4.4 Flow Cytometry
			3.5 Freezing Cell Cultures for Cryogenic Storage
			3.6 Thawing of Cryogenically Stored Cells
		4 Notes
		References
	Chapter 3: Isolation of Rat Glomeruli and Propagation of Mesangial Cells to Study the Kidney in Health and Disease
		1 Introduction
			1.1 Whole Kidney Analyses Are Not Representative of Glomerular Changes
			1.2 Mesangial Cells
		2 Materials
			2.1 Preparation of Rat Glomeruli Suspensions
				2.1.1 Isolation
				2.1.2 Tissue Dissociation (Optional)
			2.2 Propagation of Glomerular Mesangial Cells
		3 Methods
			3.1 Isolation of Intact Glomeruli
				3.1.1 Isolation
				3.1.2 Applications
			3.2 Propagation of Mesangial Cells
				3.2.1 Propagation
				3.2.2 Characterization
		4 Notes
		References
	Chapter 4: In Vitro Models to Evaluate Molecular Permeability of the Kidney Filtration Barrier
		1 Introduction
		2 Materials
			2.1 Equipment and General Materials
			2.2 Isolation and Decellularization of Glomeruli
			2.3 GBM Membrane Fabrication
			2.4 FITC-Ficoll Synthesis
			2.5 Diffusional Permeability Assay
			2.6 Sieving Coefficient Assay
			2.7 HPLC Sample Preparation, Testing, and Analysis
		3 Methods
			3.1 Isolation and Decellularization of Glomeruli
			3.2 GBM Fabrication
			3.3 Sterilization
			3.4 FITC-Ficoll Synthesis
			3.5 Diffusional Permeability Assay
			3.6 Sieving Coefficient Assay
			3.7 HPLC Sample Preparation, Testing, and Analysis
		4 Notes
		References
	Chapter 5: Generating Human Glomeruli from Pluripotent Stem Cells for Disease Modelling and Drug Screening
		1 Introduction
		2 Materials
			2.1 iPSC-Derived Kidney Organoid Generation
			2.2 iPSC-Derived Kidney Organoid Dissociation
			2.3 iPSC-Derived Kidney Organoid Glomeruli (OrgGlom) Isolation
			2.4 iPSC-Derived Kidney Organoid Glomeruli (OrgGlom) Culture
			2.5 iPSC-Derived Kidney Organoid Glomeruli (OrgGlom) Fixation
			2.6 iPSC-Derived Kidney Organoid Glomeruli (OrgGlom) Immunofluorescent Staining
		3 Methods
			3.1 iPSC-Derived Kidney Organoid Generation
			3.2 iPSC-Derived Kidney Organoid Dissociation
			3.3 iPSC-Derived Kidney Organoid Glomeruli (OrgGlom) Isolation
			3.4 iPSC-Derived Kidney Organoid Glomeruli (OrgGlom) Culture
			3.5 iPSC-Derived Kidney Organoid Glomeruli (OrgGlom) Fixation
			3.6 iPSC-Derived Kidney Organoid Glomeruli (OrgGlom) and Organoid Podocyte (OrgPod) Immunofluorescent Staining
		4 Notes
		References
	Chapter 6: Large-Scale Production of Kidney Organoids from Human Pluripotent Stem Cells
		1 Introduction
		2 Materials
			2.1 General Materials and Considerations for hPSC and Organoid Culture (See Notes 1 and 2)
			2.2 hPSC Culture
			2.3 Organoid Generation
			2.4 Gene Expression and Immunohistochemical Analyses
		3 Methods
			3.1 hPSC Culture
				3.1.1 Coating of Culture Dishes
				3.1.2 hPSC Passaging
			3.2 Organoid Generation
				3.2.1 Day 0 - Starting Differentiation
				3.2.2 Day 2 - Half Medium Change
				3.2.3 Day 3 - Transfer to Stage II Medium
				3.2.4 Organoid Maintenance
			3.3 Organoid Analysis
		4 Notes
		References
	Chapter 7: Construction of a Multitubular Perfusable Kidney-on-Chip for the Study of Renal Diseases
		1 Introduction
		2 Materials
			2.1 Making of Feature Mold
			2.2 PDMS Scaffolding
			2.3 Collagen Molding
			2.4 Coating and Cell Seeding
			2.5 Staining
		3 Methods
			3.1 Making of the PDMS Feature Mold
				3.1.1 Prepare the Wafer and Laminator
				3.1.2 Apply and Bond the Dry Film to the Wafer
				3.1.3 Expose Dry Film to UV
				3.1.4 Develop the Wafer
				3.1.5 Silanize the Wafer
			3.2 Fabrication of the PDMS Scaffold
				3.2.1 Prepare PDMS at a 1:10 Ratio Curing Agent/Base
				3.2.2 Prepare Substrate for PDMS
				3.2.3 Pour and Cure PDMS
				3.2.4 Assemble the PDMS Layers
				3.2.5 Bond the PDMS to the Glass Slide
			3.3 Collagen Molding
				3.3.1 Treat the Surface of the Chip with APTES (Fig. 3b Part 1)
				3.3.2 Treat the Surface of the Chip with Glutaraldehyde
				3.3.3 Prepare Wires
				3.3.4 Insert Wires
				3.3.5 Prepare the Collagen
				3.3.6 Allow Collagen to Polymerize
			3.4 Coating and Cell Seeding
				3.4.1 Prepare the Working Environment
				3.4.2 Prepare the Coating Solution
				3.4.3 Check for the Absence of Bubbles in the Chip Channels
				3.4.4 Coat with Laminin
				3.4.5 Prepare the Working Environment for Cell Seeding
				3.4.6 Prepare Cells
				3.4.7 Check the Chip Configuration
				3.4.8 Seed Cells
				3.4.9 Let Cells Adhere
				3.4.10 Remove Wires and Tubing
				3.4.11 Flushing (Optional)
				3.4.12 Seal the Wire Insertion with PDMS (Optional for Later Perfusion)
			3.5 Cell Culture, Fixation, and Staining
				3.5.1 Changing Medium
				3.5.2 Fixing the Cells
				3.5.3 Staining
		4 Notes
		References
	Chapter 8: A ``Kidney-on-the-Chip´´ Model Composed of Primary Human Tubular, Endothelial, and White Blood Cells
		1 Introduction
		2 Materials
			2.1 General Materials
			2.2 Cell Culture and Maintenance
			2.3 Peripheral Blood Mononuclear Cell (PBMC) Isolation
			2.4 OrganoPlate Technology
			2.5 ECM Preparation
			2.6 Induced PBMC Migration
			2.7 Barrier Integrity Assay (BIA)
		3 Methods
			3.1 RPC Culture
			3.2 HUVEC Cell Culture
			3.3 PBMC Cell Isolation
			3.4 ECM Preparation
			3.5 OrganoPlate Preparation
			3.6 Cell Seeding and Cultivation in the OrganoPlate
				3.6.1 Seeding RPCs in Channel 1
				3.6.2 Seeding HUVECs in Channel 3 (Dry)
				3.6.3 Seeding HUVECs in Channel 3 (Wet)
				3.6.4 Seeding PBMCs in Channel 3 (with HUVECs)
			3.7 Induced PBMC Migration
			3.8 Barrier Integrity Assay (BIA)
		4 Notes
		References
	Chapter 9: Standardized Protocol for the Preparation of Precision-Cut Kidney Slices: A Translational Model of Renal Fibrosis
		1 Introduction
			1.1 History of Precision-Cut Tissue Slices
			1.2 Precision-Cut Kidney Slices in Short
		2 Materials
			2.1 Specialist Equipment
			2.2 Krebs-Henseleit Buffer (KHB)
			2.3 William´s E Medium (WEM)
			2.4 Tissue Collection
		3 Methods
			3.1 Preparation of Krebs-Henseleit Buffer (KHB)
			3.2 Preparation for Slicing Procedure
			3.3 Collection of Kidney Tissue
				3.3.1 Rodent Kidney Tissue
				3.3.2 Human Kidney Tissue (See Note 3)
			3.4 Preparation of Renal Tissue Cores for Slicing
			3.5 Preparation of Precision-Cut Kidney Slices
			3.6 Incubation of the Slices
			3.7 After an Experiment
			3.8 Characterization - Viability, Morphology, and Function
				3.8.1 Adenosine Triphosphate (ATP) and Protein Content
				3.8.2 Lactate Dehydrogenase (LDH)
				3.8.3 Glutathione
				3.8.4 Morphology
				3.8.5 Functionality
		4 Notes
		References
	Chapter 10: Culture of Three-Dimensional Madin-Darby Canine Kidney (MDCK) Cysts for In Vitro Drug Testing in Polycystic Kidney...
		1 Introduction
		2 Materials
			2.1 Cell Line Maintenance
			2.2 Extracellular Matrix Preparation
			2.3 Drugs
			2.4 Equipment
		3 Methods
			3.1 Establishing MDCK Cells in Culture
			3.2 Passaging MDCK Cells
			3.3 Preparation of Collagen Matrix Solution
			3.4 Cyst Culture in Collagen Matrix
			3.5 Effect of Drugs on Established Cysts
			3.6 Effect of Drugs on Cyst Initiation
			3.7 Imaging of Cysts
			3.8 Quantification of Cysts
		4 Notes
		References
	Chapter 11: Zebrafish Model to Study Podocyte Function Within the Glomerular Filtration Barrier
		1 Introduction
		2 Materials
			2.1 Preparation of Assay Plate
			2.2 Plating of the Fish
			2.3 Imaging of GFP Fluorescence of the Vitamin D-Binding Protein in the Retinal Vessel Plexus of Tg(l-fabp:DBP:eGFP) Zebrafish...
			2.4 Collection of Zebrafish Larvae and Preparation of Fish for Further Analyses
				2.4.1 Fixation of Zebrafish Larvae
		3 Methods
			3.1 Prepare Plate for Assay (If Automated Imaging System Is Used)
			3.2 Plating of the Fish
			3.3 Imaging of GFP Fluorescence of Vitamin D-Binding Protein in the Retinal Vessel Plexus of Tg(l-fabp:DBP:eGFP) Zebrafish (``...
				3.3.1 For Analysis in a Standard Fluorescence Microscope
				3.3.2 If Automated Imaging Is Used
			3.4 Collection of Zebrafish Larvae and Preparation of Fish for Further Analyses
			3.5 Analysis of Retinal Vessel Plexus Fluorescence
		4 Notes
		References
Part II: Imaging in Kidney Research
	Chapter 12: Magnetic Resonance Imaging (MRI) Analysis of Tissue Sodium Concentration in Chronic Kidney Disease
		1 Introduction
		2 Materials
			2.1 Human Subjects
			2.2 Magnetic Resonance Imaging
		3 Methods
			3.1 Performance of MRI
			3.2 Image and Data Analysis
			3.3 Outcomes and Interpretation
		4 Notes
		References
	Chapter 13: Determination of Interstitial Collagen Deposition and Related Topography Using the Second Harmonic Generation-Base...
		1 Introduction
		2 Materials
		3 Methods
			3.1 Removal of Paraffin from FFPE Tissue Slides (See Note 1)
			3.2 Optimization of HistoIndex Scanning and Analysis of Images (See Notes 3 and 4)
			3.3 Basic FibroIndex Outputs Explained
				3.3.1 Collagen and Tissue Area
				3.3.2 Collagen Topology
		4 Notes
		References
	Chapter 14: Quantitative Imaging of Podocyte Foot Processes in the Kidney Using Confocal and STED Microscopy
		1 Introduction
		2 Materials
			2.1 Tissue
			2.2 Fixation (Fresh or Fresh-Frozen Tissue)
			2.3 De-paraffinization and Rehydration (FFPE Tissue)
			2.4 Hydrogel Embedding (Optional)
			2.5 Sectioning
			2.6 Optical Clearing
			2.7 Staining Buffers
			2.8 Primary Antibodies
			2.9 Secondary Antibodies
			2.10 Mounting Media
			2.11 Mounting
			2.12 Confocal/STED Microscopy
			2.13 Image Analysis
		3 Methods
			3.1 Protocol 1 (Fresh or Fresh-Frozen Tissue to Be Imaged with STED Microscopy)
				3.1.1 Fixation
				3.1.2 Hydrogel Embedding (Optional; See Note 2)
				3.1.3 Sectioning
				3.1.4 SDS De-lipidation
				3.1.5 Staining
				3.1.6 Mounting
				3.1.7 STED Imaging
				3.1.8 Image Analysis
			3.2 Protocol 2 (Fresh or Fresh-Frozen Tissue to Be Imaged with Confocal Microscopy)
				3.2.1 Fixation (Fresh or Fresh-Frozen Tissue)
				3.2.2 Sectioning
				3.2.3 SDS De-lipidation
				3.2.4 Staining
				3.2.5 Mounting
				3.2.6 Confocal Imaging
				3.2.7 Image Analysis
			3.3 Protocol 3 (FFPE Tissue to Be Imaged with STED or Confocal Microscopy)
				3.3.1 Fixation and Paraffinization
				3.3.2 De-paraffinization and Rehydration
				3.3.3 Sectioning
				3.3.4 SDS De-lipidation
				3.3.5 Staining
				3.3.6 Mounting
				3.3.7 Confocal/STED Microscopy
				3.3.8 Image Analysis
		4 Notes
		References
	Chapter 15: Synthesis and Expression of a Targeted, Ferritin-Based Tracer for PET Imaging of Kidney Glomeruli
		1 Introduction
		2 Materials
			2.1 General Materials and Equipment
			2.2 CF Synthesis and Validation
			2.3 Recombinant Human Ferritin Expression
			2.4 Radiolabeled CF Synthesis, Imaging, and Validation
			2.5 Computational Requirements for Image Analysis
		3 Methods
			3.1 Cationization of Ferritin
				3.1.1 Quality Assurance
			3.2 Preparation of Human Recombinant Ferritin (See Note 9)
				3.2.1 Recombination with Affinity Tag
				3.2.2 Quality Assurance
			3.3 Radiolabeling Cationic Ferritin with Isotope (Cu64)
				3.3.1 Quality Assurance
			3.4 Dynamic PET Imaging
			3.5 Image Processing
			3.6 PET/MRI
		4 Notes
		References
Part III: Analytical and Functional Measurements in the Kidney
	Chapter 16: Multiplex In Situ Hybridization in the Study of Acute Kidney Injury
		1 Introduction
		2 Materials
			2.1 Unilateral Ischemia-Reperfusion Injury Model
			2.2 Sample Collection and Fixation
			2.3 RNAscope 2.5HD Detection Kit (BROWN); BaseScope Detection Kit v2 (RED); RNAscope Multiplex FL Kit v2 with C4 Ancillary  Kit
			2.4 Chromogenic RNAscope Amplification and Signal Detection
			2.5 BaseScope Amplification and Signal Detection
			2.6 Fluorescent RNAscope (RNAscope Multiplex Fluorescent Reagent Kit v2) with C4 Extension
			2.7 Antibody Labeling
			2.8 EdU Staining
			2.9 Lectin Staining
		3 Methods
			3.1 Unilateral Ischemia-Reperfusion Injury Model
			3.2 Sample Collection and Fixation
			3.3 RNAscope and BaseScope Tissue Preparation and Hybridization
			3.4 Chromogenic RNAscope, Amplification, and Signal Detection
			3.5 BaseScope Amplification and Signal Detection
			3.6 Fluorescent RNAscope (RNAscope Multiplex Fluorescent Reagent Kit v2) with C4 Extension
			3.7 Antibody Staining
			3.8 EdU Staining
			3.9 Lectin Staining
			3.10 Analysis with QuPath Software
		4 Notes
		References
	Chapter 17: Spatial Transcriptomics in Kidney Tissue
		1 Introduction
		2 Materials
			2.1 Visium TO Reagents
			2.2 Visium GEx Reagents
			2.3 Additional Kits and Reagents
			2.4 Additional Consumables
			2.5 Quantification and Quality Control
			2.6 Sequencing Reagents
			2.7 Instruments and Equipment
			2.8 Buffers
		3 Methods
			3.1 Freezing of Kidney Tissue
			3.2 Cryosectioning of Kidney Tissue
			3.3 Visium TO
				3.3.1 H&E Staining
				3.3.2 Bright-Field Imaging
				3.3.3 Tissue Permeabilization
				3.3.4 Fluorescent cDNA Synthesis and Tissue Removal
			3.4 Visium TO Fluorescent Imaging
			3.5 Cryosectioning of Kidney Tissue
			3.6 Visium GEx
				3.6.1 First Strand cDNA Synthesis, Second Strand cDNA Synthesis, and Denaturation and Isolation of cDNA
				3.6.2 qPCR for Cycle Number Determination
				3.6.3 cDNA Amplification and SPRIselect (0.6x)
				3.6.4 cDNA QC and Quantification
			3.7 Visium GEx Library Preparation
				3.7.1 Fragmentation, End Repair, and A-Tailing and SPRIselect (0.55x) and (0.7x)
				3.7.2 Adaptor Ligation and SPRIselect (0.7x)
				3.7.3 Sample Index PCR and SPRIselect (0.55x) and (0.7x)
				3.7.4 Post-library Construction QC
			3.8 Visium GEx Sequencing
				3.8.1 Library Pooling
				3.8.2 Library Pool Quantification
				3.8.3 NextSeq Sequencing
			3.9 Mapping of the Transcripts to the H&E Image
		4 Notes
		References
	Chapter 18: Assessment of Kidney Mitochondrial Function by High-Resolution Respirometry, Transmission Electron Microscopy, and...
		1 Introduction
		2 Materials
			2.1 Animals and Cell Cultures
			2.2 Mitochondrial Isolation for Tissue
			2.3 High-Resolution Respirometry
			2.4 Enzymatic Activity of the Electron Transport System Complexes
			2.5 Protein Assays
			2.6 Cell Management
			2.7 Cell Respirometry
			2.8 Mitochondrial Mass Measurement
			2.9 Membrane Potential Measurement (Δψ) in Cultured Cells
			2.10 Histology
				2.10.1 General Materials
				2.10.2 Fresh Frozen Tissue Preparation
				2.10.3 Hematoxylin and Eosin (H & E) Staining
				2.10.4 Periodic Acid Schiff (PAS) Staining
				2.10.5 Oil Red O (ORO) Stain
				2.10.6 Nicotinamide Adenine Dinucleotide Tetrazolium Reductase (NADH-TR) Stain
				2.10.7 Cytochrome C Oxidase (COX) Stain
				2.10.8 Analysis of Histological Results
				2.10.9 Transmission Electron Microscopy (TEM)
		3 Methods
			3.1 Mitochondrial Isolation from Tissue
			3.2 Respirometry
				3.2.1 High-Resolution Respirometry
				3.2.2 Definition of the Steady Respiratory States
			3.3 Enzymatic Activity of the Electron Transport System Complexes
				3.3.1 Complex I (Nicotinamide Adenine Dinucleotide [NADH]: Ubiquinone Oxidoreductase) Activity
				3.3.2 Complex II (Succinate Dehydrogenase) Activity
				3.3.3 Complex III (Ubiquinol: Cytochrome C Oxidoreductase) Activity
				3.3.4 Complex IV (Cytochrome C Oxidase) Activity
			3.4 Cell-Based Analyses
				3.4.1 Cell Management
				3.4.2 Cellular Respirometry with Intact Cells
				3.4.3 Definition of the Respiratory Parameters in Intact Cells
				3.4.4 Evaluation of Complex IV (CIV) in Intact Cells
			3.5 Mitochondrial Mass Measurement
			3.6 Membrane Potential Measurement (Δψ) in Cultured Cells
			3.7 Kidney Histology, Enzyme Histochemistry, and Transmission Electron Microscopy (TEM)
				3.7.1 Fresh Frozen Tissue Section Preparation
				3.7.2 Hematoxylin and Eosin (H & E) Staining to Identify the Renal Morphology
				3.7.3 Periodic Acid Schiff (PAS) Stain to Detect Polysaccharides and Other Carbohydrate Macromolecules
				3.7.4 Oil Red O (ORO) Stain to Detect Lipid Deposition
				3.7.5 Nicotinamide Adenine Dinucleotide Tetrazolium Reductase (NADH-TR) Stain to Infer the Oxidative Metabolism of Complex I
				3.7.6 Cytochrome C Oxidase (COX) Stain to Infer the Oxidative Metabolism of Complex IV
				3.7.7 Analysis of Histochemical Results
				3.7.8 Transmission Electron Microscopy (TEM)
		4 Notes
		References
	Chapter 19: Transdermal Measurement of Glomerular Filtration Rate in Preclinical Research
		1 Introduction
		2 Materials
			2.1 Animal Preparation
			2.2 GFR Measurement
			2.3 Data Evaluation
		3 Methods
			3.1 Animal Preparation
			3.2 Preparing and Attaching the Transdermal GFR Monitor
			3.3 FITC-Sinistrin Injection
			3.4 Measurement of GFR
			3.5 Reading and Evaluation of Data
			3.6 Analysis and GFR Calculation in Standard MBlab Software
		4 Notes
		References
	Chapter 20: The BioHybrid Assay: A Novel Method for Determining Calcification Propensity
		1 Introduction
		2 Materials
			2.1 Isolation of Primary hpVSMCs
			2.2 Characterisation of hpVSMCs
			2.3 Seeding of hpVSMCs
			2.4 Calcification Induction with Patient Serum or Plasma
			2.5 Real-Time Detection of Calcification Progression
			2.6 Data Analysis
		3 Methods
			3.1 Isolation of hpVSMCs
			3.2 Culture of hpVSMCs
			3.3 Characterisation of hpVSMCs
			3.4 Calcification Induction with Patient Serum
			3.5 Real-Time Detection of Calcification Progression
			3.6 Data Analysis
		4 Notes
		References
	Chapter 21: Quantification of Calciprotein Particles (CPPs) in Serum/Plasma Samples Using a Fluorescent Bisphosphonate
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Other Supplies
			2.3 Equipment
		3 Methods
			3.1 Incubation with OsteoSense
			3.2 Preparation of Spin Columns
			3.3 Gel Filtration
			3.4 Measurement of the Fluorescence Intensity
		4 Notes
		References
	Chapter 22: Monitoring of Rho GTPase Activity in Podocytes
		1 Introduction
		2 Materials
			2.1 Preparation of GST-RBD and GST-CRIB Beads
			2.2 Pull-Down Assay
			2.3 SDS-PAGE and Immunoblotting
		3 Methods
			3.1 Preparation of GST-RBD and GST-CRIB Beads
			3.2 Pull-Down Assay
			3.3 SDS-PAGE and Immunoblotting
		4 Notes
		References
Index