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

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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

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.

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

assessment-of-kidney-function-in-mouse-models-of-glomerular-disease

Research

JoVE Journal

Medicine

17.4K Views.  University of Exeter. 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.

Video: Assessment of Kidney Function in Mouse Models of Glomerular Disease

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Biology

Transdermal Measurement of Glomerular Filtration Rate in Mice

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of acute kidney injury and chronic kidney disease. The measurement system consists of a miniaturized fluorescence detector that is directly attached to the skin on the back of conscious, freely moving animals, and measures the excretion kinetics of the exogenous GFR tracer, fluorescein-isothiocyanate (FITC) conjugated sinistrin (an inulin analog). This system has been described in detail in rats. However, because of their smaller size, measurement of transcutaneous GFR in mice presents additional technical challenges. In this paper we therefore provide the first detailed practical guide to the use of transdermal GFR monitors in mice based on the combined experience of three different investigators who have been performing this assay in mice over a number of years.

Here we describe a protocol to measure glomerular filtration rate (GFR) in conscious, freely moving mice using a transdermal GFR monitor.

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of ...

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations.
In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Fr&#246;mter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p &#60; 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 &#177; 0.002 in untreated MWFs to 0.045 &#177; 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

Here, we present a protocol using 2-photon microscopy in Munich Wistar Fromter rats with surface glomeruli to quantifythe effects of prolonged ureteral obstruction on glomerular dynamics and function.

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface ...

A Murine Model of Hemodialysis Access-Related Hand Dysfunction

Chronic kidney disease is a major public health problem, and the prevalence of end-stage renal disease (ESRD) requiring chronic renal replacement therapies such as hemodialysis continues to increase. Autogenous arteriovenous fistula (AVF) placement remains a primary vascular access option for ESRD patients. Unfortunately, approximately half of the hemodialysis patients experience dialysis access-related hand dysfunction (ARHD), ranging from subtle paresthesia to digital gangrene. Notably, the underlying biologic drivers responsible for ARHD are poorly understood, and no adequate animal model exists to elucidate the mechanisms and/or develop novel therapeutics for the prevention/treatment of ARHD. Herein, we describe a new mouse model in which an AVF is created between the left common iliac artery and vein, thereby facilitating the assessment of limb pathophysiology. The microsurgery includes vessel isolation, longitudinal venotomy, creation of arteriovenous anastomosis, and venous reconstruction. Sham surgeries include all the critical steps except for AVF creation. Iliac AVF placement results in clinically relevant alterations in central hemodynamics, peripheral ischemia, and impairments in hindlimb neuromotor performance. This novel preclinical AVF model provides a useful platform that recapitulates common neuromotor perturbations reported by hemodialysis patients, allowing researchers to investigate the mechanisms of ARHD pathophysiology and test potential therapeutics.

This protocol details the surgical steps of murine common iliac arteriovenous fistula creation. We developed this model to study hemodialysis access-related limb pathophysiology.

Chronic kidney disease is a major public health problem, and the prevalence of end-stage renal disease (ESRD) requiring chronic renal replacement ...

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of pathways involved in parietal epithelial cell activation could therefore be a tool to attenuate the progression of glomerular diseases. This article describes a method to culture and analyze parietal epithelial cell outgrowth of encapsulated glomeruli isolated from mouse kidney. After dissecting isolated mouse kidneys, the tissue is minced, and glomeruli are isolated by sieving. Encapsulated glomeruli are collected, and single glomeruli are cultured for 6 days to obtain glomerular outgrowth of parietal epithelial cells. During this period, parietal epithelial cell proliferation and migration can be analyzed by determining the cell number or the surface area of outgrowing cells. This assay can therefore be used as a tool to study the effects of an altered gene expression in transgenic- or knockout-mice or the effects of culture conditions on parietal epithelial cell growth characteristics and signaling. Using this method, important pathways involved in the process of parietal epithelial cell activation and consequently in glomerulosclerosis can be studied.

This article describes a method for culturing and analyzing glomerular parietal epithelial cell outgrowths of encapsulated glomeruli isolated from mouse kidney. This method can be used to study pathways involved in parietal epithelial cell proliferation and migration.

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...

Fluorescent Labeling of Glomeruli in Cleared Mouse Kidneys for Microscopy

An Efficient and Fast Method for Labeling and Analyzing Mouse Glomeruli

The glomeruli are fundamental units in the kidney; hence, studying the glomeruli is pivotal for understanding renal function and pathology. Biological imaging provides intuitive information; thus, it is of great significance to label and observe the glomeruli. However, the glomeruli observation methods currently in use require complicated operations, and the results may lose label details or three-dimensional (3D) information. The clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) tissue clearing technology has been widely used in renal research, allowing for more accurate detection and deeper detection depth. We found that mouse glomeruli can be rapidly and effectively labeled by tail vein injection of medium molecular weight FITC-Dextran followed by the CUBIC clearing method. The cleared mouse kidney could be scanned by a light-sheet microscope (or a confocal microscope when sliced) to obtain three-dimensional image stacks of all the glomeruli in the entire kidney. Processed with appropriate software, the glomeruli signals could be easily digitized and further analyzed to measure the number, volume, and frequency of the glomeruli.

Author Spotlight: Aiding Research in Kidney Biology by Labeling Glomeruli in Cleared Tissues 

This study presents an easy-to-use, complete, and simple set of methods to label and analyze glomeruli from CUBIC-cleared mouse kidneys. Data such as glomerulus number and volume can be obtained easily and reliably using fluorescein isothiocyanate (FITC)-Dextran, light sheet fluorescence microscopy (LSFM), or common confocal microscopy and software such as Imaris.

The glomeruli are fundamental units in the kidney; hence, studying the glomeruli is pivotal for understanding renal function and pathology. Biological ...