Techniques to Assess Kidney Function in Mouse Models of Glomerular Disease

Summary

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

Abstract

The use of murine models to mimic human kidney disease is becoming increasingly common. This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

Introduction

The use of murine models to mimic human kidney disease is becoming increasingly common. Such murine models include 1) spontaneous models such as spontaneously hypertensive rats (SHR)¹, streptozotocin (STZ)-induced diabetic rats and mice², and the db/db type II diabetic mice³, 2) genetically engineered models such as primary podocyte-specific focal segmental glomerular sclerosis (FSGS) models⁴, the podocyte-specific vascular endothelial growth factor A (VEGF-A) knock-out (VEGF-A KO) model⁵, and Alport syndrome models⁶, and 3) a....

Protocol

All experiments were conducted in accordance with UK legislation and local ethical committee approval. Animal studies were approved by University of Bristol research ethics committee.

Assessment of glomerular phenotype in mouse models of glomerular injury

1. Urinary albumin creatinine ratio (uACR)

1. Urine should be collected at baseline and at regular intervals.
2. Set up mouse metabolic cages with water and enrichment diet. Place mice in individual c.......

Discussion

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. The critical steps in each method allow for detailed functional, structural, and mechanistic analysis of glomerular function, including assessment of the permeability of the kidneys as a whole (uACR and plasma creatinine measurements), the permeability of individual glomeruli (glomerular L_p

Materials

Name	Company	Catalog Number	Comments
Metabolic Cages	Harvard Apparatus	52-6731
Mouse Albumin ELISA Quantitation Set	Bethyl Laboratories	E90-134
Creatinine Companion	Exocell	1012 Strip Plate
Glass Capillary Tubes	Harvard Apparatus	EC1 64-0770
Glomerular Permeability Rig			Built at the Univeristy of Bristol - not comercially available
100 μm Stainless Steel Sieve	Cole-Parmer	WZ-59984-18
70 μm Stainless Steel Sieve	Cole-Parmer	WZ-59984-21
Periodic Acid-Schiff (PAS) Staining System	Sigma-Aldrich	395B-1KT
Hematoxylin	Sigma-Aldrich	H3136
Poly-Prep Slides	Sigma-Aldrich	P0425-72EA
Nephrin (1243-1256) Antibody	Acris	BP5030
Anti-Podocin	Sigma-Aldrich	P0372-200UL
Anti-CD31	BD Biosciences	550274
NP40 Cell Lysis Buffer	ThermoFisher Scientific	FNN0021
Halt Protease and Phosphatase Inhibitor Cocktail	ThermoFisher Scientific	78437X4
TRIzol	ThermoFisher Scientific	15596018
Dnase I	New England Biolabs	M0303S
M-MLV Reverse Transcriptase	New England Biolabs	M053S
Bovine Serum Albumin	Sigma-Aldrich	A2058