Assessment of Kidney Function in Mouse Models of Glomerular Disease

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.

1. Urinary Albumin Creatinine Ratio (uACR)

NOTE: The uACR is used to assess the permeability of the GFB to albumin. The presence of albumin in the urine indicates increased permeability across the GFB, which is normalized to creatinine to control for variations in urine flow rate. Albuminuria is a common marker for chronic kidney disease.

1. Collect urine at the experimental baseline and at regular intervals (once weekly to once monthly) up until the experimental endpoint.
2. Set up mouse metabolic cages with water and enrichment diet. Place mice (male, aged 6 – 8 weeks) in individual cages for 6 h in a quiet room.
3. Return mice to regular housing and collect urine from the empty cages. A minimum of 50 µL is required.
   NOTE: If the mouse does not produce urine in the given time, repeat on another day in a warmer room.
4. Centrifuge the urine at 500 x g for 10 min. Collect the urine and retain sediment to assess podocyte loss. Store the urine at -20 °C in the short-term at this point.
5. Dilute the urine into 1% bovine serum albumin (BSA) in 1x Tris buffered saline (TBS pH 7.5) at a 1:500 to 1:10000 dilution, depending on the severity of the albuminuria.
   NOTE: The end volume should be >400 µL. Optimize to determine the right dilution at each time point.
6. Quantify the urinary albumin concentration using a mouse albumin enzyme linked immunosorbent assay (ELISA) per the manufacturer's instructions.
   1. Briefly, coat the ELISA plate, which is optimized for protein binding, with 100 µL of the anti-mouse albumin primary antibody (10 µg/mL in 0.5 M carbonate-bicarbonate pH 9.6) for 1 h at room temperature.
   2. Wash excess antibody from the wells five times with 1x TBS plus 0.05% Tween (pH 8.0), and add 200 µL of blocking solution (1x TBS with 1% BSA pH 8.0) overnight at room temperature.
7. Wash plate five times and add 100 µL of the standards (serial dilution: 2000 µg/mL to 15.63 µg/mL in 1x TBS with 1% BSA, pH 8.0), the blank, and the diluted samples in triplicate. Leave for 1 h at room temperature then wash plate five times.
   1. Add 100 µL of HRP detection antibody to each well (10 ng/mL in 1xTBS with 1% BSA, pH 8.0) and incubate at room temperature for 1 h.
   2. Wash the plate five times and add 100 µL of enzyme substrate solution to each well. Leave the plate in the dark to develop for 15 min and stop the reaction by adding 100 µL of 0.18 M sulfuric acid (H₂SO₄).
      CAUTION: H₂SO₄ is corrosive.
8. Determine the albumin concentration of each sample by reading the plate at an absorbance of 450 nm. Use the standard curve to quantify the albumin present in each sample. If the technical repeats have a CV value greater than 5%, repeat the assay for those samples.
9. Alternatively, assess the urinary albumin concentration using electrophoresis. Add 5 µL of 4x protein sample loading buffer to 15 µL of urine. Heat samples to 95 °C for 10 min and then load into a 4 – 12% Tris precast gel.
   1. Run the gel at 100 V and stain overnight in Coomassie blue per manufacturer's protocol.
   2. After imaging the gel, use densitometry to assess fold change albumin concentration relative to control.
      NOTE: If no bands are observed for albumin when expected, assess the protein concentration of the urine and adjust the volume of urine added to the gel.
10. Dilute the raw urine sample in dH₂O at 1:1, 1:5, and 1:10.
    NOTE: The end volume should be >70 µL. Optimize to determine the right dilution of each sample.
11. Quantify the urinary creatinine concentration using a chemical assay per the kit instructions. Briefly, load 20 µL of creatinine standards, blank, or diluted urine to a 96 well plate in triplicate.
12. Determine the creatinine concentration of each sample by reading the plate at an absorbance of 490 nm before and after the addition of the acid solution (CAUTION, corrosive; avoid contact with skin).
    NOTE: The difference between the absorbance values is directly proportional to the creatinine concentration in each sample. A standard curve is generation from the standards. If the technical repeats have a CV value greater than 5%, repeat the assay for those samples.
13. Generate the uACR (µg/mg). Normalize the data to the baseline value of each mouse for graphical representation.

2. Tissue and Blood Collection

NOTE: The kidney and glomerular tissue can be used to assess structural, protein, and mRNA expression markers of renal disease. The blood can be used to assess markers of renal function, such as creatinine, which can be up-regulated in renal disease, indicating a reduction in the filtration capacity of the glomeruli.

1. Prepare the following solutions: fresh 2.5% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.3), 4% paraformaldehyde in 1x phosphate buffered saline (PBS), mammalian Ringer's solution (115 mM sodium chloride (NaCl), 10 mM sodium acetate (CH₃COONa), 1.2 mM sodium phosphate (Na₂HPO₄), 25 MM sodium bicarbonate (NaHCO₃), 1.2 mM magnesium sulfate (MgSO₄), 1 mM calcium chloride (CaCl₂), 5.5 mM D(+)glucose, pH 7.4) with 1% BSA, and 1x PBS.
   CAUTION: 2.5% glutaraldehyde: toxic, sensitizer, irritant; use in a fume cabinet. 0.1 M sodium cacodylate: toxic, use in a fume cabinet. 4% PFA: fixative, use in fume cabinet
2. Prepare the following materials: isoflurane, small ethylenediaminetetraacetic acid (EDTA)-coated blood tubes, 23 – 25G needles, 5 mL EDTA coated syringes, 10 mL glass vials, 10 mL plastic vials, 0.5 mL plastic tubes, disposable tissue molds, dry ice, liquid N₂, mouse surgical tools, and optimal cutting medium (OCT).
3. Place the mouse under deep anesthesia, which is verified by the mouse being non-responsive to a needle prick to the foot pad, using an isoflurane chamber, or equivalent routes of terminal anesthesia such as injectable anesthetic agents (pentobarbital, 50 mg/kg intraperitoneal [IP]; avertin, 240 mg/kg IP), or carbon dioxide (CO₂) exposure (75% CO₂/25% O₂).
4. Cull mouse via cardiac puncture into the left ventricle and collect as much blood as possible. Transfer to the EDTA-coated blood tube for up to 4 h. If preferred, mice can be culled via cervical dislocation with care taken not to rupture the jugular vein.
5. Dissect out the kidneys through the abdomen and wash in ice cold 1x PBS.
6. To examine the cortical glomeruli, remove one pole of kidney cortex and cut into 1 mm³ pieces. To examine the deep juxta-medullary glomeruli, repeat the same technique with tissue from the medulla. Place in 5 mL of 2.5% glutaraldehyde solution in a glass EM vial. Store at 4 °C.
   CAUTION: 2.5% glutaraldehyde solution: toxic, sensitizer, irritant; use in a fume cabinet
   NOTE: process within 1 month for best results.
7. For histology, remove the upper third of a kidney, to ensure both cortical and juxta-medullary glomeruli will be present, and fix in 5 mL of 4% paraformaldehyde at 4 °C for 24 h. Transfer to 5 mL of 70% EtOH for 24 hours before embedding in paraffin.
   CAUTION: 4% PFA: fixative, use in fume cabinet
8. For immunofluorescence, place a third of the kidney, to ensure both cortical and medullary glomeruli will be present, into the tissue mold and coat in OCT. Place on dry ice to freeze and store at -80 °C.
9. For protein and RNA, place 3 x 2 mm³ pieces of kidney cortex into 0.5 mL plastic tubes and snap freeze in liquid N₂. Store at -80 °C. For long-term tissue storage for RNA, place tissue in 5 volumes of RNA stabilization solution and store at -80 °C.
10. For isolation of glomeruli, slice up the remaining kidney tissue and place in 5 mL of mammalian Ringer's solution with 1% BSA on ice. Prepare to sieve glomeruli immediately.

3. Plasma Creatinine

NOTE: Plasma creatinine can be up-regulated in renal disease, indicating a reduction in the filtration capacity of the glomeruli. The blood urea nitrogen (BUN) levels can also be assessed, although the protocol is not described here.

1. Centrifuge the blood sample at 500 x g for 15 min at 4 °C.
2. Collect the plasma, which can be stored at -20 °C in the short-term at this point.
3. Quantify the plasma creatinine concentration using a chemical creatinine assay per the instructions above for urinary creatinine in protocol 1.11.
4. Determine the creatinine concentration of each sample by reading the plate at an absorbance of 490 nm before and after the addition of the acid solution.
   NOTE: The difference between these absorbance values is directly proportional to the creatinine concentration in each sample. A standard curve is generation from the standards. If the technical repeats have a CV value greater than 5%, repeat the assay for those samples.

4. Isolation of Glomeruli

NOTE: Glomeruli can be isolated to assess the permeability of individual glomeruli ex vivo, as well as the expression of specific protein and mRNA markers of glomerular disease.

1. Take the kidney tissue placed in mammalian Ringer's solution with 1% BSA and dissect the glomeruli using a standard sieving technique¹³. Briefly, stack the 70 µm (bottom), 100 µm, 125 µm, 175 µm, 250 µm, and 425 µm (top) sieves on to a glass beaker.
2. Mash the kidney, using a syringe plunger, into the 425 µm sieve and push through using ice cold mammalian Ringer's solution with 1% BSA. As the bits of kidney are pushed through, remove the top sieve and proceed to do the same on the next. Repeat until only the 100 µm and 70 µm sieves remain.
3. Transfer the glomerular harvest retained by the 100 µm and 70 µm sieves to 10 mL of fresh mammalian Ringer's solution with 1% BSA, on ice.
   NOTE: If the number of glomeruli per mL Ringer's solution is few, reduce the volume of Ringer's solution used to collect the glomeruli from the last two sieves.
4. Remove 5 mL of the solution containing glomeruli into two separate tubes (2.5 mL each) and centrifuge at 1,000 x g for 10 min at 4 °C. Remove the supernatant and snap freeze the glomeruli in liquid N₂ before storing at -80 °C for protein and RNA extraction at a later date.
5. Place the remaining solution containing glomeruli in a water bath at 37 °C for measurement of the glomerular L_pA/V_i ex vivo. Complete within 3 h of removing the kidney.

5. Glomerular Water Permeability (L_pA/V_i)

NOTE: The glomerular L_pA/V_i assay enables the ex vivo measurement of the permeability of individual glomeruli in a fast a reproducible manner. An increase in the glomerular L_pA/V_i indicates disruption of the GFB, which is suggestive of renal disease.

1. Set up the glomerular L_pA/V_i rig as described in Salmon et al¹⁰. Please refer to Figure 1 for a detailed diagram of the set up.
2. Prepare the following solutions: mammalian Ringer's solution with 1% BSA (pH 7.4) and mammalian Ringer's solution with 8% BSA (pH 7.4). Warm both to 37 °C.
3. Pull micropipettes from glass capillary tubes (optical density: 1.2 mm). Generate a 5 – 8 µm aperture tip by cutting the micropipette under a microscope.
4. Use the glomerular L_pA/V_i rig to catch intact individual glomeruli that are free of Bowman's capsule and tubular fragments onto the micropipette using suction. A detailed summary of the oncometric assay is found in Salmon et al¹⁰. In brief, once a glomerulus is caught and secured on the suction micropipette, begin recording the video of the glomerulus under the microscope.
5. Firstly, equilibrate the glomerulus in the 1% BSA Ringer's solution for 30 s before switching the perifusate to the concentrated 8% BSA Ringer's solution for 10 s. Then switch the perifusate back to 1% BSA Ringer's solution and stop the recording.
6. Wash the glomerulus away and repeat the process for 10 – 15 glomeruli per mouse. Ensure the perifusate flow rates are identical and not to fast (10 mL/min) so as not to distort the glomerular structure.
7. Measure the initial rate of glomerular shrinkage to calculate the glomerular water permeability (L_pA) normalized to the glomerular volume (V_i). Detailed information regarding the analysis can be found in Salmon et al¹⁰.

6. Periodic Acid Schiff (PAS) Stain

NOTE: The PAS stain will highlight the basement membranes of glomerular capillary loops and the tubular epithelium. It enables detailed visualization of the glomerular cells, mesangial matrix and potential expansion, and potential changes of the GBM (i.e., thickening and irregularities).

1. Section the paraffin-embedded, PFA-fixed kidney cortex using a microtome at 5 µm thickness onto poly-L-lysine coated slides. Dry at 37 °C for 1 h. Ensure the section does not contain any folds or holes, which can distort the morphology under the microscope.
2. Deparaffinize slides by incubating twice in xylene (CAUTION, irritant; use in fume cabinet) for 3 min each, twice in 100% EtOH for 3 min each, and then once in 95%, 70%, and 50% EtOH for 3 min each, all at room temperature. Re-hydrate the slides in dH₂O.
3. Incubate the slides in periodic acid solution (CAUTION, irritant; use in fume cabinet) (1 g/dL) for 5 min, and then rinse the slides in several changes of dH₂O. Use a container with 100 mL dH₂O at room temperature.
   CAUTION: periodic acid solution: irritant; use in fume cabinet
4. Incubate slides in Schiff's reagent (Parasoaniline HCl 6 g/L and sodium metabisulfite 4% in HCl 0.25 mol/L) for 15 min at room temperature. Wash slides in running tap water for 5 min.
5. Counterstain with Hematoxylin for 3 s before thoroughly rinsing slides in running tap water for 15 min.
   NOTE: Some optimization may be required to determine the optimal time for Hematoxylin staining.
6. Dehydrate slides using the reverse of the deparaffinization protocol in step 6.2. Finish with xylene.
7. Air dry slides and mount with xylene-based mounting media.
8. Image on a light microscope at 400X magnification to assess glomerular structures. Evaluate the following: thickening and irregularities of the GBM, collapsing of capillary loops, fibrotic tissue, sclerosis, cellular proliferation (endothelial, podocyte, and mesangial, or inflammatory cells infiltrating the tuft).
   NOTE: For a comprehensive evaluation of glomerular pathophysiology, lesions elsewhere in the kidney should be evaluated, such as in the tubules.

7. Transmission Electron Microscopy (TEM)

NOTE: TEM allows the examination of ultra-structural abnormalities in the kidney, such as the GBM, podocyte foot processes, and endothelial fenestrations, which are not visible with light microscopy. This is important in models where renal damage is not so pronounced (i.e., no albuminuria and major structural abnormalities).

1. Take the 2.5% gluteraldehdye- fixed diced kidney and post-fix in 1% osmium tetroxide for 1 h. Wash in 50 mL of 0.1 M cacodylate buffer (pH 7.3) and then 50 mL of dH₂O (3 x 15 min changes).
   CAUTION: 2.5% glutaraldehyde: toxic, sensitizer, irritant; use in a fume cabinet. 0.1 M sodium cacodylate: toxic, use in a fume cabinet
2. Dehydrate with EtOH and embed in Araldite resin.
3. Cut sections at 50 – 100 nm thickness and stain with 3% (aqueous) uranyl acetate and Reynolds' lead citrate solution.
4. Take digital micrographs over several areas of the glomerulus at 940X, 1250X, and 6200X to be sure the podocytes, GEnCs, GBM, and mesangium can be identified.
5. Use ImageJ to analyze the blinded glomeruli. Set the scale for each 6200X micrograph by drawing a line between two points of known distance, such as a ruler. Go to analyze, and then set scale, where the length of the line will be displayed in pixels. Type in the known distance and units of measure. Use the protocols listed below for the measurement of each parameter.
   NOTE: This analysis requires around 1 day per mouse. For adequate statistical power, use the average measurements from 3 glomeruli from 3 mice.
   1. For GBM, insert a fixed digital grid (10 x 10) over the 6200X micrograph and measure the thickness of the GBM at the point where the grid lines cross the GBM. Measure from the basal endothelial cell membrane to the basal podocyte foot process cell membrane in a perpendicular tangent to the endothelial cell membrane using the straight line tool. Determine the mean measurement for each glomerulus from 10 individual measurements.
   2. For the endothelial fenestration number, measure the length of the GBM present in the 6200X micrograph and count the number of endothelial fenestrations per unit length of GBM. Take an average from at least 4 micrographs per glomerulus.
   3. For the podocyte foot process width, insert a fixed digital grid (10 x 10) over the 6200X micrograph. Measure the width of the podocyte foot processes that cross the grid lines. Measure the width at the widest part of the foot process where it meets the GBM; ensure the line is perpendicular to the tangent of the podocyte basal membrane. Determine the mean measurement for each glomerulus from 10 individual measurements.
   4. For podocyte slit width, insert a fixed digital grid (10 x 10) over the 6200X micrograph. Measure the width of the podocyte slit diaphragms that cross the grid lines. This is the point where the foot processes are closest together, at the widest part of each foot process, from podocyte membrane to membrane. Ensure the measurement is perpendicular to the tangent of the podocyte basal membrane. Determine the mean measurement for each glomerulus from 10 individual measurements.
   5. For the number of podocyte foot processes, measure the length of the GBM present in the 6200X micrograph and count the number of podocyte foot processes per unit length of GBM. Take an average from at least 4 micrographs per glomerulus.
   6. For the sub-podocyte space coverage, see detailed method in Neal et al.¹⁴.
6. Using the 940X micrographs, examine glomeruli for the presence of abnormal structure, deposits, and infiltrates by eye.

8. Immunofluorescence for Podocyte and Endothelial Markers

NOTE: Immunostaining allows visualization of the protein expression patterns, such as endothelial capillary loops, which can collapse in glomerular disease.

1. Place the OCT-mold containing frozen kidney at -20 °C for 2 h prior to sectioning. Ensure the cut surface of the kidney is carefully placed against the bottom of the OCT-mold to enable well orientated tissue sections.
2. Using a cryostat, section tissue at a 5 µm thickness on to poly-L-lysine coated slides.
   NOTE: Ensure there are no folds or holes in the tissue section, which can distort the morphology.
3. Upon removal from the cryostat, fix slides in 4% PFA for 10 min. Wash slides 3 x 5 min in a container with 100 mL of dH₂O.
   CAUTION: 4% PFA: fixative, use in fume cabinet
4. To minimize the amount of antibody used, draw around sections with a hydrophobic pen. Do not let the sections dry.
5. Incubate in blocking solution (3% BSA and 5% normal serum in 1x PBS) for 1 h at room temperature.
6. Remove the blocking solution with an aspirator and incubate sections with primary antibody (Nephrin, podocin, or PECAM-1) diluted 1:250 (3% BSA in 1x PBS). Place slides in a humidified chamber at 4 °C overnight. If a humid chamber is not available, lightly place a small strip of parafilm over the antibody-coated slide. Take care when removing the next day as to not disrupt the section.
7. Wash slides 3 x 5 min in 1x PBS.
8. Incubate with appropriate fluorescent secondary antibody dilution 1:1000 (3% BSA in 1x PBS) for 2 h at room temperature in the dark.
9. Wash slides 3 x 5 min in 1x PBS. Mount with fluorescent mounting media containing DAPI.
10. Image slides with a fluorescent microscope at 400X magnification to view the glomeruli. Ensure this is blinded to avoid bias.
11. Use ImageJ to analyze the staining intensity, normalized to the glomerular area, and the pattern of staining, i.e., number of capillary loops normalized to the glomerular area, in a blinded manner.

9. Protein Extraction and Western Blotting

NOTE: Western blotting allows us to assess the expression proteins known to be dysregulated in renal disease. For example, a reduction in podocin and nephrin expression indicates podocyte loss.

1. Extract protein from kidney cortex and sieved glomeruli; the protocol is the same for each and the volume of lysis buffer is adjusted for the amount of tissue.
2. Thaw kidney/glomeruli on ice before adding NP-40 lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris pH 8) containing protease and phosphatase inhibitors. Homogenize the sample for 30 s.
3. Incubate the homogenized samples on ice for 30 min, vortexing at regular intervals.
4. Centrifuge the samples at 10,000 x g for 15 min at 4 °C.
5. Remove the supernatant to a fresh tube on ice.
   NOTE: expect to recover approximately 1 mg protein per sample.
6. Denature the proteins using the standard 4x Laemmli buffer. Boil the mixture at 95 – 100 °C for 5 min.
7. Assess the expression of glomerular cell marker proteins (Nephrin, Podocin, PECAM-1, etc.) and the phosphorylation and expression of proteins known/hypothesized to be altered in the kidney/glomeruli of the disease model using Western blotting (standard method; Mahmood and Yang¹⁵).
   NOTE: The protocol will vary depending on the size and abundance of the protein of interest.

10. RNA Extraction and Polymerase Chain Reaction (PCR)

NOTE: mRNA expression analysis allows us to determine how genes are regulated in renal disease, such as changes in gene expression and alternative splicing.

1. Whilst the kidney cortex is still frozen, thoroughly grind in 3 mL of phenol reagent using a pestle and mortar. If using glomerular extracts, add 1 mL of phenol reagent and homogenize the sample for 30 s.
   CAUTION: TRIzol reagent: irritant; use in fume cabinet
2. Perform an RNA extraction using the method described by Chomczynski and Sacchi¹⁶.
   NOTE: Commercial RNA extraction kits are available as an alternative to this method.
3. Assess the quantity and quality of RNA obtained using one of the various methods available. RNA is aliquoted and stored at -80 °C at this point. Avoid repeat freeze thawing.
   NOTE: If new to this method, check the quality of the RNA before proceeding to the next step by running the RNA on an agarose gel to ensure a clear 28S and 18S ribosomal band. Expect to recover between 2 to 5 µg of RNA using this method.
4. DNase treat 1 µg of RNA (make volume up to 10 µL with RNase-free water plus 1 µL of DNase and 1 µL of DNase buffer) for 1 h at 37 °C. Stop the reaction with 1 µL of DNase stop solution at 65 °C for 10 min.
5. Add 0.5 µL of oligo (dT) and random primers. Incubate at 70 °C for 10 min.
6. Immediately quench on ice for 5 min.
7. Add the following; MMLV reverse transcriptase enzyme (400 U; replace with DEPC H₂O in the RT – control sample), MMLV buffer (1x), dNTP mix (0.5 mM), and ribonuclease inhibitor (40 U); make up to 50 µL with DEPC water.
8. Incubate reaction mix at 37 °C for 1 h followed by 95 °C for 5 min to deactivate the enzyme.
   NOTE: To generate a higher yield of cDNA, incubate at 37 °C for up to 3 h.
9. Assess the quantity and quality of cDNA using the various methods available.
10. Use PCR to assess the mRNA expression and splicing patterns of genes hypothesized to be dysregulated in the glomerular disease model. The protocol will vary depending on the gene of interest.