Name: assessment of kidney function in mouse models of glomerular disease
Uploaded: 2018-06-30T14:00:00
Duration: 9 min 16 s
Description: Watch this Scientific Journal Video about Assessment of Kidney Function in Mouse Models of Glomerular Disease at JoVE.com

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

<ol>
	<li>Humtstrom, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+structural+kidney+damage+in+spontaneously+hypertensive+rats.">Development of structural kidney damage in spontaneously hypertensive rats.</a> Journal of Hypertension. 30 (6), 1087-1091 (2012).</li><li>Kitada, M., Ogura, Y., Koya, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+diabetic+nephropathy:+their+utility+and+limitations.">Rodent models of diabetic nephropathy: their utility and limitations.</a> International Journal of Nephrology and Renovascular Disease. 14 (9), 279-290 (2016).</li><li>Tesch, G. H., Lim, A. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+FSGS:+lessons+for+pathogenesis+and+treatment.">Animal models of FSGS: lessons for pathogenesis and treatment.</a> Seminars in Nephrology. 23 (2), 161-171 (2003).</li><li>Stevens, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-A165b+protects+against+proteinuria+in+a+mouse+model+with+progressive+depletion+of+all+endogenous+VEGF-A+splice+isoforms+from+the+kidney.">VEGF-A165b protects against proteinuria in a mouse model with progressive depletion of all endogenous VEGF-A splice isoforms from the kidney.</a> Journal of Physiology. 595 (19), 6281-6298 (2017).</li><li>Cosgrove, D., Kalluri, R., Miner, J. H., Segal, Y., Borza, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choosing+a+mouse+model+to+study+the+molecular+pathobiology+of+Alport+glomerulonephritis.">Choosing a mouse model to study the molecular pathobiology of Alport glomerulonephritis.</a> Kindey International. 71 (7), 615-618 (2007).</li><li>Kujal, P., Vernerova, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5/6+nephrectomy+as+an+experimental+model+of+chronic+renal+failure+and+adaptation+to+reduced+nephron+number.">5/6 nephrectomy as an experimental model of chronic renal failure and adaptation to reduced nephron number.</a> Ceskoslenska Fysiologuie. 57 (4), 104-109 (2008).</li><li>Chevalier, R. L., Forbes, M. S., Thornhill, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ureteral+obstruction+as+a+model+of+renal+interstitial+fibrosis+and+obstructive+nephropathy.">Ureteral obstruction as a model of renal interstitial fibrosis and obstructive nephropathy.</a> Kidney International. 75 (11), 1145-1152 (2009).</li><li>Oltean, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF165b+overexpression+restores+normal+glomerular+water+permeability+in+VEGF164-overexpressing+adult+mice.">VEGF165b overexpression restores normal glomerular water permeability in VEGF164-overexpressing adult mice.</a> American Journal of Physiology-Renal Physiology. 303 (7), F1026-F1036 (2012).</li><li>Salmon, A. H., Neal, C. R., Bates, D. O., Harper, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+growth+factor+increases+the+ultrafiltration+coefficient+in+isolated+intact+Wistar+rat+glomeruli.">Vascular endothelial growth factor increases the ultrafiltration coefficient in isolated intact Wistar rat glomeruli.</a> Journal of Physiology. 570 (Pt 1), 141-156 (2006).</li><li>Zheng, F., Striker, G. E., Esposito, C., Lupia, E., Striker, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain+differences+rather+than+hyperglycemia+determine+the+severity+of+glomerulosclerosis+in+mice.">Strain differences rather than hyperglycemia determine the severity of glomerulosclerosis in mice.</a> Kidney International. 54 (6), 1999-2007 (1998).</li><li>Betz, B., Conway, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+update+on+the+use+of+animal+models+in+diabetic+nephropathy+research.">An update on the use of animal models in diabetic nephropathy research.</a> Current Diabetes Reports. 16 (18), (2016).</li><li>Savin, V. J., Terreros, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filtration+in+single+isolated+mammalian+glomeruli.">Filtration in single isolated mammalian glomeruli.</a> Kidney International. 20 (2), 188-197 (1981).</li><li>Neal, C. R., Crook, H., Bell, E., Harper, S. J., Bates, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+glomeruli+by+electron+microscopy+reveals+a+distinct+restrictive+urinary+subpodocyte+space.">Three-dimensional reconstruction of glomeruli by electron microscopy reveals a distinct restrictive urinary subpodocyte space.</a> Journal of the American Society of Nephrology. 16 (5), 1223-1235 (2005).</li><li>Mahmood, T., Yang, P. 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T., Liu, D. L., Deng, J. J., Niu, R., Liu, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+arctiin+on+glomerular+filtration+barrier+damage+in+STZ-induced+diabetic+nephropathy+rats.">Effect of arctiin on glomerular filtration barrier damage in STZ-induced diabetic nephropathy rats.</a> Phytotherapy Research. 27 (10), 1474-1480 (2013).</li><li>Oltean, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+growth+factor-A165b+is+protective+and+restores+endothelial+glycocalyx+in+diabetic+nephropathy.">Vascular endothelial growth factor-A165b is protective and restores endothelial glycocalyx in diabetic nephropathy.</a> Journal of the American Society of Nephrology. 26 (8), 1889-1904 (2015).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Podocyte-specific+overexpression+of+GLUT1+surprisingly+reduces+mesangial+matric+expansion+in+diabetic+nephropathy+in+mice.">Podocyte-specific overexpression of GLUT1 surprisingly reduces mesangial matric expansion in diabetic nephropathy in mice.</a> American Journal of Physiology-Renal Physiology. 299 (1), F91-F98 (2010).</li></ol>

The authors have nothing to disclose.

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 LpA/Vi), examination of the structural alterations (PAS, Trichrome blue, and EM), protein localization (IF), and glomerular gene expression (RT-PCR and Western blotting). These methods are key to the full assessment of glomerular function in mouse models of renal disease.
When assessing the permeability of the GFB, many studies have opted to use the conventional uACR or 24 h albumin excretion rate as an effective measure17,18. Although these techniques allow assessment of the GFB permeability as a whole, it does not allow for individual glomerular permeability assessment and variation amongst glomeruli. Previous studies have found measurement of the glomerular LpA/Vi to be a more sensitive measure of changes to the GFB permeability5,9. Indeed, in the representative results demonstrated in this paper, at 14 weeks post induction of VEGF-A KO, VEGF-A KO X Neph-VEGF-A165b mice have a significantly lower uACR compared to VEGF-A KO mice; however, this result is not reflected in the glomerular LpA/Vi measurements, where VEGF-A165b did not significantly prevent increases in the GFB permeability (Figure 1 and Figure 2)5. This shows the importance of using multiple assays to assess both the kidney permeability and the permeability of individual glomeruli. Furthermore, the glomerular LpA/Vi oncometric assay suggests that the permeability of individual glomeruli from the same kidney can vary greatly, especially in disease models5,10,19. One limitation to measuring the glomerular LpA/Vi is that it can only be performed at the experimental end-point; thus, regular uACR measurements are required to give an indication of the experimental end-point.
In addition to assessing the functional phenotype, the present method also encourages assessment of the structural and ultrastructural phenotype. This can be done using a selection of stains such as PAS, trichrome, and silver stains; each to assess different aspects of the glomerular morphology. In acute models of glomerular disease, which is often the case in mouse models, is can be difficult to detect any major structural abnormalities using these stains unless you are a trained renal pathologist. Therefore, carrying out EM is suggested to assess the ultrastructure of the GFB, which allows the quantitative measurement of parameters such as the GBM, endothelial fenestrae size and number, and podocyte characteristics. Such measurements require minimal training to perform and enables the investigator to determine the cell-types/structures affected in a disease model. In the example shown in the representative results, the VEGF-A KO mouse was found to be a mild model of glomerular disease, thus, no major structural abnormalities were present upon PAS staining. However, podocyte-specific VEGF-A KO did induce changes to the GBM, podocytes, and endothelial cells when examining the glomerular ultra-structure5. Unfortunately, the preparation of the kidney for EM described in the present method does not enable detection of the endothelial glycocalyx, which is also known to have significant effects on the permeability of the GFB19. In order to accurately measure the glycocalyx depth, the kidney should be perfuse-fixed with 2.5% glutaraldehyde with 1% Alcian blue for endothelial glycocalyx labelling, as described in Oltean et al19.
Once the functional and structural phenotype have been assessed, the expression/activation patterns of different genes and pathways can then be assessed specifically in the glomeruli. Prior ultra-structural assessment could give some information regarding the cell types/glomerular structures involved, indicating whether podocyte- or endothelial-specific genes/pathways should be examined. For example, in the representative results from the VEGF-A KO mice, a reduction in the endothelial fenestrae number was observed (Figure 3D); therefore, the glomerular protein expression of an endothelial marker known to be involved in the VEGF-A pathway was examined; VEGFR-2 (Figure 4B)5. In addition to the expression of proteins in the glomeruli, their localization can also be visualized using IF. In a study by Zhang et al20, podocyte-specific overexpression of GLUT1 was confirmed in the podocytes by IF co-localizing the increased GLUT1 with podocin.
In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper to assess kidney function in mouse models of glomerular disease enables the glomerular phenotype to be fully evaluated from multiple aspects. 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 vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. This method can be easily applied to future investigations into glomerular function both in the assessment of disease phenotypes and potential therapeutics.
In conclusion, this generic and adaptable protocol describes a full kidney work-up for 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.

The use of murine models to mimic human kidney disease is becoming increasingly common. Such murine models include spontaneous models such as spontaneously hypertensive rats (SHR)1, streptozotocin (STZ)-induced diabetic rats and mice2, and the db/db type II diabetic mice3, 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 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 is 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 is analyzed. 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 are isolated from the mouse kidney cortex, and then the protein/RNA are 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>
	<tbody>
		<tr>
			<td>Metabolic Cages</td>
			<td>Harvard Apparatus</td>
			<td>52-6731</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris buffered saline (10x)</td>
			<td>Sigma-Aldrich</td>
			<td>T5912-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A2058</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Albumin ELISA Quantitation Set</td>
			<td>Bethyl Laboratories</td>
			<td>E90-134</td>
			<td></td>
		</tr>
		<tr>
			<td>TMB ELISA Substrate solution</td>
			<td>ThermoFisher Scientific</td>
			<td>34028</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulphuric acid</td>
			<td>Sigma-Aldrich</td>
			<td>339741</td>
			<td></td>
		</tr>
		<tr>
			<td>SPECTRstar nano</td>
			<td>BMG Labtech</td>
			<td>SPECTRstar nano or equivalent</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAlater stabilisation solution</td>
			<td>ThermoFisher Scientific</td>
			<td>AM7020</td>
			<td></td>
		</tr>
		<tr>
			<td>4-15% precast protein gel</td>
			<td>BIORAD</td>
			<td>4568084</td>
			<td></td>
		</tr>
		<tr>
			<td>4x Laaemmli Sample buffer</td>
			<td>BIORAD</td>
			<td>161-0747</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Trans-Blot cell</td>
			<td>BIORAD</td>
			<td>1703930</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Tris running buffer</td>
			<td>BIORAD</td>
			<td>1610732</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue Dye</td>
			<td>ThermoFisher Scientific</td>
			<td>20278</td>
			<td></td>
		</tr>
		<tr>
			<td>Creatinine Companion</td>
			<td>Exocell</td>
			<td>1012 Strip Plate</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde Solution</td>
			<td>Sigma-Aldrich</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Cacodylate</td>
			<td>Sigma-Aldrich</td>
			<td>C0250</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Phosphate Buffered Saline</td>
			<td>ThermoFisher Scientific</td>
			<td>AM9625</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>342483</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M2643</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C5670</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA Blood Collection tubes</td>
			<td>BD</td>
			<td>367835</td>
			<td></td>
		</tr>
		<tr>
			<td>23-25G Needle</td>
			<td>BD</td>
			<td>PMC0735</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml Glass Vial</td>
			<td>Thomas Scientific</td>
			<td>0914X10</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 10 ml Polypropylene Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>10110101</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 ml Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>10681894</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Tissue Molds</td>
			<td>ThermoFisher Scientific</td>
			<td>22-363-553</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Surgical Disection Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>13-005-204</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimal Cutting Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>23-730-571</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>ThermoFisher Scientific</td>
			<td>AAJ19943K2</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillary Tubes</td>
			<td>Harvard Apparatus</td>
			<td>EC1 64-0770</td>
			<td></td>
		</tr>
		<tr>
			<td>Glomerular Permeability Rig</td>
			<td>Built at the Univeristy of Bristol - not comercially available</td>
			<td>Citation of LpA rig: Salmon et al. 2006; J. Physiol</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Sieves</td>
			<td>Cole-Parmer</td>
			<td>WZ-59984</td>
			<td></td>
		</tr>
		<tr>
			<td>Periodic Acid-Schiff (PAS) Staining System</td>
			<td>Sigma-Aldrich</td>
			<td>395B-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Sigma-Aldrich</td>
			<td>H3136</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>MerckMillipore</td>
			<td>108298</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-Prep Slides</td>
			<td>Sigma-Aldrich</td>
			<td>P0425-72EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>8030</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide solution</td>
			<td>Sigma-Aldrich</td>
			<td>75632</td>
			<td></td>
		</tr>
		<tr>
			<td>Aradite Resin</td>
			<td>Agar Scientific</td>
			<td>CY212</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl Acetate</td>
			<td>Agar Scientific</td>
			<td>AGR1260A</td>
			<td></td>
		</tr>
		<tr>
			<td>Lead Citrate</td>
			<td>Agar Scientific</td>
			<td>AGR1210</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>ThermoFisher Scientific</td>
			<td>e.g. 957000H</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic Pen</td>
			<td>Abcam</td>
			<td>ab2601</td>
			<td></td>
		</tr>
		<tr>
			<td>Nephrin (1243-1256) Antibody</td>
			<td>Acris</td>
			<td>BP5030</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Podocin</td>
			<td>Sigma-Aldrich</td>
			<td>P0372-200UL</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD31</td>
			<td>BD Biosciences</td>
			<td>550274</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor Secondary Antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>A32732</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield Mounting Medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>NP40 Cell Lysis Buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>FNN0021</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease and Phosphatase Inhibitor Cocktail</td>
			<td>ThermoFisher Scientific</td>
			<td>78437X4</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Transfer Buffer</td>
			<td>BIORAD</td>
			<td>1610734</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF Membrane</td>
			<td>ThermoFisher Scientific</td>
			<td>LC2002</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP-Conjugated Secondary Antibodies</td>
			<td>Abcam</td>
			<td>ab6721</td>
			<td></td>
		</tr>
		<tr>
			<td>ECF Substrate for Western Blotting</td>
			<td>Fisher</td>
			<td>10713387</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol</td>
			<td>ThermoFisher Scientific</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>New England Biolabs</td>
			<td>M0303S</td>
			<td></td>
		</tr>
		<tr>
			<td>M-MLV Reverse Transcriptase</td>
			<td>New England Biolabs</td>
			<td>M053S</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo dT</td>
			<td>ThermoFisher Scientific</td>
			<td>18418012</td>
			<td></td>
		</tr>
		<tr>
			<td>Random Primers</td>
			<td>ThermoFisher Scientific</td>
			<td>48190011</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP</td>
			<td>ThermoFisher Scientific</td>
			<td>18427088</td>
			<td></td>
		</tr>
		<tr>
			<td>Ribonuclease Inhibitor</td>
			<td>ThermoFisher Scientific</td>
			<td>10777019</td>
			<td></td>
		</tr>
		<tr>
			<td>DEPC Water</td>
			<td>ThermoFisher Scientific</td>
			<td>AM9915G</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent Light Miscroscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image J Analysis Software</td>
			<td>Image J</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Thermocycler</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TEM Microscope</td>
			<td>Britannica</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of kidney function in mouse models of glomerular disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these 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.

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-A165b isoform in the podocytes in a constitutive manner). Upon measurement of the urinary albumin creatinine ratio at weeks 0, 4, 10, and 14 after doxycycline induction of VEGF-A KO, VEGF-A KO mice developed progressive albuminuria by 10 weeks compared to WT littermate controls. The absolute values can be seen in Figure 2A, and the normalized to the baseline of each mouse values in Figure 2B. However, albuminuria in not observed in the VEGF-A X Neph-VEGF-A165b mice (Figure 2), indicating that VEGF-A165b is protective in the model of albuminuria5.
The glomerular LpAVi was measured in individual glomeruli sieved from WT, VEGF-A KO, and VEGF-A X Neph-VEGF-A165b kidneys. An example of how a glomerulus is caught and the shrinkage observed when perifused with 8% BSA is shown in Figure 3A. This shrinkage is then used to determine the glomerular LpA/Vi for each glomerulus (Figure 3B). VEGF-A KO mice had a significantly increased glomerular LpA/Vi at 14 weeks post VEGF-KO induction compared to WT control glomeruli. Although lower in VEGF-A X Neph-VEGF-A165b mice, the increased glomerular LpA/Vi was not prevented by over-expression of VEGF-A165b at 14 weeks5.
PAS staining of kidney cortex sections 14 weeks after induction of VEGF-A KO did not reveal any glomerular structural abnormalities in the VEGF-A KO or VEGF-A X Neph-VEGF-A165b mice (Figure 4A). However, upon analysis of the glomerular ultra-structure via EM, VEGF-A KO mice had developed an increased GBM width, decreased number of endothelial fenestrae, decreased SPS coverage, and increased average podocyte slit width (Figure 4C-4F). The average podocyte foot process width and number of slits remained unchanged (Figure 3B and 3G). Over-expression of VEGF-A165b in the VEGF-A KO mice prevented the changes to the GBM and slit width (Figure 4C and 4F). However, VEGF-A165b had no effect on the altered fenestrae number and SPS coverage (Figure 4D and 4E)5.
RT-PCR performed on RNA extracted from sieved glomeruli revealed that the human VEGF-A165b mRNA is only present in the VEGF-A KO X Neph-VEGF-A165b mice (Figure 5A). When extracting protein from sieved glomeruli and assessing the levels of proteins via Western blotting, the glomerular protein expression of VEGFR-2 was found to be decreased in VEGF-A KO mice, which was prevented by over-expression of VEGF-A165b (Figure 5B and 5C)5.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57764/57764fig1.jpg" /> 
Figure 1. Diagrammatic set up of the glomerular permeability (LpA) rig. (A) The glomerulus is caught on (B) the micropipette within a holder using suction, which is clamped onto a mount for stability. (C) Rectangular microslide. (D)&#160;4X&#160;objective of a microscope with a video camera. (E) 1% BSA solution warmed to 37 &#176;C. (F) 8% BSA solution warmed to 37 &#176;C. (G) Remote tap bearing two perifusate-containing lines, which permits rapid perifusate exchange. (H) Route of perifusate towards the microslide, which then bathes the glomerulus. <a href="https://www.jove.com/files/ftp_upload/57764/57764fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57764/57764fig2.jpg" /> 
Figure 2. Urinary albumin creatinine ratio. (A) The uACR values at weeks 0, 4, 10, and 14 after induction of VEGF-A KO in WT, VEGF-A KO, and VEGF-A X Neph-VEGF-A165b mice. (B) The same uACR values normalized to the baseline value (week 0) of each individual mouse. The uACR significantly increased in VEGF-A KO mice at weeks 10 and 14 compared to WT littermate controls, which was prevented in the VEGF-A X Neph-VEGF-A165b mice (*p &#60;0.05; Two-way ANOVA, correction for comparison between pairs; n= 4 - 12 mice per time point; error bars: standard error of the mean [SEM]). This figure has been modified from Stevens et al5. <a href="https://www.jove.com/files/ftp_upload/57764/57764fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57764/57764fig3.jpg" /> 
Figure 3. Measurement of glomerular water permeability. (A) The glomerulus is caught on the micropipette via suction; the perifusate is switched from 1% BSA (Ai) to 8% BSA (Aii), and glomerular shrinkage is observed. (B) Measurements taken before and after the 8% BSA switch are used to determine the glomerular LpA/Vi. (C) VEGF-A KO mice develop an increased glomerular LpA/Vi at 14 weeks post induction of VEGF-A KO compared to WT controls. This was not significantly prevented in VEGF-A X Neph-VEGF-A165b mice (*p &#60;0.05; One way ANOVA, Bonferroni correction for comparison between pairs; n = 4 - 9 mice, 15 - 30 glomeruli; error bars: SEM). <a href="https://www.jove.com/files/ftp_upload/57764/57764fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57764/57764fig4.jpg" /> 
Figure 4. Glomerular structural analysis. (A) PAS staining of the kidney cortex did not indicate any structural abnormalities in the glomeruli from WT, VEGF-A KO, and VEGF-A X Neph-VEGF-A165b mice (Ai - iii). EM revealed ultra-structural abnormalities in the VEGF-A KO glomeruli (Aiv - vi). (B) The average FPW did not change between groups. (C) The GBM increased in the VEGF-A KO glomeruli, which was prevented by VEGF-A165b. (D) The number of fenestrae was decreased in VEGF-A KO glomeruli, which remained unaltered by VEGF-A165b. (E) The SPS coverage was decreased in VEGF-A KO glomeruli, which also remained unchanged by VEGF-A165b. (F) The average slit width was increased in VEGF-A KO glomeruli, which was prevented by VEGF-A165b. (G) The slit number was unchanged between the three groups (*p &#60;0.05; One way ANOVA, Bonferroni correction for comparison between pairs; n = 3 mice, 9 glomeruli; error bars: SEM). This figure has been modified from Stevens et al5. <a href="https://www.jove.com/files/ftp_upload/57764/57764fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57764/57764fig5.jpg" /> 
Figure 5. mRNA and protein expression of markers. (A) RT-PCR showed that human VEGF-A165b mRNA expression was only evident in the sieved glomeruli from VEGF-A KO X Neph-VEGF-A165b mice. (B) Western blotting indicated that VEGFR-2 protein expression was down-regulated in VEGF-A KO glomeruli, which was prevented in VEGF-A KO X Neph-VEGF-A165b glomeruli (*p &#60;0.05; One way ANOVA, Bonferroni correction for comparison between pairs; n = 3-&#160; 6 mice; error bars: SEM). This figure has been modified from Stevens et al5. <a href="https://www.jove.com/files/ftp_upload/57764/57764fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Assessment of Kidney Function in Mouse Models of Glomerular Disease at JoVE.com

Assessment of Kidney Function in Mouse Models of Glomerular Disease (Video) | JoVE

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.

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

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