Name: techniques to assess kidney function in mouse models of glomerular disease
Uploaded: 2017-12-18T16:05:27
Description: 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 ...

This work was supported by the British Heart Foundation, Richard Bright VEGF Research Trust and the MRC.

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)
<ol>
	<li>Urine should be collected at baseline and at regular intervals.</li>
	<li>Set up mouse metabolic cages with water and enrichment diet. Place mice in individual c...

<ol>
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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 Lp</s...

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) acquired models such as the 5/6 nephrectomy7 and the unilateral ureteral obstruction (UUO) model8. In order to assess the different aspects of glomerular function in these models, several techniques are available. The purpose of this method paper is to demonstrate a comprehensive work-up that should be performed in mouse models of kidney disease in order to fully assess glomerular function.
The rationale behind the use of this method is that it enables the glomerular phenotype to be fully evaluated from multiple aspects. This includes assessing the glomerular permeability, both to protein and to water, the glomerular structural abnormalities, and changes in the expression/splicing of mRNAs and proteins essential for normal glomerular function. By using this method, the researcher is able to determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This is vital information on the mechanism of disease, which is required when examining potential therapeutic avenues in these models.
In the literature, it is a common occurrence to be presented with a mouse model of glomerular disease where the phenotype is determined by an increased level of albumin in the urine. However, there is evidence to suggest that a single method to determine glomerular function is not always effective; measuring the urinary albumin excretion rate or the urinary albumin creatinine ratio (uACR) only provides information on total renal function, and not of the individual glomeruli. Previous studies have demonstrated that the permeability can vary in different glomeruli from the same kidney5,9,10. In addition, assessment of the permeability of individual glomeruli is a more sensitive way of assessing glomerular function; the technique of measuring the individual glomerular water permeability (LpA/Vi) has shown to be more sensitive to changes in glomerular function than measuring the uACR9. This assay would be beneficial in mouse models that are resistant to proteinuria, such as those on a c57BL/6 background11. The advantage of the present method paper is that it examines both the total renal permeability to albumin as well as the individual glomerular permeability to water.
Examination of glomerular structural abnormalities is often assessed by a battery of stains such as Periodic Acid Schiff (PAS), trichrome, and silver stains. These enable a trained renal pathologist to evaluate the level of renal disease via a scoring method. Although all good methods, changes to the glomerular macro-structure are not always observed in acute kidney injury models12. This method proposes that in addition to carrying out the renal histology techniques described above, the glomerular ultra-structure should also be assessed via electron microscopy (EM). A stained glomerulus can look relatively normal under a regular light microscope; however, upon assessment with EM, small changes in the glomerular basement membrane (GBM) width, podocyte foot process effacement, endothelial fenestrations, and the sub-podocyte space coverage can be analysed. Therefore, it is vital that both the glomerular ultra-structure and micro-structure is assessed to determine the mechanism of glomerular dysfunction.
In addition to assessing glomerular structural abnormalities, changes in mRNA and protein expression and splicing, as well as protein activation (e.g phosphorylation), should be examined to further elucidate the mechanisms of glomerular disease. When looking at glomerular disease, or, for example, when KO/over-expressing a gene specifically in glomerular cells, such as in the podocyte-specific VEGF-A KO mouse5, it is important that the protein and mRNA changes are examined only within the glomerular cells, and not the whole kidney. This protocol describes a method in which the glomeruli can be isolated from the mouse kidney cortex, and then the protein/RNA isolated. This allows specific analysis of the protein/mRNA dysregulation in the glomeruli of the disease model.
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 methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

<table border="0" cellpadding="0" cellspacing="0" style="width:707px;" width="707">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Metabolic Cages</td>
			<td style="width:133px;">Harvard Apparatus</td>
			<td style="width:136px;">52-6731</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:247px;">Mouse Albumin ELISA Quantitation Set</td>
			<td style="width:133px;">Bethyl Laboratories</td>
			<td style="width:136px;">E90-134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Creatinine Companion</td>
			<td style="width:133px;">Exocell</td>
			<td style="width:136px;">1012 Strip Plate</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Glass Capillary Tubes</td>
			<td style="width:133px;">Harvard Apparatus</td>
			<td style="width:136px;">EC1 64-0770</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:247px;">Glomerular Permeability Rig</td>
			<td style="width:133px;"></td>
			<td style="width:136px;"></td>
			<td style="width:191px;">Built at the Univeristy of Bristol - not comercially available</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">100 &#956;m Stainless Steel Sieve</td>
			<td style="width:133px;">Cole-Parmer</td>
			<td style="width:136px;">WZ-59984-18</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">70 &#956;m Stainless Steel Sieve</td>
			<td style="width:133px;">Cole-Parmer</td>
			<td style="width:136px;">WZ-59984-21</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Periodic Acid-Schiff (PAS) Staining System</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:136px;">395B-1KT</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Hematoxylin</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:136px;">H3136</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Poly-Prep Slides</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:136px;">P0425-72EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Nephrin (1243-1256) Antibody</td>
			<td style="width:133px;">Acris</td>
			<td style="width:136px;">BP5030</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Anti-Podocin</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:136px;">P0372-200UL</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:247px;">Anti-CD31</td>
			<td style="width:133px;">BD Biosciences</td>
			<td align="right" style="width:136px;">550274</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:247px;">NP40 Cell Lysis Buffer</td>
			<td style="width:133px;">ThermoFisher Scientific</td>
			<td style="width:136px;">FNN0021</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:247px;">Halt Protease and Phosphatase Inhibitor Cocktail</td>
			<td style="width:133px;">ThermoFisher Scientific</td>
			<td style="width:136px;">78437X4</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:247px;">TRIzol</td>
			<td style="width:133px;">ThermoFisher Scientific</td>
			<td align="right" style="width:136px;">15596018</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:247px;">Dnase I</td>
			<td style="width:133px;">New England Biolabs</td>
			<td style="width:136px;">M0303S</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:247px;">M-MLV Reverse Transcriptase</td>
			<td style="width:133px;">New England Biolabs</td>
			<td style="width:136px;">M053S</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Bovine Serum Albumin</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:136px;">A2058</td>
			<td></td>
		</tr>
	</tbody>
</table>

techniques to assess kidney function in mouse models of glomerular disease

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.

Progressive depletion of podocyte VEGF-A results in albuminuria, which is rescued by the constitutive expression of the human VEGF-A165b splice isoform
Urine was collected using metabolic cages from wild type (WT), inducible podocyte-specific VEGF-A knock out (VEGF-A KO), and VEGF-A KO X Neph-VEGF165b mice (VEGF-A KO mice that over-express the human VEGF-A<...

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.

Techniques to Assess Kidney Function in Mouse Models of Glomerular Disease

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 ...