Computational protocol: Progression of Alport Kidney Disease in Col4a3 Knock Out Mice Is Independent of Sex or Macrophage Depletion by Clodronate Treatment

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[…] Urine was sampled weekly and analyzed for albumin, creatinine, NGAL, and KIM-1. Albumin was measured using the Albuwell M kit (Exocell, Philadelphia, PA, USA) and normalized to creatinine levels analyzed by Aution urine analysis system (Arkray, Kyoto, Japan).Mouse KIM-1 was analyzed with an immunoassay using an anti-mouse rat monoclonal and an anti-mouse goat polyclonal as the capture and detection reagent, respectively (R&D Systems, Minneapolis, MN, USA) on an SI6000 from Mesoscale Discovery (MSD, Rockville, MD, USA). 30 μL of capture antibody (4 μg/mL in PBS) was incubated overnight on MSD standard plates at 4°C. The plate was washed 3X with PBS followed by the addition of 25 μl of urine (1:4 dilution in MSD diluent 5) and incubated for 1h at room temp. Incubation with the secondary antibody for 1h was followed by a wash and application of MSD Sulfo-Tagged anti-goat antibody for 1h. After another wash, 150 μl of MSD Read Buffer T was added, and the plates read on an MSD SI 6000. Data were analyzed on MSD Discovery Workbench software. NGAL and Albumin were assayed at 1:1000 and 1:100 dilutions, respectively as per manufacturer’s instruction using kits from Bioporto (Hellerup, Denmark) and Abnova (Taiwan, Republic of China), respectively. KIM-1 and NGAL were normalized to creatinine analyzed using the Urinary Detection Kit from Arbor Assays (Ann Arbor, MI, USA) at a 1:25 dilution in H2O and assayed as per manufacturer’s instructions. Plates were read on a SpectraMax M5 from Molecular Devices (Sunnyvale, CA, USA), analyzed with SoftMax Pro v5.Blood was sampled every two weeks from V. sublingualis into Microtainer tubes (BD biosciences, San Jose, CA, USA) under isoflurane anesthesia. Serum was used to measure blood urea nitrogen by Spotchem EZ Automated analyzer and Spotchem ll reagent strip (Arkray). [...] Kidneys were fixed in 10% buffered formalin for 48h at room temp and processed for embedding in paraffin using standard procedures. Immunohistochemical staining for F4/80 was performed using an automated Ventana Discovery XT Platform (Ventana medical systems, Tucson, AZ, USA). Sections, pretreated with protease (Ventana), were incubated with Peroxidazed 1 (Biocare medical, Concord, CA, USA) and stained with monoclonal rat anti-mouse F4/80 antibodies (1:100; #MCA497G, ABD Serotec, Oxford, UK) for 48 min. Reaction was detected with the OmniMap anti-Rt HRP (Ventana) and ChromoMap DAB Kit (Ventana), followed by counterstaining with hematoxylin. For α-SMA staining, sections were treated with 0.5% H2O2 in methanol for 20 min, followed by 20 min incubation with monoclonal mouse anti-human α-SMA antibodies (1:25; #M0851, DAKO, Glostrup, Denmark), detection by ARK™ Peroxidase kit (DAKO), and counterstaining with hematoxylin. For Sirius red staining, Picrosirius Red solution and 0.04% Light Green solution (EMS, Hatfield, PA, USA) were used according to the manufacturer’s recommendations. Sections were stained with hematoxylin and eosin (H&E) and Periodic acid-Schiff (PAS) using standard protocols. Digital images were obtained with a ScanScope XT system (Leica, Nussloch, Germany). For Immunofluorescence staining, sections were incubated overnight at 4°C with monoclonal rat-anti mouse F4/80 antibodies (1:20; #MCA497G, ABD Serotec, Oxford, UK) and polyclonal goat-anti mouse CD206 antibodies (1:100; #AF2535, R&D systems, Minneapolis, MN, USA) followed by 1h incubation with donkey anti-rat IgG Alexa fluor 549 (1:500, Jackson Immunoresearch, Westgrove, PA, USA) and donkey anti-goat IgG Alexa fluor 488 (1:500, Life technologies, Gaithersburg, MD, USA) and counterstaining with hematoxylin. Digital images were obtained with LSM700 confocal microscope (Carl Zeiss Microscopy, Gottingen, Germany). [...] For the quantification of Sirius red and chromogenic staining, area %, defined as stained area per total surface area of entire kidney including cortex and medulla, was obtained with Image Scope software (Leica) using the Positive Pixel Count algorithm. For the analysis of F4/80/CD206 staining, five representative images from kidneys sections were analyzed by ImageJ (NIH, Bethesda, MD, USA). Colocalization Threshold plugin of ImageJ was used to calculate the F4/80+/CD206+ area. F4/80+/CD206- was calculated as a difference between F4/80+ and F4/80+/CD206+ area. The data were presented as Area %. […]

Pipeline specifications

Software tools SoftMax Pro, chromoMap, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Diseases Kidney Diseases, Nephritis, Hereditary, Genetic Diseases, Inborn, Renal Insufficiency
Chemicals Clodronic Acid