This method can help answer key questions in field of glomerular disease regarding the functional and structural phenotype of the glomerulus and allows for the mechanistic analysis of kidney pathology. The main advantage of this technique is that it is adaptable to all rated models of glomerular disease. For assessment of the urinary albumin creatinine ratio, place one six to eight week old male mouse into a metabolic mouse cage containing water and enrichment diet food in a quiet room.
After six hours, return the mouse to their regular housing and collect a minimum of 50 microliters of the baseline urine sample from each cage, repeating the urine collection from once weekly to once monthly. At the experimental endpoint, harvest the kidneys from the abdomen of each animal and wash the organs in ice cold PBS. To examine the cortical glomeruli, remove and dice one pole of the kidney cortex into one cubic millimeter pieces.
To examine the deep juxtamedullary glomeruli, remove and dice a small section of the medulla into one cubic millimeter pieces. Then, place the fragments from each section of tissue into individual electron microscopy vials containing five milliliters of 2.5%glutaraldehyde solution and store the samples at four degrees Celsius. For histology of the cortical and juxtamedullary glomeruli, remove and fix the upper third of the kidney in five milliliters of 4%paraformaldehyde at four degrees Celsius for 24 hours.
Then, transfer the fixed tissue into five milliliters of 70%ethanol for 24 hours before embedding in paraffin. For immunofluoro chemical analysis of the cortical and medullary glomeruli, place a third of the kidney into a tissue mold and submerge the tissue in optimal cutting temperature compound. Then, place the mold on dry ice until the compound is frozen and store the samples at 80 degrees Celsius until they are sectioned.
For protein analysis, place three by two cubic millimeter pieces of kidney cortex into 0.5 milliliter plastic tubes and snap freeze the samples in liquid nitrogen for storage at 80 degrees Celsius. For long term tissue storage for RNA analysis, after snap freezing three by two cubic millimeter kidney pieces as just demonstrated, add five volumes of RNase stabilization solution for 80 degree Celsius storage. For glomeruli isolation, slice the remaining kidney tissue and place the tissue pieces in five milliliters of mammalian ringer's solution supplemented with 1%bovine serum albumin, or BSA, on ice for immediate sieving.
To isolate the glomeruli, stack sieves with ascending micron pore sizes onto a glass beaker and place the kidney slices onto the top 425 micron pore sized sieve. Next, use a syringe plunger and fresh ice cold mammalian ringers solution supplemented with 1%BSA to mash the kidney tissue through the first sieve. As the bits of kidney are pushed through, remove the top sieve and continue to push the kidney fragments through each sieve in turn.
When only the 100 and 70 micron sieves remain, transfer the glomerular harvest retained by the last two sieves into 50 milliliter conical tube with 10 milliliters of fresh mammalian ringer's solution supplemented with 1%BSA on ice. Split three milliliters of the glomeruli suspension between two 1.5 milliliter microcentrifuge tubes and pellet the glomeruli by centrifugation. After removing the supernatant, snap freeze the glomeruli in liquid nitrogen for 80 degrees Celsius storage for later protein and RNA extraction.
Then, place the remaining glomeruli solution into a 37 degree Celsius water bath for injection into a glomerular water permeability rig on a light microscope stage. Upon capture of an intact, individual glomerulus, free a Bowman's capsule and tubular fragments by micropipette, begin recording, and perfuse the glomerulus with fresh ringer's solution supplemented with 1%BSA. After 30 seconds, switch the perfusate to a concentrated 8%BSA ringer's solution.
After 10 seconds of perfusion, switch the perfusate back to 1%BSA ringer's solution and stop the recording. For detailed visualization of the glomerular cells and residual matrix, dry the fixed, paraffin-embedded kidney cortex samples at 37 degrees Celsius for one hour, followed by deparaffinization with consecutive xylene and descending ethanol immersions. Rehydrate the samples in distilled water for five minutes and incubate the slides in periodic acid solution in a fume cabinet.
After five minutes, rinse the slides with three five minute washes in 100 milliliters of distilled water, followed by a 15 minute incubation in Schiff's reagent. At the end of the incubation, wash the slides in running tap water for five minutes and counter stain with hematoxylin for three seconds before thoroughly rinsing in running tap water for another 15 minutes. Then, dehydrate the slides with ascending ethanol immersions and two consecutive three minute xylene immersions.
After air drying, the slides can be mounted with xylene-based mounting medium for assessment of their glomerular structure on a light microscope at a 400x magnification. Vascular endothelial growth factor, or VEGF-A, knockout mice develop progressive albuminuria by 10 weeks, compared to wild-type litter mate controls, whereas VEGF165b overexpressing knockout animals are protected from this condition. VEGF-A knockout mice have a significantly increased glomerular water permeability compared to wild-type controls, which is partially rescued by VEGF-A165b overexpression.
Periodic acid Schiff staining of kidney cortex sections in VEGF-A knockout mice does not reveal any glomerular structural abnormalities by light microscopy analysis. Upon electron microscopy analysis, however, the knockout mice demonstrate an increased glomerular basement membrane width, a decreased number of endothelial fenestrae, a decreased subpodocyte space coverage, and an increased average podocyte slit width, while the average number of slits remained unchanged. VEGF165b overexpression in the VEGF-A knockout animals rescues the changes in glomerular basement membrane and slit width.
However, VEGF165b overexpression does not effect the altered fenestrae numbers or subpodocyte space coverage. RNA extracted from sieved glomeruli confirms human VEGF165b mRNA expression in the VEGF165b overexpressing knockout mice, correlating with a decreased expression of VEGFR2 in the VEGF-A knockout mice that is rescued by VEGF165b overexpression in the knock in animals. After watching this video, you should have a good understanding of how to complete a full kidney workup for the evaluation of a murine model of glomerular disease, including an assessment of the glomerular permeability to both albumin and water.
